Journal of Experimental Botany - recent issues

New insights on the effects of heat stress on crops

Halford, N. G. — Fri, 30 Oct 2009 06:38:02 PDT

Symbolism of plants: examples from European-Mediterranean culture presented with biology and history of art: DECEMBER: Cross-roses and rose crosses

Kandeler, R., Ullrich, W. R. — Fri, 30 Oct 2009 06:38:02 PDT

Symbolism of plants: examples from European-Mediterranean culture presented with biology and history of art: EPILOGUE

Kandeler, R., Ullrich, W. R. — Fri, 30 Oct 2009 06:38:02 PDT

Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls

Fri, 30 Oct 2009 06:38:02 PDT

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO₂-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO₂-Tyr in hypocotyls was 0.56 µmol mg^–1 protein and 0.19 pmol mg^–1 protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

The alternative respiratory pathway allows sink to cope with changes in carbon availability in the sink-limited plant Erythronium americanum

Gandin, A., Lapointe, L., Dizengremel, P. — Fri, 30 Oct 2009 06:38:02 PDT

Mechanisms that allow plants to cope with a recurrent surplus of carbon in conditions of imbalance between source and sink activity has not received much attention. The response of sink growth and metabolism to the modulation of source activity was investigated using elevated CO₂ and elevated O₃ growth conditions in Erythronium americanum. Sink activity was monitored via slice and mitochondrial respiratory rates, sucrose hydrolysis activity, carbohydrates, and biomass accumulation throughout the growth season, while source activity was monitored via gas exchanges, rubisco and phosphoenolpyruvate carboxylase activities, carbohydrates, and respiratory rates. Elevated CO₂ increased the net photosynthetic rate by increasing substrate availability for rubisco. Elevated O₃ decreased the net photosynthetic rate mainly through a reduction in rubisco activity. Despite this modulation of the source activity, neither plant growth nor starch accumulation were affected by the treatments. Sucrose synthase activity was higher in the sink under elevated CO₂ and lower under elevated O₃, thereby modulating the pool of glycolytic intermediates. The alternative respiratory pathway was similarly modulated in the sink, as seen with both the activity and capacity of the pathway, as well as with the alternative oxidase abundance. In this sink-limited species, the alternative respiratory pathway appears to balance carbon availability with sink capacity, thereby avoiding early feedback-inhibition of photosynthesis in conditions of excess carbon availability.

Polyamine oxidase activity contributes to sustain maize leaf elongation under saline stress

Rodriguez, A. A., Maiale, S. J., Menendez, A. B., Ruiz, O. A. — Fri, 30 Oct 2009 06:38:02 PDT

The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H₂O₂, ·O₂^–, and HO· production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 µM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 µM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl₂ (an ·O₂^– dismutating agent) or the HO· scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25–30% of the maize leaf blade elongation.

Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain

Waters, B. M., Uauy, C., Dubcovsky, J., Grusak, M. A. — Fri, 30 Oct 2009 06:38:02 PDT

The NAM-B1 gene is a NAC transcription factor that affects grain nutrient concentrations in wheat (Triticum aestivum). An RNAi line with reduced expression of NAM genes has lower grain protein, iron (Fe), and zinc (Zn) concentrations. To determine whether decreased remobilization, lower plant uptake, or decreased partitioning to grain are responsible for this phenotype, mineral dynamics were quantified in wheat tissues throughout grain development. Control and RNAi wheat were grown in potting mix and hydroponics. Mineral (Ca, Cu, Fe, K, Mg, Mn, P, S, and Zn) and nitrogen (N) contents of organs were determined at regular intervals to quantify the net remobilization from vegetative tissues and the accumulation of nutrients in grain. Total nutrient accumulation was similar between lines, but grain Fe, Zn, and N were at lower concentrations in the NAM knockdown line. In potting mix, net remobilization of N, Fe, and Zn from vegetative tissues was impaired in the RNAi line. In hydroponics with ample nutrients, net remobilization was not observed, but grain Fe and Zn contents and concentrations remained lower in the RNAi line. When Fe or Zn was withheld post-anthesis, both lines demonstrated remobilization. These results suggest that a major effect of the NAM genes is an increased efflux of nutrients from the vegetative tissues and a higher partitioning of nutrients to grain.

Axillary bud outgrowth potential is determined by parent apical bud activity

Thomas, R. G., Hay, M. J. M. — Fri, 30 Oct 2009 06:38:03 PDT

Axillary buds within a plant shoot system are known to differ in their ability to respond to treatments favouring their development. This ability is referred to as their outgrowth potential. Using two species of prostrate nodally-rooting herbs, dicotyledonous Trifolium repens and monocotyledonous Tradescantia fluminensis, grown throughout in a strictly vegetative state, this study tested two hypotheses. Hypothesis 1: that each axillary bud exhibits an outgrowth potential that is directly related to the growth rate of its parent apical bud, and Hypothesis 2: that the growth rate attained by an axillary bud depends upon both its outgrowth potential and the local supply of stimulatory root-derived signal (NRS) available to it. Activation levels (growth rates) of apical buds were varied by differential exposure to nodal roots and the outgrowth responses of axillary buds recently emerged from them were then measured under standardized conditions of NRS supply. Hypothesis 1 was shown to be correct for both species. Hypothesis 2, tested only in T. repens, was supported by results showing that an axillary bud's outgrowth potential and the NRS supply to it each independently influenced its growth rate, there being no significant interaction between the two. These results emphasize the significant role the physiological state/activity of apical buds has on the outgrowth potential of axillary buds formed within them. The fact that similar relationships were observed on axillary buds on stems of differing developmental maturity and branching hierarchy, and in two taxonomically diverse species, suggests they might be widespread among morphologically similar species.

Effects of {beta}-1,3-glucan from Septoria tritici on structural defence responses in wheat

Fri, 30 Oct 2009 06:38:03 PDT

The accumulation of the pathogenesis-related (PR) proteins β-1,3-glucanase and chitinase and structural defence responses were studied in leaves of wheat either resistant or susceptible to the hemibiotrophic pathogen Septoria tritici. Resistance was associated with an early accumulation of β-1,3-glucanase and chitinase transcripts followed by a subsequent reduction in level. Resistance was also associated with high activity of β-1,3-glucanase, especially in the apoplastic fluid, in accordance with the biotrophic/endophytic lifestyle of the pathogen in the apoplastic spaces, thus showing the highly localized accumulation of defence proteins in the vicinity of the pathogen. Isoform analysis of β-1,3-glucanase from the apoplastic fluid revealed that resistance was associated with the accumulation of an endo-β-1,3-glucanase, previously implicated in defence against pathogens, and a protein with identity to ADPG pyrophosphatase (92%) and germin-like proteins (93%), which may be involved in cell wall reinforcement. In accordance with this, glycoproteins like extensin were released into the apoplast and callose accumulated to a greater extent in cell walls, whereas lignin and polyphenolics were not found to correlate with defence. Treatment of a susceptible wheat cultivar with purified β-1,3-glucan fragments from cell walls of S. tritici gave complete protection against disease and this was accompanied by increased gene expression of β-1,3-glucanase and the deposition of callose. Collectively, these data indicate that resistance is dependent on a fast, initial recognition of the pathogen, probably due to β-1,3-glucan in the fungal cell walls, and this results in the accumulation of β-1,3-glucanase and structural defence responses, which may directly inhibit the pathogen and protect the host against fungal enzymes and toxins.

The 'trade-off' between synthesis of primary and secondary compounds in young tomato leaves is altered by nitrate nutrition: experimental evidence and model consistency

Bot, J. L., Benard, C., Robin, C., Bourgaud, F., Adamowicz, S. — Fri, 30 Oct 2009 06:38:03 PDT

Plants allocate internal resources to fulfil essential, yet possibly conflicting, demands such as defence or growth, as hypothesized by the ‘growth–differentiation balance theory’ (GDB). This trade-off was examined in young tomato plants grown for 25 d using the nutrient film technique with seven nitrate concentrations ([NO₃]). The modification of primary (growth-related: organic acids, carbohydrates) and secondary (defence-related: phenolics) metabolite concentrations in leaves was assessed. Then a simple model was devised to simulate the trade-off between growth and secondary metabolism in response to N nutrition. N affected growth and metabolite concentrations in the leaves. Dry biomass, leaf area, and concentrations of nitrate and organic acid (malic, citric) increased with rising [NO₃], up to a threshold, above which they remained constant. Starch, sucrose, and organic N concentrations were invariant with [NO₃]. Glucose, fructose, and phenolic (chlorogenic acid, rutin, and kaempferol-rutinoside) concentrations were highest at lowest [NO₃]. They declined progressively with rising [NO₃] until a threshold, above which they remained constant. Model predictions are in phase with experimental phenolic concentration data although the simulated metabolic rates differ from the GDBH proposals depicted in the literature. From the model output it is shown that a careful definition of the C reserve compounds is required.

Biochemical and proteomic analysis of 'Dixiland' peach fruit (Prunus persica) upon heat treatment

Fri, 30 Oct 2009 06:38:03 PDT

Shipping of peaches to distant markets and storage require low temperature; however, cold storage affects fruit quality causing physiological disorders collectively termed ‘chilling injury’ (CI). In order to ameliorate CI, different strategies have been applied before cold storage; among them heat treatment (HT) has been widely used. In this work, the effect of HT on peach fruit quality as well as on carbon metabolism was evaluated. When fruit were exposed to 39 °C for 3 d, ripening was delayed, with softening inhibition and slowing down of ethylene production. Several differences were observed between fruit ripening at ambient temperature versus fruit that had been heat treated. However, the major effects of HT on carbon metabolism and organoleptic characteristics were reversible, since normal fruit ripening was restored after transferring heated peaches to ambient temperature. Positive quality features such as an increment in the fructose content, largely responsible for the sweetness, and reddish coloration were observed. Nevertheless, high amounts of acetaldehyde and low organic acid content were also detected. The differential proteome of heated fruit was characterized, revealing that heat-induced CI tolerance may be acquired by the activation of different molecular mechanisms. Induction of related stress proteins in the heat-exposed fruits such as heat shock proteins, cysteine proteases, and dehydrin, and repression of a polyphenol oxidase provide molecular evidence of candidate proteins that may prevent some of the CI symptoms. This study contributes to a deeper understanding of the cellular events in peach under HT in view of a possible technological use aimed to improve organoleptic and shelf-life features.

Differences in C metabolism of ash species and provenances as a consequence of root oxygen deprivation by waterlogging

Jaeger, C., Gessler, A., Biller, S., Rennenberg, H., Kreuzwieser, J. — Fri, 30 Oct 2009 06:38:03 PDT

The waterlogging tolerance and the physiological responses to this stress were tested in seedlings of Fraxinus angustifolia, an ash tree inhabiting riparian forests, and two provenances of the closely related Fraxinus excelsior, one derived from a riparian forest (FER) and one from a mountainous region (FEM). Besides visible damage, physiological parameters reflecting adaptations of plants to waterlogging such as net CO₂ assimilation, alcoholic fermentation, and the concentrations of metabolites related to flooding responses were studied. Consistent with the higher flooding tolerance of F. angustifolia and FER compared with FEM, net assimilation remained unaffected in F. angustifolia, was slightly reduced in FER, but was strongly affected in FEM. Altered carbohydrate concentrations in the roots of the seedlings suggest differences in the ability to supply alcoholic fermentation with substrate during prolonged periods of soil anoxia. Another difference between the seedlings was connected to the -aminobutyric acid (GABA) shunt which resulted in alanine accumulation in the flooding-tolerant trees, but strong GABA accumulation in the more sensitive FEM seedlings. This finding indicates differences in GABA conversion into alanine which might result in an accumulation of phytotoxic levels of intermediates. Such provenance-specific differences in Common ash suggest that the selection of appropriate provenances is essential for forest management in flood-prone areas.

Brassinosteroids interact negatively with jasmonates in the formation of anti-herbivory traits in tomato

Fri, 30 Oct 2009 06:38:04 PDT

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpyxjai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA–BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.

Variability among species in the apoplastic pH signalling response to drying soils

Sharp, R. G., Davies, W. J. — Fri, 30 Oct 2009 06:38:04 PDT

After the imposition of soil drying treatments, an elevation of xylem sap pH is one of the earliest observable responses in many herbaceous model plant species. It is theorized that alkalization of sap results in a concurrent elevation in abscisic acid (ABA) concentration delivered to transpiring tissues by preventing Henderson–Hasselbalch-regulated partitioning between the apoplast and symplast. However, here it is demonstrated that the sap alkalzation response to soil drying is far from universal in higher plant species. Tests were conducted to determine how universal the pH response to drying soil was in a range of perennial species from a diverse range of plant families. The response was not found in the majority of the 22 species tested. Four species exhibited significant increases in pH, but the majority showed no significant change in xylem sap pH. There was no evolutionary relationship between the species that showed alkalization under drought stress. However, the species that alkalized sap also exhibited good control over internal water status and were the most isohydric species of those tested. None of the species exhibiting anisohydric responses alkalized xylem sap under drought stress. Regardless of alkalization response, plants still retain the ability to respond to changes in xylem sap pH when manipulated by alkaline buffer foliar sprays. This finding indicates that plants have conserved the ability to respond to changes in xylem pH and redistribute ABA, even if they do not currently utilize the mechanism when exposed to drought stress. It was found in Buddleja davidii, Euonymus fortunei, and Hydrangea serrata that the xylem sap pH response to water deficits mirrored the natural pH changes that occur as sap is transported to the leaves, indicating that plants need to be able to have naturally occurring alkalization processes in place for them to be up-regulated under drought stress.

Regulation of oleosin expression in developing peanut (Arachis hypogaea L.) embryos through nucleosome loss and histone modifications

Fri, 30 Oct 2009 06:38:04 PDT

Nucleosome loss and histone modifications are important mechanisms for transcriptional regulation. Concomitant changes in chromatin structures of two peanut (Arachis hypogaea L.) oleosin genes, AhOleo17.8 and AhOleo18.5, were examined in relation to transcriptional activity. Spatial and temporal expression analyses showed that both AhOleo17.8 and AhOleo18.5 promoters can adopt three conformational states, an inactive state (in vegetative tissues), a basal activated state (in early maturation embryos), and a fully activated state (in late maturation embryos). Chromatin immunoprecipitation assays revealed an increase of histone H3 acetylation levels at the proximal promoters and coding regions of AhOleo17.8 and AhOleo18.5 associated with basal transcription in early maturation embryos. Meanwhile, a decrease of histone H3K9 dimethylation levels at coding regions of oleosins was observed in early maturation embryos. However, a dramatic decrease in the histone acetylation signal was observed at the core promoters and the coding regions of the two oleosins in the fully activated condition in late maturation embryos. Although a small decrease of histone H3 levels of oleosins chromatin was detected in early maturation embryos, a significant loss of histone H3 levels occurred in late maturation embryos. These analyses indicate that the histone eviction from the proximal promoters and coding regions is associated with the high expression of oleosin genes during late embryos maturation. Moreover, the basal expression of oleosins in early maturation embryos is accompanied by the increase of histone H3 acetylation and decrease of histone H3K9me2.

Arabidopsis L-type lectin receptor kinases: phylogeny, classification, and expression profiles

Bouwmeester, K., Govers, F. — Fri, 30 Oct 2009 06:38:04 PDT

In plants, lectin receptor kinases are considered to play crucial roles during development and in the adaptive response to various stimuli. Arabidopsis lectin receptor kinases can be divided into three type-classes based on sequence similarity of their extracellular lectin motifs. The current study focuses on the legume-like lectin receptor kinases (LecRKs), which are regarded as ideal candidates for monitoring cell wall integrity and are possibly functional in adaptive responses. An inventory of the Arabidopsis LecRK gene family is presented here. It consists of 45 members including three that were recently identified; two encode N-terminal truncated variants one of which has two in tandem kinase domains. Phylogenetic trees derived from full-length amino acid sequence alignments were highly concordant to phylograms that were purely based on lectin motifs or kinase domains. The phylograms allowed reclassification of the LecRK genes and hence a new proposal for gene nomenclature was suggested. In addition, a comprehensive expression analysis was executed by exploring public repositories. This revealed that several LecRK genes are differentially expressed during plant growth and development. Moreover, multiple LecRKs appear to be induced upon treatment with elicitors and pathogen infection. Variation in gene expression was also analysed in seedlings of diverse Arabidopsis accessions. Taken together, this study provides a genome-wide overview of the LecRK gene family and an up-to-date classification using a novel and systematic gene nomenclature.

Functional diversity in gravitropic reaction among tropical seedlings in relation to ecological and developmental traits

Almeras, T., Derycke, M., Jaouen, G., Beauchene, J., Fournier, M. — Fri, 30 Oct 2009 06:38:04 PDT

Gravitropism is necessary for plants to control the orientation of their axes while they grow in height. In woody plants, stem re-orientations are costly because they are achieved through diameter growth. The functional diversity of gravitropism was studied to check if the mechanisms involved and their efficiency may contribute to the differentiation of height growth strategies between forest tree species at the seedling stage. Seedlings of eight tropical species were grown tilted in a greenhouse, and their up-righting movement and diameter growth were measured over three months. Morphological, anatomical, and biomechanical traits were measured at the end of the survey. Curvature analysis was used to analyse the up-righting response along the stems. Variations in stem curvature depend on diameter growth, size effects, the increase in self-weight, and the efficiency of the gravitropic reaction. A biomechanical model was used to separate these contributions. Results showed that (i) gravitropic movements were based on a common mechanism associated to similar dynamic patterns, (ii) clear differences in efficiency (defined as the change in curvature achieved during an elementary diameter increment for a given stem diameter) existed between species, (iii) the equilibrium angle of the stem and the anatomical characters associated with the efficiency of the reaction also differed between species, and (iv) the differences in gravitropic reaction were related to the light requirements: heliophilic species, compared to more shade-tolerant species, had a larger efficiency and an equilibrium angle closer to vertical. This suggests that traits determining the gravitropic reaction are related to the strategy of light interception and may contribute to the differentiation of ecological strategies promoting the maintenance of biodiversity in tropical rainforests.

Transcriptomic profiling of heat-stress response in potato periderm

Fri, 30 Oct 2009 06:38:04 PDT

Potato (Solanum tuberosum L.) periderm is composed of the meristematic phellogen that gives rise to an external layer of suberized phellem cells (the skin) and the internal parenchyma-like phelloderm. The continuous addition of new skin layers and the sloughing of old surface layers during tuber maturation results in smooth, shiny skin. However, smooth-skin varieties frequently develop unsightly russeting in response to high soil temperatures. Microscopic observation of microtubers exposed to high temperatures (37°C) suggested heat-enhanced development and accumulation of suberized skin-cell layers. To identify the genes involved in the periderm response to heat stress, skin and phelloderm samples collected separately from immature tubers exposed to high soil temperatures (33°C) and controls were subjected to transcriptome profiling using a potato cDNA array. As expected, the major functional group that was differentially expressed in both skin and phelloderm consisted of stress-related genes; however, while the major up-regulated phelloderm genes coded for heat-shock proteins, many of the skin's most up-regulated sequences were similar to genes involved in the development of protective/symbiotic membranes during plant–microbe interactions. The primary activities regulated by differentially expressed peridermal transcription factors were response to stress (33%) and cell proliferation and differentiation (28%), possibly reflecting the major processes occurring in the heat-treated periderm and implying the integrated activity of the stress response and tissue development. Accumulating data suggest that the periderm, a defensive tissue, responds to heat stress by enhancing the production and accumulation of periderm/skin layers to create a thick protective cover. Skin russeting may be an indirect outcome; upon continued expansion of the tuber, the inflexible skin cracks while new layers are produced below it, resulting in a rough skin texture.

The amylose extender mutant of maize conditions novel protein-protein interactions between starch biosynthetic enzymes in amyloplasts

Liu, F., Makhmoudova, A., Lee, E. A., Wait, R., Emes, M. J., Tetlow, I. J. — Fri, 30 Oct 2009 06:38:04 PDT

The amylose extender (ae^–) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein–protein interactions in amyloplasts of ae^– mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein–protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae^– amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae^– endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae^–), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.

Symbolism of plants: examples from European-Mediterranean culture presented with biology and history of art: NOVEMBER: Chicory

Kandeler, R., Ullrich, W. R. — Wed, 30 Sep 2009 16:47:59 PDT

Phagotrophy in the origins of photosynthesis in eukaryotes and as a complementary mode of nutrition in phototrophs: relation to Darwin's insectivorous plants

Raven, J. A., Beardall, J., Flynn, K. J., Maberly, S. C. — Wed, 30 Sep 2009 16:47:59 PDT

Darwin performed innovative observational and experimental work on the apparently paradoxical occurrence of carnivory in photosynthetic flowering plants. The nutritional use of particulate organic material which also supplies other elements is now known to be widespread in free-living algae as well as in organisms with endosymbiotic algae and with kleptoplastids. In addition to this direct nutritional role, phagotrophy, in the broad sense of internalization of photosynthetic organisms by a eukaryote, is essential for the occurrence of present-day endosymbiotic algae and kleptoplastid-containing protists, and was essential for the origin of plastids themselves. The endosymbiotic phenomena involving photosynthetic organisms clearly played a major role in combining genomes providing different metabolic functions, but, in our opinion, this does not demand a re-appraisal of evolution by natural selection. That the balance of physiological optimization for competition for resources and minimization of losses (e.g. through predation) is a fine one, and thus subject to a complex selective process, is illustrated by the diversity of mixotrophic strategies in extant phytoplankton.

Is it good noise? The role of developmental instability in the shaping of a root system

Forde, B. G. — Wed, 30 Sep 2009 16:47:59 PDT

Root architecture plays a major part in determining a root system's ability to function effectively and efficiently in its essential roles of anchorage and the capture of soil resources. The characteristics of root development that are conventionally considered to be the main determinants of root architecture are the rate, angle, and duration of root growth and the pattern of root branching. In this review, the case is made that there is an additional trait that has been largely ignored but which has a significant influence on root architecture, namely the degree to which stochasticity (or ‘developmental instability’) affects the developmental process. Although the intrinsic variability in the development and growth of lateral roots has been recognized for some time, in almost every study of root development this remarkable facet of root behaviour tends to be hidden beneath the veil of statistical averaging. Progress in other fields is providing intriguing insights into the phenomenon of developmental instability, how it is generated at the molecular and cellular levels and the genetic mechanisms by which it is buffered. This review will consider the existence of developmental instability in roots, its underlying causes, its effects on root architecture, and the evidence that it is under genetic control. The hypothesis will be advanced that developmental instability in roots is an adaptive trait, and its potential relevance to root function will be discussed in both an ecological and an agronomic context.

Rubisco activase and wheat productivity under heat-stress conditions

Wed, 30 Sep 2009 16:47:59 PDT

Rubisco activase (RCA) constrains the photosynthetic potential of plants at high temperatures (heat stress). Endogenous levels of RCA could serve as an important determinant of plant productivity under heat-stress conditions. Thus, in this study, the possible relationship between expression levels of RCA and plant yield in 11 European cultivars of winter wheat following prolonged exposure to heat stress was investigated. In addition, the effect of a short-term heat stress on RCA expression in four genotypes of wheat, five genotypes of maize, and one genotype of Arabidopsis thaliana was examined. Immunoblots prepared from leaf protein extracts from control plants showed three RCA cross-reacting bands in wheat and two RCA cross-reacting bands in maize and Arabidopsis. The molecular mass of the observed bands was in the range between 40 kDa and 46 kDa. Heat stress affected RCA expression in a few genotypes of wheat and maize but not in Arabidopsis. In wheat, heat stress slightly modulated the relative amounts of RCA in some cultivars. In maize, heat stress did not seem to affect the existing RCA isoforms (40 kDa and 43 kDa) but induced the accumulation of a new putative RCA of 45–46 kDa. The new putative 45–46 kDa RCA was not seen in a genotype of maize (ZPL 389) that has been shown to display an exceptional sensitivity to heat stress. A significant, positive, linear correlation was found between the expression of wheat 45–46 kDa RCA and plant productivity under heat-stress conditions. Results support the hypothesis that endogenous levels of RCA could play an important role in plant productivity under supraoptimal temperature conditions.

{alpha}' Subunit of soybean {beta}-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds

Wed, 30 Sep 2009 16:47:59 PDT

The ' and β subunits of soybean β-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The ' subunit accumulated in rice seeds at a higher level than the β subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the ' subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the ' subunit and the β subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the β subunit accumulated in low electron density regions in the periphery of PB-II, whereas the ' subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated ' subunits lacking one cysteine residue in the N-terminal mature region ('Cys1) or five cysteine residues in the pro and N-terminal mature regions ('Cys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing 'Cys 5 and 'Cys1. 'Cys5 was localized only in the low-density regions, whereas 'Cys1 was found in both low- and high-density regions. These results suggest that the ' subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

Partial phenotypic reversion of ABA-deficient flacca tomato (Solanum lycopersicum) scions by a wild-type rootstock: normalizing shoot ethylene relations promotes leaf area but does not diminish whole plant transpiration rate

Dodd, I. C., Theobald, J. C., Richer, S. K., Davies, W. J. — Wed, 30 Sep 2009 16:47:59 PDT

To evaluate the role of root-synthesized ABA in regulating growth and stomatal behaviour under well-watered conditions, isogenic wild-type (WT) and ABA-deficient flacca (flc) tomato (Solanum lycopersicum) were reciprocally and self-grafted just below the cotyledonary node. Since flc scions had lower leaf water potentials due to higher transpiration rates, a subset of all graft combinations was grown under a shoot misting treatment to minimize differences in shoot water status. Misting did not alter the relative effects of the different graft combinations on leaf area. WT scions had the greatest leaf area and lowest whole plant transpiration rate irrespective of the rootstock, implying that shoot ABA biosynthesis was sufficient to account for a WT shoot phenotype. In WT scions, the rootstock had no effect on detached leaf ethylene evolution or xylem concentrations of ABA or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In flc scions, although the WT rootstock suppressed stomatal conductance of individual leaves, there was no detectable effect on whole plant transpiration rate. However, leaf area of flc/WT (scion/rootstock) plants increased 1.6-fold compared to flc self-grafts. WT rootstocks increased xylem ABA concentration in flc scions (relative to flc self-grafts) up to 3-fold, and resulted in xylem ACC concentrations and detached leaf ethylene evolution similar to WT scions. Since the WT rootstock normalized shoot ethylene relations but only partially restored the leaf area of flc scions (relative to that of WT scions), shoot ABA biosynthesis can directly promote leaf area via an unknown, ethylene-independent, mechanism.

Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode

Wed, 30 Sep 2009 16:47:59 PDT

Root-knot nematodes (RKNs) are sedentary biotrophic parasites that induce the differentiation of root cells into feeding cells that provide the nematodes with the nutrients necessary for their development. The development of new control methods against RKNs relies greatly on the functional analysis of genes that are crucial for the development of the pathogen or the success of parasitism. In the absence of genetic transformation, RNA interference (RNAi) allows for phenotype analysis of nematode development and nematode establishment in its host after sequence-specific knock-down of the targeted genes. Strategies used to induce RNAi in RKNs are so far restricted to small-scale analyses. In the search for a new RNAi strategy amenable to large-scale screenings the possibility of using RNA viruses to produce the RNAi triggers in plants was tested. Tobacco rattle virus (TRV) was tested as a means to introduce double-stranded RNA (dsRNA) triggers into the feeding cells and to mediate RKN gene silencing. It was demonstrated that virus-inoculated plants can produce dsRNA and siRNA silencing triggers for delivery to the feeding nematodes. Interestingly, the knock-down of the targeted genes was observed in the progeny of the feeding nematodes, suggesting that continuous ingestion of dsRNA triggers could be used for the functional analysis of genes involved in early development. However, the heterogeneity in RNAi efficiency between TRV-inoculated plants appears as a limitation to the use of TRV-mediated silencing for the high-throughput functional analysis of the targeted nematode genes.

Accumulation of soluble sugars in peel at high temperature leads to stay-green ripe banana fruit

Yang, X., Pang, X., Xu, L., Fang, R., Huang, X., Guan, P., Lu, W., Zhang, Z. — Wed, 30 Sep 2009 16:47:59 PDT

Bananas (Musa acuminata, AAA group) fail to develop a yellow peel and stay green when ripening at temperatures >24 °C. The identification of the mechanisms leading to the development of stay-green ripe bananas has practical value and is helpful in revealing pathways involved in the regulation of chlorophyll (Chl) degradation. In the present study, the Chl degradation pathway was characterized and the progress of ripening and senescence was assessed in banana peel at 30 °C versus 20 °C, by monitoring relevant gene expression and ripening and senescence parameters. A marked reduction in the expression levels of the genes for Chl b reductase, SGR (Stay-green protein), and pheophorbide a oxygenase was detected for the fruit ripening at 30 °C, when compared with fruit at 20 °C, indicating that Chl degradation was repressed at 30 °C at various steps along the Chl catabolic pathway. The repressed Chl degradation was not due to delayed ripening and senescence, since the fruit at 30 °C displayed faster onset of various ripening and senescence symptoms, suggesting that the stay-green ripe bananas are of similar phenotype to type C stay-green mutants. Faster accumulation of high levels of fructose and glucose in the peel at 30 °C prompted investigation of the roles of soluble sugars in Chl degradation. In vitro incubation of detached pieces of banana peel showed that the pieces of peel stayed green when incubated with 150 mM glucose or fructose, but turned completely yellow in the absence of sugars or with 150 mM mannitol, at either 20 °C or 30 °C. The results suggest that accumulation of sugars in the peel induced by a temperature of 30 °C may be a major factor regulating Chl degradation independently of fruit senescence.

Aquaporin gene expression and apoplastic water flow in bur oak (Quercus macrocarpa) leaves in relation to the light response of leaf hydraulic conductance

Voicu, M. C., Cooke, J. E. K., Zwiazek, J. J. — Wed, 30 Sep 2009 16:47:59 PDT

It has previously been shown that hydraulic conductance in bur oak leaves (Quercus macrocarpa Michx.), measured with the high pressure flow meter technique (HPFM), can significantly increase within 30 min following exposure to high irradiance. The present study investigated whether this increase could be explained by an increase in the cell-to-cell pathway and whether the response is linked to changes in the transcript level corresponding to aquaporin genes. Four cDNA sequences showing high similarity to members of the aquaporin gene family from other plant species were characterized from bur oak leaves and the expression levels of these cDNA sequences were examined in leaves by quantitative real-time PCR (QRT-PCR). No change was found in the relative transcript abundance corresponding to these four putative aquaporin genes in leaves with light-induced high hydraulic conductance (exposed to high irradiance) compared to leaves with low hydraulic conductance (exposed to low irradiance). However, in sun leaves that were exposed to different light levels prior to leaf collection (full sunlight, shade, and covered with aluminium foil for 16 h), the relative transcript levels of two of the putative aquaporin genes increased several-fold in shaded leaves compared to the sun-exposed or covered leaves. When the leaves were pressure-infiltrated with the apoplastic tracer dye trisodium 3-hydroxy-5,8,10-pyrenetrisulphonate (PTS₃, 0.02%), there was no change in the PTS₃ concentration of leaf exudates collected in ambient light or in high irradiance, but there was a small apoplastic acidification. There was also no change in PTS₃ concentration between the leaves infiltrated under high irradiance with 0.02% PTS₃ or with 0.1 mM HgCl₂ in 0.02% PTS₃. The results suggest that the putative aquaporin genes that were identified in the present study probably do not play a role in the light responses of hydraulic conductance at the transcript level, but they may function in regulating water homeostasis in leaves adapted to different light conditions. In addition, it is shown that high irradiance induced changes in the pH of the apoplast and that there does not appear to be a significant shift to the cell-to-cell mediated water transport in bur oak leaves exposed to high irradiance as measured by the apoplastic tracer dye.

Rubisco in planta kcat is regulated in balance with photosynthetic electron transport

Eichelmann, H., Talts, E., Oja, V., Padu, E., Laisk, A. — Wed, 30 Sep 2009 16:47:59 PDT

Site turnover rate (k_cat) of Rubisco was measured in intact leaves of different plants. Potato (Solanum tuberosum L.) and birch (Betula pendula Roth.) leaves were taken from field-growing plants. Sunflower (Helianthus annuus L.), wild type (wt), Rubisco-deficient (–RBC), FNR-deficient (–FNR), and Cyt b₆f deficient (–CBF) transgenic tobacco (Nicotiana tabacum L.) were grown in a growth chamber. Rubisco protein was measured with quantitative SDS-PAGE and FNR protein content with quantitative immunoblotting. The Cyt b₆f level was measured in planta by maximum electron transport rate and the photosystem I (PSI) content was assessed by titration with far-red light. The CO₂ response of Rubisco was measured in planta with a fast-response gas exchange system at maximum ribulose 1,5-bisphosphate concentration. Reaction site k_cat was calculated from V_m and Rubisco content. Biological variation of k_cat was significant, ranging from 1.5 to 4 s^–1 in wt, but was >6 s^–1 at 23 °C in –RBC leaves. The lowest k_cat of 0.5 s^–1 was measured in –FNR and –CBF plants containing sufficient Rubisco but having slow electron transport rates. Plotting k_cat against PSI per Rubisco site resulted in a hyperbolic relationship where wt plants are on the initial slope. A model is suggested in which Rubisco Activase is converted into an active ATP-form on thylakoid membranes with the help of a factor related to electron transport. The activation of Rubisco is accompanied by the conversion of the ATP-form into an inactive ADP-form. The ATP and ADP forms of Activase shuttle between thylakoid membranes and stromally-located Rubisco. In normal wt plants the electron transport-related activation of Activase is rate-limiting, maintaining 50–70% Rubisco sites in the inactive state.

Metabolic responses to salt stress of barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differ in salinity tolerance

Widodo, , Patterson, J. H., Newbigin, E., Tester, M., Bacic, A., Roessner, U. — Wed, 30 Sep 2009 16:47:59 PDT

Plants show varied cellular responses to salinity that are partly associated with maintaining low cytosolic Na⁺ levels and a high K⁺/Na⁺ ratio. Plant metabolites change with elevated Na⁺, some changes are likely to help restore osmotic balance while others protect Na⁺-sensitive proteins. Metabolic responses to salt stress are described for two barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differed in salinity tolerance under the experimental conditions used. After 3 weeks of salt treatment, Clipper ceased growing whereas Sahara resumed growth similar to the control plants. Compared with Clipper, Sahara had significantly higher leaf Na⁺ levels and less leaf necrosis, suggesting they are more tolerant to accumulated Na⁺. Metabolite changes in response to the salt treatment also differed between the two cultivars. Clipper plants had elevated levels of amino acids, including proline and GABA, and the polyamine putrescine, consistent with earlier suggestions that such accumulation may be correlated with slower growth and/or leaf necrosis rather than being an adaptive response to salinity. It is suggested that these metabolites may be an indicator of general cellular damage in plants. By contrast, in the more tolerant Sahara plants, the levels of the hexose phosphates, TCA cycle intermediates, and metabolites involved in cellular protection increased in response to salt. These solutes remain unchanged in the more sensitive Clipper plants. It is proposed that these responses in the more tolerant Sahara are involved in cellular protection in the leaves and are involved in the tolerance of Sahara leaves to high Na⁺.

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

Wed, 30 Sep 2009 16:47:59 PDT

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC₅₀ value ~1 µM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the T_i plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 µM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

A single gene, AIN, in Medicago truncatula mediates a hypersensitive response to both bluegreen aphid and pea aphid, but confers resistance only to bluegreen aphid

Klingler, J. P., Nair, R. M., Edwards, O. R., Singh, K. B. — Wed, 30 Sep 2009 16:47:59 PDT

Biotic stress in plants frequently induces a hypersensitive response (HR). This distinctive reaction has been studied intensively in several pathosystems and has shed light on the biology of defence signalling. Compared with microbial pathogens, relatively little is known about the role of the HR in defence against insects. Reference genotype A17 of Medicago truncatula Gaertn., a model legume, responds to aphids of the genus Acyrthosiphon with necrotic lesions resembling a HR. In this study, the biochemical nature of this response, its mode of inheritance, and its relationship with defence against aphids were investigated. The necrotic lesion phenotype and resistance to the bluegreen aphid (BGA, Acyrthosiphon kondoi Shinji) and the pea aphid (PA, Acyrthosiphon pisum (Harris)) were analysed using reference genotypes A17 and A20, their F₂ progeny and recombinant inbred lines. BGA-induced necrotic lesions co-localized with the production of H₂O₂, consistent with an oxidative burst widely associated with hypersensitivity. This HR correlated with stronger resistance to BGA in A17 than in A20; these phenotypes cosegregated as a semi-dominant gene, AIN (Acyrthosiphon-induced necrosis). In contrast to BGA, stronger resistance to PA in A17, compared with A20, did not cosegregate with a PA-induced HR. The AIN locus resides in a cluster of sequences predicted to encode the CC-NBS-LRR subfamily of resistance proteins. AIN-mediated resistance presents a novel opportunity to use a model plant and model aphid to study the role of the HR in defence responses to phloem-feeding insects.

An inducible, modular system for spatio-temporal control of gene expression in stomatal guard cells

Xiong, T. C., Hann, C. M., Chambers, J. P., Surget, M., Ng, C. K.-Y. — Wed, 30 Sep 2009 16:47:59 PDT

Stomata, flanked by pairs of guard cells, are small pores on the leaf surfaces of plants and they function to control gas exchange between plants and the atmosphere. Stomata will open when water is available to allow for the uptake of carbon dioxide for photosynthesis. During periods of drought, stomata will close to reduce desiccation stress. As such, optimal functioning of stomata will impact on water use efficiency by plants. The development of an inducible, modular system for robust and targeted gene expression in stomatal guard cells is reported here. It is shown that application of ethanol vapour to activate the gene expression system did not affect the ability of stomata to respond to ABA in bioassays to determine the promotion of stomatal closure and the inhibition of stomatal opening. The system that has been developed allows for robust spatio-temporal control of gene expression in all cells of the stomatal lineage, thereby enabling molecular engineering of stomatal function as well as studies on stomatal development.

Function of Arabidopsis hexokinase-like1 as a negative regulator of plant growth

Karve, A., Moore, B. d. — Wed, 30 Sep 2009 16:47:59 PDT

A recent analysis of the hexokinase (HXK) gene family from Arabidopsis revealed that three hexokinase-like (HKL) proteins lack catalytic activity, but share about 50% identity with the primary glucose (glc) sensor/transducer protein AtHXK1. Since the AtHKL1 protein is predicted to bind glc, although with a relatively decreased affinity, a reverse genetics approach was used to test whether HKL1 might have a related regulatory function in plant growth. By comparing phenotypes of an HKL1 mutant (hkl1-1), an HXK1 mutant (gin2-1), and transgenic lines that overexpress HKL1 in either wild-type or gin2-1 genetic backgrounds, it is shown that HKL1 is a negative effector of plant growth. Interestingly, phenotypes of HKL1 overexpression lines are generally very similar to those of gin2-1. These are quantified, in part, as reduced seedling sensitivity to high glc concentrations and reduced seedling sensitivity to auxin-induced lateral root formation. However, commonly recognized targets of glc signalling are not apparently altered in any of the HKL1 mutant or transgenic lines. In fact, most, but not all, of the observed phenotypes associated with HKL1 overexpression occur independently of the presence of HXK1 protein. The data indicate that HKL1 mediates cross-talk between glc and other plant hormone response pathways. It is also considered Whether a possibly decreased glc binding affinity of HKL1 could possibly be a feedback mechanism to limit plant growth in the presence of excessive carbohydrate availability is further considered.

Identification of genes related to germination in aged maize seed by screening natural variability

Revilla, P., Butron, A., Rodriguez, V. M., Malvar, R. A., Ordas, A. — Wed, 30 Sep 2009 16:47:59 PDT

Ageing reduces vigour and viability in maize inbred lines due to non-heritable degenerative changes. Besides non-heritable genetic changes due to chromosome aberrations and damage in the DNA sequence, heritable changes during maize conservation have been reported. Genetic variability among aged seeds of inbred lines could be used for association studies with seed germination. The objective of this study was to identify genes related to germination in aged seeds. The sweet corn inbred line P39 and the field corn inbred line EP44 were used as plant material. Bulks of living and dead seeds after 20 and 22 years of storage were compared by using simple sequence repeats (SSRs) and, when the bulks differed for a marker, the individual grains were genotyped. Differences between dead and living seeds could be explained by residual variability, spontaneous mutation, or ageing. Variability was larger for chromosome 7 than for other chromosomes, and for distal than for proximal markers, suggesting some relationships between position in the genome and viability in aged seed. Polymorphic SSRs between living and dead seeds were found in six known genes, including pathogenesis-related protein 2, superoxide dismutase 4, catalase 3, opaque endosperm 2, and metallothionein1 that were related to germination, along with golden plant 2. In addition, five novel candidate genes have been identified; three of them could be involved in resistance to diseases, one in detoxification of electrophillic compounds, and another in transcription regulation. Therefore, genetic variability among aged seeds of inbreds was useful for preliminary association analysis to identify candidate genes.

An integrated strategy to identify key genes in almond adventitious shoot regeneration

Wed, 30 Sep 2009 16:47:59 PDT

Plant genetic transformation usually depends on efficient adventitious regeneration systems. In almond (Prunus dulcis Mill.), regeneration of transgenic adventitious shoots was achieved but with low efficiency. Histological studies identified two main stages of organogenesis in almond explants that were induced for adventitious shoot regeneration; a dedifferentiation stage (early) and a shoot initiation stage (late). Histological observation revealed that the limitation in the recovery of transformed shoots is primarily a function of the low organogenic competence of the transformed tissues rather than transformation efficiency. To identify key genes involved in organogenesis, shoot-induced leaves and suppression-subtractive hybridization were used, to build a cDNA library from each organogenic stage. cDNA clones from both libraries were randomly picked, PCR-amplified, and arrayed on glass slides. For transcript profiling, microarray hybridization was performed using cDNA pools from both the early and the late stages. Statistically significant differential expression was found for 128 cDNA clones (58 early, and 70 late), representing 92 unique gene functions. Genes encoding proteins related to protein synthesis and processing and nitrogen and carbon metabolism were differentially expressed in the early stage, whilst genes encoding proteins involved in plant cell rescue and defence and interaction with the environment were mostly found in the late stage. The LTP/-amylase inhibitor/trypsin gene was more strongly expressed at an early stage, as confirmed by quantitative RT-PCR, while a gibberellic acid stimulated protein gene seems to be a good marker for the late stage. These results are discussed on the basis of the putative roles of the annotated differentially regulated genes in almond organogenesis.

Onion epidermis as a new model to study the control of growth anisotropy in higher plants

Suslov, D., Verbelen, J.-P., Vissenberg, K. — Wed, 30 Sep 2009 16:47:59 PDT

To elucidate the role of cellulose microfibrils in the control of growth anisotropy, a link between their net orientation, in vitro cell wall extensibility, and anisotropic cell expansion was studied during development of the adaxial epidermis of onion (Allium cepa) bulb scales using polarization confocal microscopy, creep tests, and light microscopy. During growth the net cellulose alignment across the whole thickness of the outer epidermal wall changed from transverse through random to longitudinal and back to transverse relative to the bulb axis. Cell wall extension in vitro was always higher transverse than parallel to the net cellulose alignment. The direction of growth anisotropy was perpendicular to the net microfibril orientation and changed during development from longitudinal to transverse to the bulb axis. The correlation between the degree of growth anisotropy and the net cellulose alignment was poor. Thus the net cellulose microfibril orientation across the whole thickness of the outer periclinal epidermis wall defines the direction but not the degree of growth anisotropy. Strips isolated from the epidermis in the directions perpendicular and transverse to a net cellulose orientation can be used as an extensiometric model to prove a protein involvement in the control of growth anisotropy.

Chlorella saccharophila cytochrome f and its involvement in the heat shock response

Zuppini, A., Gerotto, C., Moscatiello, R., Bergantino, E., Baldan, B. — Wed, 30 Sep 2009 16:47:59 PDT

Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes.

Molecular dissection of the pea shoot apical meristem

Wed, 30 Sep 2009 16:47:59 PDT

The shoot apical meristem (SAM) is responsible for the development of all the above-ground parts of a plant. Our understanding of the SAM at the molecular level is incomplete. This study investigates the gene expression repertoire of SAMs in the garden pea (Pisum sativum). To this end, 10 346 EST sequences representing 7610 unique genes were generated from SAM cDNA libraries. These sequences, together with previously reported pea ESTs, were used to construct a 12K oligonucleotide array to identify genes with differential SAM expression, as compared to axillary meristems, root apical meristems, or non-meristematic tissues. A number of genes were identified, predominantly expressed in specific cell layers or domains of the SAM and thus are likely components of the gene networks involved in stem cell maintenance or the initiation of lateral organs. Further in situ hybridization analysis confirmed the spatial localization of some of these genes within the SAM. Our data also indicate the diversification of some gene expression patterns and hence functions in legume crop plants. A number of transcripts highly expressed in all three meristems have also been uncovered and these candidates may provide valuable insight into molecular networks that underpin the maintenance of meristematic functionality.

Symbolism of plants: examples from European-Mediterranean culture presented with biology and history of art: OCTOBER: Roses

Kandeler, R., Ullrich, W. R. — Wed, 02 Sep 2009 11:01:17 PDT

Plant cell walls throughout evolution: towards a molecular understanding of their design principles

Sarkar, P., Bosneaga, E., Auer, M. — Wed, 02 Sep 2009 11:01:17 PDT

Throughout their life, plants typically remain in one location utilizing sunlight for the synthesis of carbohydrates, which serve as their sole source of energy as well as building blocks of a protective extracellular matrix, called the cell wall. During the course of evolution, plants have repeatedly adapted to their respective niche, which is reflected in the changes of their body plan and the specific design of cell walls. Cell walls not only changed throughout evolution but also are constantly remodelled and reconstructed during the development of an individual plant, and in response to environmental stress or pathogen attacks. Carbohydrate-rich cell walls display complex designs, which together with the presence of phenolic polymers constitutes a barrier for microbes, fungi, and animals. Throughout evolution microbes have co-evolved strategies for efficient breakdown of cell walls. Our current understanding of cell walls and their evolutionary changes are limited as our knowledge is mainly derived from biochemical and genetic studies, complemented by a few targeted yet very informative imaging studies. Comprehensive plant cell wall models will aid in the re-design of plant cell walls for the purpose of commercially viable lignocellulosic biofuel production as well as for the timber, textile, and paper industries. Such knowledge will also be of great interest in the context of agriculture and to plant biologists in general. It is expected that detailed plant cell wall models will require integrated correlative multimodal, multiscale imaging and modelling approaches, which are currently underway.

Lateral root emergence: a difficult birth

Peret, B., Larrieu, A., Bennett, M. J. — Wed, 02 Sep 2009 11:01:17 PDT

Lateral root initiation takes place deep within the parental root, requiring new primordia to break through the overlying tissues before they emerge into the soil. Lateral root emergence has been well described at the cellular level but, until recently, the molecular mechanisms involved were unclear. Scientists in the 19th and 20th centuries hypothesized that the cell wall of the overlying tissues was modified by enzymes released by cells within the primordium. Recent studies in the model plant Arabidopsis thaliana revealed the existence of a complex transcellular signalling network regulated by auxin that controls cell wall remodelling in cells overlying lateral root primordia. In the first part of this review, early observations on the cell biology of lateral root formation and emergence are summarized, and in the following two sections recent observations in Arabidopsis that led to the identification of the molecular mechanism regulating lateral root emergence are described.

Receptor-mediated signalling in plants: molecular patterns and programmes

Tor, M., Lotze, M. T., Holton, N. — Wed, 02 Sep 2009 11:01:17 PDT

A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed.

Low temperature induces different cold sensitivity in two poplar clones (Populusxcanadensis Monch 'I-214' and P. deltoides Marsh. 'Dvina')

Wed, 02 Sep 2009 11:01:17 PDT

Changes of stem diameter were continuously monitored during winter in two field-grown poplar clones, using automatic point dendrometers. The objective of this study was to find an analytical solution to seasonal synchronization of stem diameter oscillations and low air temperatures. The study identified to what extent and with what frequency low air temperature induced stem diameter variation in ‘Dvina’ (P. deltoides) and ‘I-214’ (Populusxcanadensis) poplar clones, after exposure to summer drought. The patterns of reversible stem shrinkage were related to the cycles of low air temperature. Hourly and daily evidence showed that ‘I-214’ was more sensitive to low air temperatures than ‘Dvina’. The analysis of raw data and graphic details implemented with the study of derivative tests allowed an increase in the general sensitivity of the investigation applied to describe the response of poplar clones to environmental conditions. Given these diameter fluctuation patterns, automatic point dendrometers were confirmed to be a reliable non-invasive method for testing the sensitivity of diameter variation to cold temperature. Variation in rate and duration of daily stem shrinkage in response to low air temperature in winter appeared to occur independently of the effects of water deficit suffered by plants the previous summer.

Exploring the importance of within-canopy spatial temperature variation on transpiration predictions

Bauerle, W. L., Bowden, J. D., Wang, G. G., Shahba, M. A. — Wed, 02 Sep 2009 11:01:17 PDT

Models seldom consider the effect of leaf-level biochemical acclimation to temperature when scaling forest water use. Therefore, the dependence of transpiration on temperature acclimation was investigated at the within-crown scale in climatically contrasting genotypes of Acer rubrum L., cv. October Glory (OG) and Summer Red (SR). The effects of temperature acclimation on intracanopy gradients in transpiration over a range of realistic forest growth temperatures were also assessed by simulation. Physiological parameters were applied, with or without adjustment for temperature acclimation, to account for transpiration responses to growth temperature. Both types of parameterization were scaled up to stand transpiration (expressed per unit leaf area) with an individual tree model (MAESTRA) to assess how transpiration might be affected by spatial and temporal distributions of foliage properties. The MAESTRA model performed well, but its reproducibility was dependent on physiological parameters acclimated to daytime temperature. Concordance correlation coefficients between measured and predicted transpiration were higher (0.95 and 0.98 versus 0.87 and 0.96) when model parameters reflected acclimated growth temperature. In response to temperature increases, the southern genotype (SR) transpiration responded more than the northern (OG). Conditions of elevated long-term temperature acclimation further separate their transpiration differences. Results demonstrate the importance of accounting for leaf-level physiological adjustments that are sensitive to microclimate changes and the use of provenance-, ecotype-, and/or genotype-specific parameter sets, two components likely to improve the accuracy of site-level and ecosystem-level estimates of transpiration flux.

Visible foliar injury and infrared imaging show that daylength affects short-term recovery after ozone stress in Trifolium subterraneum

Wed, 02 Sep 2009 11:01:17 PDT

Tropospheric ozone is a major air pollutant affecting plants worldwide. Plants in northern regions can display more ozone injury than plants at lower latitudes despite lower ozone levels. Larger ozone influx and shorter nights have been suggested as possible causes. However, the effects of the dim light present during northern summer nights have not been investigated. Young Trifolium subterraneum plants kept in environmentally controlled growth rooms under long day (10 h bright light, 14 h dim light) or short day (10 h bright light, 14 h darkness) conditions were exposed to 6 h of 70 ppb ozone during daytime for three consecutive days. Leaves were visually inspected and imaged in vivo using thermal imaging before and after the daily exposure. In long-day-treated plants, visible foliar injury within 1 week after exposure was more severe. Multivariate statistical analyses showed that the leaves of ozone-exposed long-day-treated plants were also warmer with more homogeneous temperature distributions than exposed short day and control plants, suggesting reduced transpiration. Temperature disruptions were not restricted to areas displaying visible damage and occurred even in leaves with only slight visible injury. Ozone did not affect the leaf temperature of short-day-treated plants. As all factors influencing ozone influx were the same for long- and short-day-treated plants, only the dim nocturnal light could account for the different ozone sensitivities. Thus, the twilight summer nights at high latitudes may have a negative effect on repair and defence processes activated after ozone exposure, thereby enhancing sensitivity.

Creating S-type characteristics in the F-type enzyme fructan:fructan 1-fructosyltransferase of Triticum aestivum L.

Wed, 02 Sep 2009 11:01:17 PDT

Invertases cleave sucrose in glucose and fructose, using water as an acceptor. Fructosyltransferases catalyse the transfer of a fructosyl residue between sucrose and/or fructan molecules. Plant fructosyltransferases (FTs) evolved from vacuolar invertases by small mutational changes, leading to differences in substrate specificity. The S-type of enzymes (invertases, sucrose:sucrose 1-fructosyltransferases or 1-SSTs, and sucrose:fructan 6-fructosyltransferases or 6-SFTs) prefer sucrose as the donor substrate while F-type enzymes (fructan:fructan 1-fructosyltransferases or 1-FFTs and fructan:fructan 6^G-fructosyltransferases or 6^G-FFTs) preferentially use fructan as the donor substrate. Recently, a functional Asp/Arg or Asp/Lys couple in the Hypervariable Loop (HVL) was suggested to be essential to keep Asp in a favourable orientation for binding sucrose as the donor substrate in S-type enzymes. However, the F-type enzyme 1-FFT of Triticum aestivum (Ta1-FFT) also contains the Asp/Arg couple in the HVL, although it prefers fructan as the donor substrate. In this paper, mutagenesis studies on Ta1-FFT are presented. In Ta1-SST, Tyr282 (the Asp281 homologue) seems to be essential in creating a tight H-bond Network (HBN) in which the Arg-residue of the Asp/Arg couple is held in a fixed position. This tight HBN is disrupted in Ta1-FFT, leading to a more flexible Arg-residue and a dysfunctional Asp/Arg couple. A single D281Y mutation in Ta1-FFT restored the tight HBN and introduced typical S-type characteristics. Conclusively, in wheat FTs Asp281 (and its homologues) is involved in donor substrate specificity.

AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis

Lin, Z., Ho, C.-W., Grierson, D. — Wed, 02 Sep 2009 11:01:17 PDT

Arabidopsis AtTRP1 is an orthologue of SlTPR1, a tomato tetratricopeptide repeat protein that interacts with the tomato ethylene receptors LeETR1 and NR in yeast 2-hybrid assays and in vitro, and modulates plant development. AtTRP1 is encoded by a single copy gene in the Arabidopsis genome, and is related to TCC1, a human protein that competes with Raf-1 for Ras binding, and distantly related to the immunophilin-like FK-binding proteins TWD1 and PAS1. The former is involved in auxin transport and the latter is translocated to the nucleus in response to auxin. AtTRP1 interacted preferentially with the Arabidopsis ethylene receptor ERS1 in yeast two-hybrid assays. This association was confirmed by in vivo co-immunoprecipitation. AtTRP1 promoter–GUS was highly expressed in vascular tissue, mature anthers, the abscission zone, and was induced by ACC. Overexpression of AtTRP1 in wild-type Arabidopsis resulted in dwarf plants with reduced fertility, altered leaf/silique morphology, and enhanced expression of the ethylene responsive gene AtChitB. Exogenous GA did not reverse the dwarf habit. Etiolated transgenic seedlings overexpressing AtTRP1 displayed enhanced sensitivity to low ACC and this was correlated with the transgene expression. Seedlings overexpressing AtTRP1 at high levels exhibited shortened and swollen hypocotyls, inhibited root growth, and an altered apical hook. Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes. These results indicated that overexpression of AtTRP1 affects cross-talk between ethylene and auxin signalling and enhances some ethylene responses and alters some auxin responses. A model for AtTRP1 action is proposed.

Identification of candidate genes affecting {Delta}9-tetrahydrocannabinol biosynthesis in Cannabis sativa

Wed, 02 Sep 2009 11:01:17 PDT

RNA isolated from the glands of a ⁹-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.

OXI1 protein kinase is required for plant immunity against Pseudomonas syringae in Arabidopsis

Petersen, L. N., Ingle, R. A., Knight, M. R., Denby, K. J. — Wed, 02 Sep 2009 11:01:17 PDT

Expression of the Arabidopsis Oxidative Signal-Inducible1 (OXI1) serine/threonine protein kinase gene (At3g25250) is induced by oxidative stress. The kinase is required for root hair development and basal defence against the oomycete pathogen Hyaloperonospora parasitica, two separate H₂O₂-mediated processes. In this study, the role of OXI1 during pathogenesis was characterized further. Null oxi1 mutants are more susceptible to both virulent and avirulent strains of the biotrophic bacterial pathogen Pseudomonas syringae compared with the wild type, indicating that OXI1 positively regulates both basal resistance triggered by the recognition of pathogen-associated molecular patterns, as well as effector-triggered immunity. The level of OXI1 expression appears to be critical in mounting an appropriate defence response since OXI1 overexpressor lines also display increased susceptibility to biotrophic pathogens. The induction of OXI1 after P. syringae infection spatially and temporally correlates with the oxidative burst. Furthermore, induction is reduced in atrbohD mutants and after application of DPI (an inhibitor of NADPH oxidases) suggesting that reactive oxygen species produced through NADPH oxidases drives OXI1 expression during this plant–pathogen interaction.

Are plant growth and photosynthesis limited by pre-drought following rewatering in grass?

Xu, Z., Zhou, G., Shimizu, H. — Wed, 02 Sep 2009 11:01:17 PDT

Although the relationship between grassland productivity and soil water status has been extensively researched, the responses of plant growth and photosynthetic physiological processes to long-term drought and rewatering are not fully understood. Here, the perennial grass (Leymus chinensis), predominantly distributed in the Euro-Asia steppe, was used as an experimental plant for an irrigation manipulation experiment involving five soil moisture levels [75–80, 60–75, 50–60, 35–50, and 25–35% of soil relative water content (SRWC), i.e. the ratio between present soil moisture and field capacity] to examine the effects of soil drought and rewatering on plant biomass, relative growth rate (RGR), and photosynthetic potential. The recovery of plant biomass following rewatering was lower for the plants that had experienced previous drought compared with the controls; the extent of recovery was proportional to the intensity of soil drought. However, the plant RGR, leaf photosynthesis, and light use potential were markedly stimulated by the previous drought, depending on drought intensity, whereas stomatal conductance (g_s) achieved only partial recovery. The results indicated that g_s may be responsible for regulating actual photosynthetic efficiency. It is assumed that the new plant growth and photosynthetic potential enhanced by pre-drought following rewatering may try to overcompensate the great loss of the plant's net primary production due to the pre-drought effect. The present results highlight the episodic effects of drought on grass growth and photosynthesis. This study will assist in understanding how degraded ecosystems can potentially cope with climate change.

Does night-time transpiration contribute to anisohydric behaviour in a Vitis vinifera cultivar?

Rogiers, S. Y., Greer, D. H., Hutton, R. J., Landsberg, J. J. — Wed, 02 Sep 2009 11:01:17 PDT

The hypothesis that vines of the Semillon wine grape variety show anisohydric behaviour was tested, i.e. that tissue hydration is unstable under fluctuating environmental conditions. Stomatal conductance and transpiration rates from leaves were measured during the day and at night. Leaf water potential (_l) in Semillon was negatively correlated to vapour pressure deficit (VPD) both predawn and during the day. Furthermore, _l fell to significantly lower values than in any of the nine other varieties examined. Night-time values of stomatal conductance (g_n) and transpiration (E_n) in Semillon were up to four times higher than in other varieties; plants enclosed in plastic bags overnight to reduce E_n resulted in better plant–soil equilibration so that predawn _l in Semillon was the same as in Grenache. These data indicate that the hypothesis is supported, and that night-time transpiration contributes significantly to the low _l values in Semillon during warm, dry nights. The other contributing factor is daytime stomatal conductance (g_day), which in Semillon leaves was higher than in other varieties, although the decline in g_day with increasing VPD was greater in Semillon than in Shiraz or Grenache. The high values of g_day were associated with high rates of transpiration (E_day) by Semillon through a day when VPD reached 4.5 kPa. When compared to other varieties, Semillon was not unusual in terms of root length density, stomatal density, xylem sap abscisic acid, or leaf electrolyte leakage. Night-time and daytime water loss and insufficient stomatal regulation therefore account for the tendency to anisohydric behaviour shown by Semillon.

The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

Wed, 02 Sep 2009 11:01:17 PDT

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene.

Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco

Zhang, G., Chen, M., Li, L., Xu, Z., Chen, X., Guo, J., Ma, Y. — Wed, 02 Sep 2009 11:01:17 PDT

A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices

Mrosk, C., Forner, S., Hause, G., Kuster, H., Kopka, J., Hause, B. — Wed, 02 Sep 2009 11:01:17 PDT

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.

Assimilation of xylem-transported 13C-labelled CO2 in leaves and branches of sycamore (Platanus occidentalis L.)

McGuire, M. A., Marshall, J. D., Teskey, R. O. — Wed, 02 Sep 2009 11:01:17 PDT

Previous reports have shown that CO₂ dissolved in xylem sap in tree stems can move upward in the transpiration stream. To determine the fate of this dissolved CO₂, the internal transport of respired CO₂ at high concentration from the bole of the tree was simulated by allowing detached young branches of sycamore (Platanus occidentalis L.) to transpire water enriched with a known quantity of ¹³CO₂ in sunlight. Simultaneously, leaf net photosynthesis and CO₂ efflux from woody tissue were measured. Branch and leaf tissues were subsequently analysed for ¹³C content to determine the quantity of transported ¹³CO₂ label that was fixed. Treatment branches assimilated an average of 35% (SE=2.4) of the ¹³CO₂ label taken up in the treatment water. The majority was fixed in the woody tissue of the branches, with smaller amounts fixed in the leaves and petioles. Overall, the fixation of internally transported ¹³CO₂ label by woody tissues averaged 6% of the assimilation of CO₂ from the atmosphere by the leaves. Woody tissue assimilation rates calculated from measurements of ¹³C differed from rates calculated from measurements of CO₂ efflux in the lower branch but not in the upper branch. The results of this study showed unequivocally that CO₂ transported in xylem sap can be fixed in photosynthetic cells in the leaves and branches of sycamore trees and provided evidence that recycling of xylem-transported CO₂ may be an important means by which trees reduce the carbon cost of respiration.

RACK1 is a negative regulator of ABA responses in Arabidopsis

Guo, J., Wang, J., Xi, L., Huang, W.-D., Liang, J., Chen, J.-G. — Wed, 02 Sep 2009 11:01:17 PDT

Receptor for Activated C Kinase 1 (RACK1) is viewed as a versatile scaffold protein in mammals. The protein sequence of RACK1 is highly conserved in eukaryotes. However, the function of RACK1 in plants remains poorly understood. Accumulating evidence suggested that RACK1 may be involved in hormone responses, but the precise role of RACK1 in any hormone signalling pathway remains elusive. Molecular and genetic evidence that Arabidopsis RACK1 is a negative regulator of ABA responses is provided here. It is shown that three RACK1 genes act redundantly to regulate ABA responses in seed germination, cotyledon greening and root growth, because rack1a single and double mutants are hypersensitive to ABA in each of these processes. On the other hand, plants overexpressing RACK1A displayed ABA insensitivity. Consistent with their proposed roles in seed germination and early seedling development, all three RACK1 genes were expressed in imbibed, germinating and germinated seeds. It was found that the ABA-responsive marker genes, RD29B and RAB18, were up-regulated in rack1a mutants. Furthermore, the expression of all three RACK1 genes themselves was down-regulated by ABA. Consistent with the view that RACK1 negatively regulates ABA responses, rack1a mutants lose water significantly more slowly from the rosettes and are hypersensitive to high concentrations of NaCl during seed germination. In addition, the expression of some putative RACK1-interacting, ABA-, or abiotic stress-regulated genes was mis-regulated in rack1a rack1b double mutants in response to ABA. Taken together, these findings provide compelling evidence that RACK1 is a critical, negative regulator of ABA responses.

A rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (Actinidia deliciosa) buds

Wed, 02 Sep 2009 11:01:17 PDT

Budbreak in kiwifruit (Actinidia deliciosa) can be poor in locations that have warm winters with insufficient winter chilling. Kiwifruit vines are often treated with the dormancy-breaking chemical hydrogen cyanamide (HC) to increase and synchronize budbreak. This treatment also offers a tool to understand the processes involved in budbreak. A genomics approach is presented here to increase our understanding of budbreak in kiwifruit. Most genes identified following HC application appear to be associated with responses to stress, but a number of genes appear to be associated with the reactivation of growth. Three patterns of gene expression were identified: Profile 1, an HC-induced transient activation; Profile 2, an HC-induced transient activation followed by a growth-related activation; and Profile 3, HC- and growth-repressed. One group of genes that was rapidly up-regulated in response to HC was the glutathione S-transferase (GST) class of genes, which have been associated with stress and signalling. Previous budbreak studies, in three other species, also report up-regulated GST expression. Phylogenetic analysis of these GSTs showed that they clustered into two sub-clades, suggesting a strong correlation between their expression and budbreak across species.

Molecular mechanism for jasmonate-induction of anthocyanin accumulation in Arabidopsis

Shan, X., Zhang, Y., Peng, W., Wang, Z., Xie, D. — Wed, 02 Sep 2009 11:01:17 PDT

Anthocyanins are important plant pigments that fulfil many physiological and ecological functions. Anthocyanin biosynthesis is controlled by numerous regulatory factors at the transcriptional level. Jasmonates (JAs) has been shown to induce anthocyanin accumulation in several plant species, however, the molecular mechanism for JA-regulated anthocyanin accumulation remains unknown. In this study, genetic, molecular, and physiological approaches were used to reveal the molecular basis of JA-regulated pigmentation in Arabidopsis. It was found that the F-box protein COI1 was required for JA-specific induced expression of the ‘late’ anthocyanin biosynthetic genes DFR, LDOX, and UF3GT. It is further demonstrated that COI1 was essential for JA-induction of transcription factors PAP1, PAP2, and GL3. It is speculated that COI1 regulates the expression of the transcription factors, including PAP1, PAP2, and GL3, which mediates the ‘late’ anthocyanin biosynthetic genes DFR, LDOX, and UF3GT, thereby modulating JA-induced anthocyanin biosynthesis in Arabidopsis.

Identification and localization of the bilitranslocase homologue in white grape berries (Vitis vinifera L.) during ripening

Wed, 02 Sep 2009 11:01:17 PDT

A homologue of the mammalian bilirubin transporter bilitranslocase (BTL) (TCDB 2.A.65.1.1), able to perform an apparent secondary active transport of flavonoids, has previously been found in carnation petals and red grape berries. In the present work, a BTL homologue was also shown in white berries from Vitis vinifera L. cv. Tocai/Friulano, using anti-sequence antibodies specific for rat liver BTL. This transporter, similarly to what found in red grape, was localized in the first layers of the epidermal tissue and in the vascular bundle cells of the mesocarp. In addition, a strong immunochemical reaction was detected in the placental tissue and particularly in peripheral integuments of the seed. The protein was expressed during the last maturation stages in both skin and pulp tissues and exhibited an apparent molecular mass of c. 31 kDa. Furthermore, the transport activity of such a carrier, measured as bromosulphophthalein (BSP) uptake, was detected in berry pulp microsomes, where it was inhibited by specific anti-BTL antibodies. The BTL homologue activity exhibited higher values, for both K_m and V_max, than those found in the red cultivar. Moreover, two non-pigmented flavonoids, such as quercetin (a flavonol) and eriodictyol (a flavanone), inhibited the uptake of BSP in an uncompetitive manner. Such results strengthen the hypothesis that this BTL homologue acts as a carrier involved also in the membrane transport of colourless flavonoids and demonstrate the presence of such a carrier in different organs and tissues.

Gene regulation in parthenocarpic tomato fruit

Wed, 02 Sep 2009 11:01:17 PDT

Parthenocarpy is potentially a desirable trait for many commercially grown fruits if undesirable changes to structure, flavour, or nutrition can be avoided. Parthenocarpic transgenic tomato plants (cv MicroTom) were obtained by the regulation of genes for auxin synthesis (iaaM) or responsiveness (rolB) driven by DefH9 or the INNER NO OUTER (INO) promoter from Arabidopsis thaliana. Fruits at a breaker stage were analysed at a transcriptomic and metabolomic level using microarrays, real-time reverse transcription-polymerase chain reaction (RT-PCR) and a Pegasus III TOF (time of flight) mass spectrometer. Although differences were observed in the shape of fully ripe fruits, no clear correlation could be made between the number of seeds, transgene, and fruit size. Expression of auxin synthesis or responsiveness genes by both of these promoters produced seedless parthenocarpic fruits. Eighty-three percent of the genes measured showed no significant differences in expression due to parthenocarpy. The remaining 17% with significant variation (P <0.05) (1748 genes) were studied by assigning a predicted function (when known) based on BLAST to the TAIR database. Among them several genes belong to cell wall, hormone metabolism and response (auxin in particular), and metabolism of sugars and lipids. Up-regulation of lipid transfer proteins and differential expression of several indole-3-acetic acid (IAA)- and ethylene-associated genes were observed in transgenic parthenocarpic fruits. Despite differences in several fatty acids, amino acids, and other metabolites, the fundamental metabolic profile remains unchanged. This work showed that parthenocarpy with ovule-specific alteration of auxin synthesis or response driven by the INO promoter could be effectively applied where such changes are commercially desirable.

Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response

Wed, 02 Sep 2009 11:01:17 PDT

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.

Developmental changes and organelle biogenesis in the reproductive organs of thermogenic skunk cabbage (Symplocarpus renifolius)

Wed, 02 Sep 2009 11:01:17 PDT

Sex-dependent thermogenesis during reproductive organ development in the inflorescence is a characteristic feature of some of the protogynous arum species. One such plant, skunk cabbage (Symplocarpus renifolius), can produce massive heat during the female stage but not during the subsequent male stage in which the stamen completes development, the anthers dehisce, and pollen is released. Unlike other thermogenic species, skunk cabbage belongs to the bisexual flower group. Although recent studies have identified the spadix as the thermogenic organ, it remains unclear how individual tissues or intracellular structures are involved in thermogenesis. In this study, reproductive organ development and organelle biogenesis were examined during the transition from the female to the male stage. During the female stage, the stamens exhibit extensive structural changes including changes in organelle structure and density. They accumulate high levels of mitochondrial proteins, including possible thermogenic factors, alternative oxidase, and uncoupling protein. By contrast, the petals and pistils do not undergo extensive changes during the female stage. However, they contain a larger number of mitochondria than during the male stage in which they develop large cytoplasmic vacuoles. Comparison between female and male spadices suggests that mitochondrial number rather than their level of activity correlates with thermogenesis. Their spadices, even in the male, contain a larger amount of mitochondria that had greater oxygen consumption, compared with non-thermogenic plants. Taken together, our data suggest that the extensive maturation process in stamens produces massive heat through increased metabolic activities. The possible mechanisms by which petal and pistil metabolism may affect thermogenesis are also discussed.

Ethylene receptor ETR2 controls trichome branching by regulating microtubule assembly in Arabidopsis thaliana

Plett, J. M., Mathur, J., Regan, S. — Wed, 02 Sep 2009 11:01:17 PDT

The single-celled trichome of Arabidopsis thaliana is a widely used model system for studying cell development. While the pathways that control the later stages of trichome development are well characterized, the early signalling events that co-ordinate these pathways are less well understood. Hormones such as gibberellic acid, salicylic acid, cytokinins, and ethylene are known to affect trichome initiation and development. To understand the role of the plant hormone ethylene in trichome development, an Arabidopsis loss-of-function ethylene receptor mutant, etr2-3, which has completely unbranched trichomes, is analysed in this study. It was hypothesized that ETR2 might affect the assembly of the microtubule cytoskeleton based on analysis of the cytoskeleton in developing trichomes, and exposures to paclitaxol and oryzalin, which respectively act either to stabilize or depolymerize the cytoskeleton. Through epistatic and gene expression analyses it is shown that ETR2 is positioned upstream of CHROMATIN ASSEMBLY FACTOR1 and TRYPTICHON and is independent of the GLABRA2 and GLABRA3 pathways. These results help extend understanding of the early events that control trichome development and identify a signalling pathway through which ethylene affects trichome branching.

Genome-wide analysis of the auxin-responsive transcriptome downstream of iaa1 and its expression analysis reveal the diversity and complexity of auxin-regulated gene expression

Lee, D. J., Park, J. W., Lee, H. W., Kim, J. — Wed, 02 Sep 2009 11:01:17 PDT

The AUXIN RESPONSE FACTORs (ARFs) and the Aux/IAA proteins regulate various auxin responses through auxin perception mediated by the F-box proteins TIR1/AFBs. ARFs are transcription factors that modulate expression of auxin response genes and are negatively regulated by the Aux/IAA proteins. To gain insight into the regulatory mechanisms of Aux/IAA-ARF action at the genome level, the transcriptome regulated downstream of iaa1, a stabilized IAA1 mutant protein, was identified using dexamethasone (DEX)-controlled nuclear translocation of iaa1 during the auxin response. The expression of the iaa1-regulated auxin-responsive genes selected from microarray data was analysed with RNA-gel blot analysis and it was shown that auxin-regulated expression of these genes was significantly inhibited by DEX treatment. While cycloheximide-inducible expression of a majority of these genes was also DEX-suppressible, expression of some genes could not be suppressed by treatment with DEX. Expression analysis in a variety of arf mutant backgrounds suggested that all iaa1-regulated auxin-response genes examined are controlled by ARFs to different extents and that the same ARF protein can regulate the expression of these genes in response to auxin in a positive or a negative manner. However, arf mutations did not affect auxin-mediated down-regulation, indicating that ARFs might not play a critical role in down-regulation. The decrease in auxin-responsive gene expression in arf7 arf19 mutants was more severe than that of tir1/afb quadruple mutants. These results show the diversity and complexity of mechanisms of Aux/IAA-ARF- and auxin-regulated gene expression. These data also provide the opportunity for functional analysis of genes mediating the auxin-response downstream of Aux/IAA-ARFs.

Enzymic characterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extension

Maris, A., Suslov, D., Fry, S. C., Verbelen, J.-P., Vissenberg, K. — Wed, 02 Sep 2009 11:01:17 PDT

Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of load-bearing cell wall components. They cleave xyloglucan chains and, often, re-form bonds to the non-reducing ends of available xyloglucan molecules in plant primary cell walls. The enzymic properties and effects on root growth of two Arabidopsis thaliana XTHs belonging to subgroup I/II, that are predominantly expressed in root hairs and in non-elongating zones of the root, were analysed here. AtXTH14 and AtXTH26 were recombinantly produced in Pichia and subsequently purified. Both proteins were found to exhibit xyloglucan endotransglucosylase (XET; EC 2.4.1.207) but not xyloglucan endohydrolase (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was at least 70x greater on xyloglucan rather than on mixed-linkage β-glucan. Differences were found in pH- and temperature-dependence as well as in acceptor–substrate preferences. Furthermore, the specific activity of XET was approximately equal for the two enzymes. Removal of N-linked sugar residues by Endo H treatment reduced XET activity to 60%. Constant-load extensiometry experiments revealed that the enzymes reduce the extension in a model system of heat-inactivated isolated cell walls. When given to growing roots, either of these XTH proteins reduced cell elongation in a concentration-dependent manner and caused abnormal root hair morphology. This is the first time that recombinant and purified XTHs added to growing roots have exhibited a clear effect on cell elongation. It is proposed that these specific XTH isoenzymes play a role in strengthening the side-walls of root-hairs and cell walls in the root differentiation zone after the completion of cell expansion.