Journal of Experimental Botany - current issue

FLC or not FLC: the other side of vernalization

Alexandre, C. M., Hennig, L. — 2008-04-24

Vernalization is the promotion of the competence for flowering by long periods of low temperatures such as those typically experienced during winters. In Arabidopsis, the vernalization response is, to a large extent, mediated by the repression of the floral repressor FLC, and the stable epigenetic silencing of FLC after cold treatments is essential for vernalization. In addition to FLC, other vernalization targets exist in Arabidopsis. In grasses, vernalization seems to be entirely independent of FLC. Here, the current understanding of FLC-independent branches of the vernalization pathway in Arabidopsis and vernalization without FLC in grasses is discussed. This review focuses on the role of AGL19, AGL24, and the MAF genes in Arabidopsis. Interestingly, vernalization acts through related molecular machineries on distinct targets. In particular, protein complexes similar to Drosophila Polycomb Repressive Complex 2 play a prominent role in establishing an epigenetic cellular memory for cold-regulated expression states of AGL19 and FLC. Finally, the similar network topology of the apparently independently evolved vernalization pathways of grasses and Arabidopsis is discussed.

On the mechanism of C4 photosynthesis intermediate exchange between Kranz mesophyll and bundle sheath cells in grasses

Sowinski, P., Szczepanik, J., Minchin, P. E. H. — 2008-04-24

C₄ photosynthesis involves cell-to-cell exchange of photosynthetic intermediates between the Kranz mesophyll (KMS) and bundle sheath (BS) cells. This was believed to occur by simple diffusion through plentiful plasmodesmatal (PD) connections between these cell types. The model of C₄ intermediates’ transport was elaborated over 30 years ago and was based on experimental data derived from measurements at the time. The model assumed that plasmodesmata occupied about 3% of the interface between the KMS and BS cells and that the plasmodesmata structure did not restrict metabolite movement. Recent advances in the knowledge of plasmodesmatal structure put these assumptions into doubt, so a new model is presented here taking the new anatomical details into account. If one assumes simple diffusion as the sole driving force, then calculations based on the experimental data obtained for C₄ grasses show that the gradients expected of C₄ intermediates between KMS and BS cells are about three orders of magnitude higher than experimentally estimated. In addition, if one takes into account that the plasmodesmata microchannel diameter might constrict the movement of C₄ intermediates of comparable Stokes’ radii, the differences in concentration of photosynthetic intermediates between KMS and BS cells should be further increased. We believe that simple diffusion-driven transport of C₄ intermediates between KMS and BS cells through the plasmodesmatal microchannels is not adequate to explain the C₄ metabolite exchange during C₄ photosynthesis. Alternative mechanisms are proposed, involving the participation of desmotubule and/or active mechanisms as either apoplasmic or vesicular transport.

Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells

2008-04-24

Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting ~1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 °C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase D, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

Regulation of shoot branching patterns by the basal root system: towards a predictive model

Thomas, R. G., Hay, M. J. M. — 2008-04-24

This study aimed to underpin the development of a generic predictive model of the regulation of shoot branching by roots in nodally rooting perennial prostrate-stemmed species using knowledge gained from physiological studies of Trifolium repens. Experiment 1 demonstrated that the net stimulatory influence from the basal rooted region of the plant on growth of newly emerging axillary buds on the primary stem decreased as their phytomeric distance from the basal root system increased. Experiment 2 found that at any one time the distribution of net root stimulus (NRS) to the apical bud on the primary stem and all lateral branches was fairly uniform within a single plant. Thus, although NRS availability was uniform throughout the shoot system at any point in time, it progressively decreased as shoot apical buds grew away from the basal root system. Based on these findings, a preliminary predictive model of the physiological regulation of branching pattern was developed. This model can explain the decline in growth rate of buds on a primary stem as it grows away from its basal root system but not the rapid progressive decline in secondary branch development on successive lateral branches. Thus knowledge of NRS availability to emerging buds is not, by itself, a sufficient basis from which to construct a predictive model. In addition, it seems that the ability of an emerging bud to become activated in response to its local NRS availability is, at least in part, directly influenced by the activation level of its parent apical bud. The experimental testing of this hypothesis, required for continued development of the model, is proceeding.

Regulatory involvement of abscisic acid in potato tuber wound-healing

Lulai, E. C., Suttle, J. C., Pederson, S. M. — 2008-04-24

Rapid wound-healing is crucial in protecting potato tubers from infection and dehydration. Wound-induced suberization and the accumulation of hydrophobic barriers to reduce water vapour conductance/loss are principal protective wound-healing processes. However, little is known about the cognate mechanisms that effect or regulate these processes. The objective of this research was to determine the involvement of abscisic acid (ABA) in the regulation of wound-induced suberization and tuber water vapour loss (dehydration). Analysis by liquid chromatography–mass spectrometry showed that ABA concentrations varied little throughout the tuber, but were slightly higher near the periderm and lowest in the pith. ABA concentrations increase then decrease during tuber storage. Tuber wounding induced changes in ABA content. ABA content in wound-healing tuber discs decreased after wounding, reached a minimum by 24 h, and then increased from the 3rd to the 7th day after wounding. Wound-induced ABA accumulations were reduced by fluridone (FLD); an inhibitor of de novo ABA biosynthesis. Wound-induced phenylalanine ammonia lyase activity was slightly reduced and the accumulation of suberin poly(phenolics) and poly(aliphatics) noticeably reduced in FLD-treated tissues. Addition of ABA to the FLD treatment restored phenylalanine ammonia lyase activity and suberization, unequivocally indicating that endogenous ABA is involved in the regulation of these wound-healing processes. Similar experiments showed that endogenous ABA is involved in the regulation of water vapour loss, a process linked to wax accumulation in wound-healing tubers. Rapid reduction of water vapour loss across the wound surface is essential in preventing desiccation and death of cells at the wound site; live cells are required for suberization. These results unequivocally show that endogenous ABA is involved in the regulation of wound-induced suberization and the processes that protect surface cells from water vapour loss and death by dehydration.

Integrated metabolite and gene expression profiling revealing phytochrome A regulation of polyamine biosynthesis of Arabidopsis thaliana

Jumtee, K., Bamba, T., Okazawa, A., Fukusaki, E., Kobayashi, A. — 2008-04-24

In this study, metabolite profiling was demonstrated as a useful tool to plot a specific metabolic pathway, which is regulated by phytochrome A (phyA). Etiolated Arabidopsis wild-type (WT) and phyA mutant seedlings were irradiated with either far-red light (FR) or white light (W). Primary metabolites of the irradiated seedlings were profiled by gas chromatography time-of-flight mass spectrometry (GC/TOF-MS) to obtain new insights on phyA-regulated metabolic pathways. Comparison of metabolite profiles in phyA and WT seedlings grown under FR revealed a number of metabolites that contribute to the differences between phyA and the WT. Several metabolites, including some amino acids, organic acids, and major sugars, as well as putrescine, were found in smaller amounts in WT compared with the content in phyA seedlings grown under FR. There were also significant differences between metabolite profiles of WT and phyA seedlings during de-etiolation under W. The polyamine biosynthetic pathway was investigated further, because putrescine, one of the polyamines existing in a wide variety of living organisms, was found to be present in lower amounts in WT than in phyA under both light conditions. The expression levels of polyamine biosynthesis-related genes were investigated by quantitative real-time RT-PCR. The gene expression profiles revealed that the arginine decarboxylase 2 (ADC2) gene was transcribed less in the WT than in phyA seedlings under both light conditions. This finding suggests that ADC2 is negatively regulated by phyA during photomorphogenesis. In addition, S-adenosylmethionine decarboxylase 2 and 4 (SAMDC2 and SAMDC4) were found to be regulated by phyA but in a different manner from the regulation of ADC2.

The AtMYB11 gene from Arabidopsis is expressed in meristematic cells and modulates growth in planta and organogenesis in vitro

2008-04-24

In plants, MYB transcription factors play important roles in many developmental processes including cell cycle progression, cell differentiation, and lateral organ polarity. It is shown here that the R2R3-MYB AtMYB11 gene is expressed in root and shoot meristems and also in young still meristematic leaf and flower primordia of Arabidopsis. Knock-out atmyb11-I mutants and RNAi plants germinate faster, show a faster hypocotyl and primary root elongation, develop more lateral and adventitious roots, show faster development of the inflorescence, and initiate more lateral inflorescences and fruits than wild-type plants. The opposite phenotype was displayed by plants overexpressing AtMYB11. De novo formation of root meristemoids and, consequently, macroscopic roots, from thin cell layers cultured in vitro was enhanced in explants from atmyb11-I and reduced in those from lines overexpressing AtMYB11. These findings indicate that AtMYB11 modulates overall growth in plants by reducing the proliferation activity of meristematic cells and delaying plant development.

Elicitor-dependent expression of the ribosome-inactivating protein beetin is developmentally regulated

Iglesias, R., Perez, Y., Citores, L., Ferreras, J. M., Mendez, E., Girbes, T. — 2008-04-24

BE27 and BE29 are two forms of beetin, a virus-inducible type 1 ribosome-inactivating protein isolated from leaves of Beta vulgaris L. Western blot analysis revealed the presence of beetin forms in adult plants but not in germ or young plants, indicating that the expression of these proteins is developmentally regulated. While beetins are expressed only in adult plants, their transcripts are present through all stages of development. In addition, the treatment of B. vulgaris leaves with mediators of plant-acquired resistance such as salicylic acid and hydrogen peroxide promoted the expression of beetin by induction of its transcript, but only in adult plants. The plant expresses three mRNAs which differ only in their 3' untranslated region. All these observations suggest a dual regulation of beetin expression, i.e. at the post-transcriptional and transcriptional levels. Additionally, total RNA isolated from leaves treated with hydrogen peroxide, which express high levels of active beetin, is not de-adenylated by endogenous beetin, nor in vitro by the addition of BE27, thus suggesting that sugar beet ribosomes are resistant to beetin.

Silencing of acidic pathogenesis-related PR-1 genes increases extracellular {beta}-(1->3)-glucanase activity at the onset of tobacco defence reactions

Riviere, M.-P., Marais, A., Ponchet, M., Willats, W., Galiana, E. — 2008-04-24

The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence or a functional redundancy in the PR-1 gene family. The data also show that there is a specific increase in apoplastic β-(1->3)-glucanase activity and a decrease in β-(1->3)-glucan deposition in PR-1-silenced lines following activation of defence reactions. Complementation of the silencing by apoplastic treatment with a recombinant PR-1a protein largely restores the wild-type β-(1->3)-glucanase activity and callose phenotype. Taken together with the immunolocalization of PR-1a to sites of β-(1->3)-glucan deposition in wild-type plants, these results are indicative of a function for PR-1a in regulation of enzymatic activity of extracellular β-(1->3)-glucanases.

Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian

Nakatsuka, T., Sato, K., Takahashi, H., Yamamura, S., Nishihara, M. — 2008-04-24

Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0–41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.

Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants

Elvira, M. I., Galdeano, M. M., Gilardi, P., Garcia-Luque, I., Serra, M. T. — 2008-04-24

Resistance conferred by the L³ gene is active against most of the tobamoviruses, including the Spanish strain (PMMoV-S), a P_1,2 pathotype, but not against certain strains of pepper mild mottle virus (PMMoV), termed P_1,2,3 pathotype, such as the Italian strain (PMMoV-I). Both viruses are nearly identical at their nucleotide sequence level (98%) and were used to challenge Capsicum chinense PI159236 plants harbouring the L³ gene in order to carry out a comparative proteomic analysis of PR proteins induced in this host in response to infection by either PMMoV-S or PMMoV-I. PMMoV-S induces a hypersensitive reaction (HR) in C. chinense PI159236 plant leaves with the formation of necrotic local lesions and restriction of the virus at the primary infection sites. In this paper, C. chinense PR protein isoforms belonging to the PR-1, β-1,3-glucanases (PR-2), chitinases (PR-3), osmotin-like protein (PR-5), peroxidases (PR-9), germin-like protein (PR-16), and PRp27 (PR-17) have been identified. Three of these PR protein isoforms were specifically induced during PMMoV-S-activation of C. chinense L³ gene-mediated resistance: an acidic β-1,3-glucanase isoform (PR-2) (M_r 44.6; pI 5.1), an osmotin-like protein (PR-5) (M_r 26.8; pI 7.5), and a basic PR-1 protein isoform (M_r 18; pI 9.4–10.0). In addition, evidence is presented for a differential accumulation of C. chinense PR proteins and mRNAs in the compatible (PMMoV-I)–C. chinense and incompatible (PMMoV-S)–C. chinense interactions for proteins belonging to all PR proteins detected. Except for an acidic chitinase (PR-3) (M_r 30.2; pI 5.0), an earlier and higher accumulation of PR proteins and mRNAs was detected in plants associated with HR induction. Furthermore, the accumulation rates of PR proteins and mRNA did not correlate with maximal accumulation levels of viral RNA, thus indicating that PR protein expression may reflect the physiological status of the plant.

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

2008-04-24

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.

Evidence for sugar signalling in the regulation of asparagine synthetase gene expressed in Phaseolus vulgaris roots and nodules

2008-04-24

A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase (asparagine synthetase) was isolated from nodule tissue of common bean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysis indicated that asparagine synthetase in bean is encoded by a small gene family. Northern analysis of RNAs from various plant organs demonstrated that PvNAS2 is highly expressed in roots, followed by nodules in which it is mainly induced during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter gusA fusion revealed that the expression of PvNAS2 in roots is confined to vascular bundles and meristematic tissues, while in root nodules its expression is solely localized to vascular traces and outer cortical cells encompassing the central nitrogen-fixing zone, but never detected in either infected or non-infected cells located in the central region of the nodule. PvNAS2 is down-regulated when carbon availability is reduced in nodules, and the addition of sugars to the plants, mainly glucose, boosted its induction, leading to the increased asparagine production. In contrast to PvNAS2 expression and the concomitant asparagine synthesis, glucose supplement resulted in the reduction of ureide content in nodules. Studies with glucose analogues as well as hexokinase inhibitors suggested a role for hexokinase in the sugar-sensing mechanism that regulates PvNAS2 expression in roots. In light of the above results, it is proposed that, in bean, low carbon availability in nodules prompts the down-regulation of the asparagine synthetase enzyme and concomitantly asparagine production. Thereby a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of purines, and then ureide generation.

Effects of cotton rootstock on endogenous cytokinins and abscisic acid in xylem sap and leaves in relation to leaf senescence

Dong, H., Niu, Y., Li, W., Zhang, D. — 2008-04-24

Leaf senescence varies greatly among cotton cultivars, possibly due to their root characteristics, particularly the root-sourced cytokinins and abscisic acid (ABA). Early-senescence (K1) and late-senescence (K2) lines, were reciprocally or self-grafted to examine the effects of rootstock on leaf senescence and endogenous hormones in both leaves and xylem sap. The results indicate that the graft of K1 scion onto K2 rootstock (K1/K2) alleviated leaf senescence with enhanced photosynthetic (Pn) rate, increased levels of chlorophyll (Chl) and total soluble protein (TSP), concurrently with reduced malondialdehyde (MDA) contents in the fourth leaf on the main-stem. The graft of K2 scion onto K1 rootstock enhanced leaf senescence with reduced Pn, Chl, and TSP, and increased MDA, compared with their respective self-grafted control plants (K1/K1 and K2/K2). Reciprocally grafted plants differed significantly from their self-grafted control plants in levels of zeatin and its riboside (Z+ZR), isopentenyl and its adenine (iP+iPA), and ABA, but not in those of dihydrozeatin and its riboside (DHZ+DHZR) in leaves in late season, which was consistent with variations in leaf senescence between reciprocally and self-grafted plants. The results suggest that leaf senescence is closely associated with reduced accumulation of Z+ZR, and iP+iPA rather than DHZ+DHZR, or enhanced ABA in leaves of cotton. Genotypic variation in leaf senescence may result from the difference in root characteristics, particularly in Z+ZR, iP+iPA, and ABA which are regulated by the root system directly or indirectly.

Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation

Das, S., DeMason, D. A., Ehlers, J. D., Close, T. J., Roberts, P. A. — 2008-04-24

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites with a broad host range which includes economically important crop species. Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume grown in many regions where root-knot nematodes are a major problem in production fields. Several sources of resistance to root-knot nematode have been identified in cowpea, including the widely used Rk gene. As part of a study to elucidate the mechanism of Rk-mediated resistance, the histological response to avirulent M. incognita feeding of a resistant cowpea cultivar CB46 was compared with a susceptible near-isogenic line (in CB46 background). Most root-knot nematode resistance mechanisms in host plants that have been examined induced a hypersensitive response (HR). However, there was no typical HR in resistant cowpea roots and nematodes were able to develop normal feeding sites similar to those in susceptible roots up to 9–14 d post inoculation (dpi). From 14–21 dpi giant cell deterioration was observed and the female nematodes showed arrested development and deterioration. Nematodes failed to reach maturity and did not initiate egg laying in resistant roots. These results confirmed that the induction of resistance is relatively late in this system. Typically in pathogen resistance HR is closely associated with an oxidative burst (OB) in infected tissue. The level of reactive oxygen species release in both compatible and incompatible reactions during early and late stages of infection was also quantified. Following a basal OB during early infection in both susceptible and resistant roots, which was also observed in mechanically wounded root tissues, no significant OB was detected up to 14 dpi, a profile consistent with the histological observations of a delayed resistance response. These results will be useful to design gene expression experiments to dissect Rk-mediated resistance at the molecular level.

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress

Hassine, A. B., Ghanem, M. E., Bouzid, S., Lutts, S. — 2008-04-24

Soil salinity and drought compromise water uptake and lead to osmotic adjustment in xero-halophyte plant species. These important environmental constraints may also have specific effects on plant physiology. Stress-induced accumulation of osmocompatible solutes was analysed in two Tunisian populations of the Mediteranean shrub Atriplex halimus L.—plants originating from a salt-affected coastal site (Monastir) or from a non-saline semi-arid area (Sbikha)—were exposed to nutrient solution containing either low (40 mM) or high (160 mM) doses of NaCl or 15% polyethylene glycol. The low NaCl dose stimulated plant growth in both populations. Plants from Monastir were more resistant to high salinity and exhibited a greater ability to produce glycinebetaine in response to salt stress. Conversely, plants from Sbikha were more resistant to water stress and displayed a higher rate of proline accumulation. Proline accumulated as early as 24 h after stress imposition and such accumulation was reversible. By contrast, glycinebetaine concentration culminated after 10 d of stress and did not decrease after the stress relief. The highest salt resistance of Monastir plants was not due to a lower rate of Na⁺ absorption; plants from this population exhibited a higher stomatal conductance and a prodigal water-use strategy leading to lower water-use efficiency than plants from Sbikha. Exogenous application of proline (1 mM) improved the level of drought resistance in Monastir plants through a decrease in oxidative stress quantified by the malondialdehyde concentration, while the exogenous application of glycinebetaine improved the salinity resistance of Sbikha plants through a positive effect on photosystem II efficiency.

Peripheral membrane proteins mediate binding of vacuolar storage proteins to membranes of the secretory pathway of developing pea cotyledons

von Lupke, A., Schauermann, G., Feussner, I., Hinz, G. — 2008-04-24

In developing pea cotyledons, storage proteins are sorted via dense vesicles into the protein storage vacuole. Formation of these unique transport vesicles is characterized by aggregation of their cargo proteins. Protein sorting into dense vesicles is pH dependent. In order to gain insight into the molecular basis of storage protein sorting, a membrane binding assay was developed which allows for a detailed biochemical analysis of binding events. Employing this assay it was possible to show that storage proteins bind in a pH-dependent manner to the membranes of the secretory pathway with a pH optimum in the range of the lumenal pH of the Golgi cisternae. Through reconstitution experiments, it was possible to demonstrate further that this recruitment occurs via the interaction of peripheral rather than intrinsic membrane proteins. Results of co-immunoprecipitation experiments point to interactions between different storage proteins in the secretory system. These results are discussed in terms of the aggregation-mediated sorting of storage proteins into maturing dense vesicles.

Phloem transdifferentiation from immature xylem cells during bark regeneration after girdling in Eucommia ulmoides Oliv

Pang, Y., Zhang, J., Cao, J., Yin, S.-Y., He, X.-Q., Cui, K.-M. — 2008-04-24

Eucommia ulmoides Oliv. (Eucommiaceae), a traditional Chinese medicinal plant, was used to study phloem cell differentiation during bark regeneration after girdling on a large scale. Here it is shown that new sieve elements (SEs) appeared in the regenerated tissues before the formation of wound cambium during bark regeneration after girdling, and they could originate from the transdifferentiation of immature/differentiating axial xylem cells left on the trunk. Assays of water-cultured twigs revealed that girdling blocked sucrose transport until the formation of new SEs, and the regeneration of the functional SEs was not dependent on the substance provided by the axis system outside the girdled areas, while exogenous indole acetic acid (IAA) applied on the wound surface accelerated SE differentiation. The experiments suggest that the immature xylem cells can transdifferentiate into phloem cells under certain conditions, which means xylem and phloem cells might share some identical features at the beginning of their differentiation pathway. This study also showed that the bark regeneration system could provide a novel method for studying xylem and phloem cell differentiation.

Respiration of thermogenic inflorescences of Philodendron melinonii: natural pattern and responses to experimental temperatures

Seymour, R. S., Gibernau, M. — 2008-04-24

The patterns of temperature and respiratory changes in the protogynous inflorescences of Philodendron melinonii (Araceae) were studied in the field in French Guiana. These are the first respiratory measurements from a member of the large subgenus Philodendron, a group previously thought to lack thermoregulatory inflorescences, in contrast to thermoregulatory Philodendron species of the subgenus Meconostigma. Heating by the male and sterile male florets was strong on the first evening of anthesis when beetles are attracted and the female florets are receptive. Heat production of the inflorescence peaked at ~0.9 W and spadix temperature reached ~39.5 °C, a level somewhat independent of ambient temperature. Thermogenesis continued throughout the night and the next day, but at a lower level, and floral temperatures fell. On the second evening, when pollen was shed, there was a small elevation in respiration and spadix temperature. Responses of cut spadix sections to experimental step changes in ambient temperature resulted in a prompt response in floral temperature and respiration rate in the direction of the change and then a much slower regulatory adjustment in the opposite direction. These responses are consistent with an immediate van 't Hoff effect, followed by up- or down-regulation of thermogenesis. However, the responses required several hours. It is concluded that the male floret tissues possess the same thermoregulatory mechanism of more precise thermoregulatory species, but a combination of small spadix size (that favours heat loss), moderate thermogenic capacity (that limits heating rate), and slow reaction time (that causes long lags between temperature change and the regulatory response) result in poor thermoregulatory performance during the second day.

A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor

Fortunati, A., Piconese, S., Tassone, P., Ferrari, S., Migliaccio, F. — 2008-04-24

A new mutant of Arabidopsis named rha1 is characterized and the gene involved cloned. In roots, the mutant shows minimal right-handed slanting, reduced gravitropic response, notable resistance to 2,4-D, but scarce resistance to IAA and NAA. The roots also show a clear resistance to the auxin transport inhibitors TIBA and NPA, and to ethylene. Other characteristics are a reduced number of lateral roots and reduced size of shoot and root in the seedlings. The gene, cloned through TAIL-PCR, was found to be a heat-shock factor that maps on chromosome 5, close to and above the RFLP marker m61. The rha1 structure, mRNA, and translation product are reported. Since, so far, no other gravitropic mutant has been described as mutated in a heat-shock factor, rha1 belongs to a new group of mutants disturbed in slanting, gravitropism, and auxin physiology. As shown through the RT-PCR analyses of its expression, the gene retains the function connected with heat shock. If the characteristics connected with auxin physiology are considered, however, it is also likely that the gene, as a transcription factor, could be involved in root circumnutation, gravitropic response, and hormonal control of differentiation. Since GUS staining under the gene promoter was localized mainly in the mature tissues, rha1 does not seem to be involved in the first steps of gravitropism, but is rather related to the general response to auxin. The alterations in slanting (mainly due to reduced chiral circumnutation) and gravitropism lead to the supposition that the two processes may have, at least in part, common origins.

Expression of the nuclear gene TaFAd is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm

Xu, P., Yang, Y., Zhang, Z., Chen, W., Zhang, C., Zhang, L., Zou, S., Ma, Z. — 2008-04-24

Alterations of mitochondrial-encoded subunits of the F_oF₁-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF_Ad that encodes the putative F_Ad subunit of the F_oF₁-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer ‘2114’ for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF_Ad is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF_Ad–GFP fusion in onion epidermal cells demonstrated TaF_Ad's mitochondrial location. TaF_Ad was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F_o was down-regulated in a manner similar to TaF_Ad in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F_o and the subunit of F₁ was unaffected. These observations implied that TaF_Ad is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

Structure-function analysis of the NB-ARC domain of plant disease resistance proteins

2008-04-24

Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine–histidine–aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)—co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

Shaping the shoot: the relative contribution of cell number and cell shape to variations in internode length between parent and hybrid apple trees

Ripetti, V., Escoute, J., Verdeil, J. L., Costes, E. — 2008-04-24

Genetic control of plant size and shape is a promising perspective, particularly in fruit trees, in order to select desirable genotypes. A recent study on architectural traits in an apple progeny showed that internode length was a highly heritable character. However, few studies have been devoted to internode cellular patterning in dicotyledonous stems, and the interplay between the two elementary cell processes that contribute to their length, i.e. cell division and elongation, is not fully understood. The present study aimed at unravelling their contributions in the genetic variation of internode length in a selection of F₁ and parent genotypes of apple tree, by exploring the number of cells and cell shape within mature internodes belonging to the main axes. The results highlighted that both the variables were homogeneous in samples collected either along a sagital line or along the pith width, and suggest that cell lengthening was homogeneous during internode development. They allowed the total number of cells to be estimated on the internode scale and opened up new perspectives for simplifying tissue sampling procedures for further investigations. Differences in internode length were observed between the genotypes, in particular between the parents, and partly resulted from a compensation between cell number and cell length. However, genetic variations in internode length primarily involved the number of cells, while cell length was more secondary. These results argue for an interplay between cellular and organismal control of internode shape that may involve the rib meristem.

Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions

Vidhyavathi, R., Venkatachalam, L., Sarada, R., Ravishankar, G. A. — 2008-04-24

Haematococcus pluvialis, a green alga, accumulates carotenoids, predominantly astaxanthin, when exposed to stress conditions. In the present work, changes in the pigment profile and expression of carotenogenic genes under various nutrient stress conditions and their regulation were studied. Nutrient stress and higher light intensity in combination with NaCl/sodium acetate (SA) enhanced total carotenoid and total astaxanthin content to 32.0 and 24.5 mg g^–1 of dry biomass, respectively. Expression of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), β-carotene ketolase (BKT), and β-carotene hydroxylase (CHY) were up-regulated under all the stress conditions studied. However, the extent of expression of carotenogenic genes varied with stress conditions. Nutrient stress and high light intensity induced expression of astaxanthin biosynthetic genes, BKT and CHY, transiently. Enhanced expression of these genes was observed with SA and NaCl/SA, while expression was delayed with NaCl. The maximum content of astaxanthin recorded in cells grown in medium with SA and NaCl/SA correlated with the expression profile of the astaxanthin biosynthetic genes. Studies using various inhibitors indicated that general carotenogenesis and secondary carotenoid induction were regulated at both the transcriptional and the cytoplasmic translational levels. The induction of general carotenoid synthesis genes was independent of cytoplasmic protein synthesis while BKT gene expression was dependent on de novo protein synthesis.

Difference in light-induced increase in ploidy level and cell size between adaxial and abaxial epidermal pavement cells of Phaseolus vulgaris primary leaves

Kinoshita, I., Sanbe, A., Yokomura, E-i. — 2008-04-24

Changes in nuclear DNA content and cell size of adaxial and abaxial epidermal pavement cells were investigated using bright light-induced leaf expansion of Phaseolus vulgaris plants. In primary leaves of bean plants grown under high (sunlight) or moderate (ML; photon flux density, 163 µmol m^–2 s^–1) light, most adaxial epidermal pavement cells had a nucleus with the 4C amount of DNA, whereas most abaxial pavement cells had a 2C nucleus. In contrast, plants grown under low intensity white light (LL; 15 µmol m^–2 s^–1) for 13 d, when cell proliferation of epidermal pavement cells had already finished, had a 2C nuclear DNA content in most adaxial pavement cells. When these LL-grown plants were transferred to ML, the increase in irradiance raised the frequency of 4C nuclei in adaxial but not in abaxial pavement cells within 4 d. On the other hand, the size of abaxial pavement cells increased by 53% within 4 d of transfer to ML and remained unchanged thereafter, whereas adaxial pavement cells continuously enlarged for 12 d. This suggests that the increase in adaxial cell size after 4 d is supported by the nuclear DNA doubling. The different responses between adaxial and abaxial epidermal cells were not induced by the different light intensity at both surfaces. It was shown that adaxial epidermal cells have a different property than abaxial ones.

Different growth responses of C3 and C4 grasses to seasonal water and nitrogen regimes and competition in a pot experiment

Niu, S., Liu, W., Wan, S. — 2008-04-24

Understanding temporal niche separation between C₃ and C₄ species (e.g. C₃ species flourishing in a cool spring and autumn while C₄ species being more active in a hot summer) is essential for exploring the mechanism for their co-existence. Two parallel pot experiments were conducted, with one focusing on water and the other on nitrogen (N), to examine growth responses to water or nitrogen (N) seasonality and competition of two co-existing species Leymus chinensis (C₃ grass) and Chloris virgata (C₄ grass) in a grassland. The two species were planted in either monoculture (two individuals of one species per pot) or a mixture (two individuals including one L. chinensis and one C. virgata per pot) under three different water or N seasonality regimes, i.e. the average model (AM) with water or N evenly distributed over the growing season, the one-peak model (OPM) with more water or N in the summer than in the spring and autumn, and the two-peak model (TPM) with more water or N in the spring and autumn than in the summer. Seasonal water regimes significantly affected biomass in L. chinensis but not in C. virgata, while N seasonality impacted biomass and relative growth rate of both species over the growing season. L. chinensis accumulated more biomass under the AM and TPM than OPM water or N treatments. Final biomass of C. virgata was less impacted by water and N seasonality than that of L. chinensis. Interspecific competition significantly decreased final biomass in L. chinensis but not in C. virgata, suggesting an asymmetric competition between the two species. The magnitude of interspecific competition varied with water and N seasonality. Changes in productivity and competition balance of L. chinensis and C. virgata under shifting seasonal water and N availabilities suggest a contribution of seasonal variability in precipitation and N to the temporal niche separation between C₃ and C₄ species.