Journal of Experimental Botany - Advance Access

Arabidopsis thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light

Koniger, M., Delamaide, J. A., Marlow, E. D., Harris, G. C. — 2008-05-08

The effects of chloroplast number and size on the capacity for blue light-dependent chloroplast movement, the ability to increase light absorption under low light, and the susceptibility to photoinhibition were investigated in Arabidopsis thaliana. Leaves of wild-type and chloroplast number mutants with mean chloroplast numbers ranging from 120 to two per mesophyll cell were analysed. Chloroplast movement was monitored as changes in light transmission through the leaves. Light transmission was used as an indicator of the ability of leaves to optimize light absorption. The ability of leaves to deal with 3 h of high light stress at 10 °C and their capacity to recover in low light was determined by measuring photochemical efficiencies of PSII using chlorophyll a fluorescence. Chloroplast movement was comparable in leaves ranging in chloroplast numbers from 120 to 30 per mesophyll cell: the final light transmission levels after exposure to 0.1 (accumulation response) and 100 µmol photons m^–2 s^–1 (avoidance response) were indistinguishable, the chloroplasts responded quickly to small increases in light intensity and the kinetics of movement were similar. However, when chloroplast numbers per mesophyll cell decreased to 18 or below, the accumulation response was significantly reduced. The avoidance response was only impaired in mutants with nine or fewer chloroplasts, both in terms of final transmission levels and the speed of movement. Only mutants lacking both blue light receptors (phot1/phot2) or those with drastically reduced chloroplast numbers and severely impacted avoidance responses showed a reduced ability to recover from high light stress.

Modulation of heat shock factors accompanies salicylic acid-mediated potentiation of Hsp70 in tomato seedlings

Snyman, M., Cronje, M. J. — 2008-05-08

In plants, salicylic acid (SA) is a signalling molecule regulating disease resistance responses such as systemic acquired resistance (SAR) and the hypersensitive response (HR), and has been implicated in both basal and acquired thermotolerance. It has been shown that SA enhances heat-induced Hsp/Hsc70 accumulation in plants. To investigate the mechanism of how SA influences the heat shock response (HSR) in plants, tomato seedlings were treated with SA alone, heat shock, or a combination of both before analyses of hsp70 mRNA, heat shock factor (Hsf)–DNA binding, and gene expression of hsp70, hsfA1, hsfA2, and hsfB1. SA alone led to activation of Hsf–DNA binding, but not induction or transcription of hsp70 mRNA. SA had no significant effect on hsfA2 and hsfB1 gene expression, but potentiated the basal levels of hsfA1. In heat-shocked plants, Hsf–DNA binding was established, and increased hsfA1, hsfA2, and hsfB1 expression was followed by accumulation of Hsp70. SA plus heat shock showed enhanced Hsf–DNA binding, enhanced induction of hsp70 mRNA transcription, and gene expression of hsfA1, hsfA2, and hsfB1, resulting in potentiated levels of Hsp/Hsc70. Since increased hsp70 and hsf gene expression coincide with increased levels of Hsp70 accumulation, it is concluded that SA-mediated potentiation of Hsp70 is due to modulation of these Hsfs by SA. In our efforts to understand the role of Hsp70 in heat-related disease susceptibility, the degree of the complexity of the cross-talk between the pathways in which SA is involved, inter alia, the plant defence response, the HSR and thermotolerance, was further underscored.

Expression pattern and activity of six glutelin gene promoters in transgenic rice

Qu, L. Q., Xing, Y. P., Liu, W. X., Xu, X. P., Song, Y. R. — 2008-05-07

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with β-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity.

Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

2008-05-07

Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.

Overexpression of phytochelatin synthase in tobacco: distinctive effects of AtPCS1 and CePCS genes on plant response to cadmium

2008-05-07

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 µM CdCl₂) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of -glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in -glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.

Identification of woolliness response genes in peach fruit after post-harvest treatments

2008-05-03

Woolliness is a physiological disorder of peaches and nectarines that becomes apparent when fruit are ripened after prolonged periods of cold storage. This disorder is of commercial importance since shipping of peaches to distant markets and storage before selling require low temperature. However, knowledge about the molecular basis of peach woolliness is still incomplete. To address this issue, a nylon macroarray containing 847 non-redundant expressed sequence tags (ESTs) from a ripe peach fruit cDNA library was developed and used. Gene expression changes of peach fruit (Prunus persica cv. O'Henry) ripened for 7 d at 21 °C (juicy fruit) were compared with those of fruit stored for 15 d at 4 °C and then ripened for 7 d at 21 °C (woolly fruit). A total of 106 genes were found to be differentially expressed between juicy and woolly fruit. Data analysis indicated that the activity of most of these genes (>90%) was repressed in the woolly fruit. In cold-stored peaches (cv. O'Henry), the expression level of selected genes (cobra, endopolygalacturonase, cinnamoyl-CoA-reductase, and rab11) was lower than in the juicy fruit, and it remained low in woolly peaches after ripening, a pattern that was conserved in woolly fruit from two other commercial cultivars (cv. Flamekist and cv. Elegant Lady). In addition, the results of this study indicate that molecular changes during fruit woolliness involve changes in the expression of genes associated with cell wall metabolism and endomembrane trafficking. Overall, the results reported here provide an initial characterization of the transcriptome activity of peach fruit under different post-harvest treatments.

Rice-arsenate interactions in hydroponics: a three-gene model for tolerance

2008-05-02

In this study, the genetic mapping of the tolerance of root growth to 13.3 µM arsenate [As(V)] using the BalaxAzucena population is improved, and candidate genes for further study are identified. A remarkable three-gene model of tolerance is advanced, which appears to involve epistatic interaction between three major genes, two on chromosome 6 and one on chromosome 10. Any combination of two of these genes inherited from the tolerant parent leads to the plant having tolerance. Lists of potential positional candidate genes are presented. These are then refined using whole genome transcriptomics data and bioinformatics. Physiological evidence is also provided that genes related to phosphate transport are unlikely to be behind the genetic loci conferring tolerance. These results offer testable hypotheses for genes related to As(V) tolerance that might offer strategies for mitigating arsenic (As) accumulation in consumed rice.

Rice-arsenate interactions in hydroponics: whole genome transcriptional analysis

Norton, G. J., Lou-Hing, D. E., Meharg, A. A., Price, A. H. — 2008-05-02

Rice (Oryza sativa) varieties that are arsenate-tolerant (Bala) and -sensitive (Azucena) were used to conduct a transcriptome analysis of the response of rice seedlings to sodium arsenate (AsV) in hydroponic solution. RNA extracted from the roots of three replicate experiments of plants grown for 1 week in phosphate-free nutrient with or without 13.3 µM AsV was used to challenge the Affymetrix (52K) GeneChip Rice Genome array. A total of 576 probe sets were significantly up-regulated at least 2-fold in both varieties, whereas 622 were down-regulated. Ontological classification is presented. As expected, a large number of transcription factors, stress proteins, and transporters demonstrated differential expression. Striking is the lack of response of classic oxidative stress-responsive genes or phytochelatin synthases/synthatases. However, the large number of responses from genes involved in glutathione synthesis, metabolism, and transport suggests that glutathione conjugation and arsenate methylation may be important biochemical responses to arsenate challenge. In this report, no attempt is made to dissect differences in the response of the tolerant and sensitive variety, but analysis in a companion article will link gene expression to the known tolerance loci available in the BalaxAzucena mapping population.

The rice StMADS11-like genes OsMADS22 and OsMADS47 cause floral reversions in Arabidopsis without complementing the svp and agl24 mutants

Fornara, F., Gregis, V., Pelucchi, N., Colombo, L., Kater, M. — 2008-05-02

During floral induction and flower development plants undergo delicate phase changes which are under tight molecular control. MADS-box transcription factors have been shown to play pivotal roles during these transition phases. SHORT VEGETATIVE PHASE (SVP) and AGAMOUS LIKE 24 (AGL24) are important regulators both during the transition to flowering and during flower development. During vegetative growth they exert opposite roles on floral transition, acting as repressor and promoter of flowering, respectively. Later during flower development they act redundantly as negative regulators of AG expression. In rice, the orthologues of SVP and AGL24 are OsMADS22, OsMADS47, and OsMADS55 and these three genes are involved in the negative regulation of brassinosteroid responses. In order to understand whether these rice genes have maintained the ability to function as regulators of flowering time in Arabidopsis, complementation tests were performed by expressing OsMADS22 and OsMADS47 in the svp and agl24 mutants. The results show that the rice genes are not able to complement the flowering-time phenotype of the Arabidopsis mutants, indicating that they are biologically inactive in Arabidopsis. Nevertheless, they cause floral reversions, which mimic the SVP and AGL24 floral overexpressor phenotypes. Yeast two-hybrid analysis suggests that these floral phenotypes are probably the consequence of protein interactions between OsMADS22 and OsMADS47 and other MADS-box proteins which interfere with the formation of complexes required for normal flower development.

Is the onset of senescence in leaf cells of intact plants due to low or high sugar levels?

van Doorn, W. G. — 2008-05-02

This review examines the hypotheses that developmental programmed cell death in leaves is mediated (i) by sugar starvation in the leaf cells or (ii) by sugar accumulation in these cells. Experimental evidence for both hypotheses is critically discussed and found to be lacking. For example, some papers show that sugars prevent senescence of cut leaves placed in darkness, and prevent low sugar levels in the leaves. In these tests, the sugars seem to replace photosynthesis, hence the results have little relevance to leaf senescence in intact plants in the light. Low nitrogen nutrition and high light results in earlier senescence than the low nitrogen treatment alone. This is accompanied by high sugar levels in the leaves. The results have led to the idea that accumulation of sugars is the cause of the additional effect, or more generally, that sugar accumulation is always the direct cause of leaf senescence. Results from over-expressing, or knocking out, hexokinase genes tend to support the high sugar hypothesis, but pleiotropic effects confound this conclusion. In addition, several experiments show the effects of treatments on senescence without the increase in leaf sugar levels. Nonetheless, sugar levels are usually measured in whole leaves. Such an overall level does not reflect the differences in the onset of senescence between tissues and cells, and can therefore not be used as an argument for or against either of the two hypotheses. It is argued that future work should determine the time line of the concentrations of various sugars in various cells and cellular compartments, in relation to senescence processes in the same cells. Taken together, the data are not decisive. It is possible that neither of the two hypotheses is correct.

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum)

Kargiotidou, A., Deli, D., Galanopoulou, D., Tsaftaris, A., Farmaki, T. — 2008-05-02

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.

Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway

Oracz, K., El-Maarouf-Bouteau, H., Bogatek, R., Corbineau, F., Bailly, C. — 2008-04-30

Freshly harvested sunflower (Helianthus annuus L.) seeds are considered to be dormant because they fail to germinate at relatively low temperatures (10 °C). This dormancy results mainly from an embryo dormancy and disappears during dry storage. Although endogenous ethylene is known to be involved in sunflower seed alleviation of dormancy, little attention had been paid to the possible role of cyanide, which is produced by the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene, in this process. The aims of this work were to investigate whether exogenous cyanide could improve the germination of dormant sunflower seeds and to elucidate its putative mechanisms of action. Naked dormant seeds became able to germinate at 10 °C when they were incubated in the presence of 1 mM gaseous cyanide. Other respiratory inhibitors showed that this effect did not result from an activation of the pentose phosphate pathway or the cyanide-insensitive pathway. Cyanide stimulated germination of dormant seeds in the presence of inhibitors of ethylene biosynthesis, but its improving effect required functional ethylene receptors. It did not significantly affect ethylene production and the expression of genes involved in ethylene biosynthesis or in the first steps of ethylene signalling pathway. However, the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was markedly stimulated in the presence of gaseous cyanide. It is proposed that the mode of action of cyanide in sunflower seed dormancy alleviation does not involve ethylene production and that ERF1 is a common component of the ethylene and cyanide signalling pathways.

Expression of the {beta}-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

Castillo, M. C., Leon, J. — 2008-04-30

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic β-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.

Genetic variability for leaf growth rate and duration under water deficit in sunflower: analysis of responses at cell, organ, and plant level

Pereyra-Irujo, G. A., Velazquez, L., Lechner, L., Aguirrezabal, L. A. N. — 2008-04-29

Plants under water deficit reduce leaf growth, thereby reducing transpiration rate at the expense of reduced photosynthesis. The objective of this work was to analyse the response of leaf growth to water deficit in several sunflower genotypes in order to identify and quantitatively describe sources of genetic variability for this trait that could be used to develop crop varieties adapted to specific scenarios. The genetic variability of the response of leaf growth to water deficit was assessed among 18 sunflower (Helianthus annuus L.) inbred lines representing a broad range of genetic diversity. Plants were subjected to long-term, constant-level, water-deficit treatments, and the response to water deficit quantified by means of growth models at cell-, leaf-, and plant-scale. Significant variation among lines was found for the response of leaf expansion rate and of leaf growth duration, with an equal contribution of these responses to the variability in the reduction of leaf area. Increased leaf growth duration under water deficit is usually suggested to be caused by changes in the activity of cell-wall enzymes, but the present results suggest that the duration of epidermal cell division plays a key role in this response. Intrinsic genotypic responses of rate and duration at a cellular scale were linked to genotypic differences in whole-plant leaf area profile to water deficit. The results suggest that rate and duration responses are the result of different physiological mechanisms, and therefore capable of being combined to increase the variability in leaf area response to water deficit.

The organ-dependent abundance of a Solanum lipid transfer protein is up-regulated upon osmotic constraints and associated with cold acclimation ability

Kielbowicz-Matuk, A., Rey, P., Rorat, T. — 2008-04-25

The expression of a gene isolated from cDNA differential screening and encoding a lipid transfer protein, designated as SsLTP1, was analysed at the protein level in two groups of Solanum species and lines differing in cold acclimation capacity. Under control conditions, the SsLTP1 was localized in all aerial organs of S. sogarandinum and S. tuberosum plants. Western analysis of subcellular extracts indicated that the protein possesses an intracellular localization. The protein abundance was found to vary as a function of organ type, the highest levels being observed in flowers, stems, and young leaves. During low temperature treatment, no change in protein level was noticed in either the S. tuberosum cv. Irga, which displays a low capacity for cold acclimation, or in a S. sogarandinum line which has lost its cold acclimation capacity. By contrast, low temperature induced a noticeable increase in SsLTP1 level in stems and leaves of S. sogarandinum and S. tuberosum cv. Ursus plants, which are able to acclimate to cold, indicating that SsLTP1 could participate in the processes leading to freezing tolerance. In other respects, SsLTP1 accumulation was observed both in cold-acclimating and in non-acclimating Solanum species when subjected to water deficit or to salt treatment. These data indicate that SsLTP1 gene expression is regulated in an organ-dependent manner and through distinct pathways under non-freezing low temperature and during osmotic treatments.

OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

Li, D., Liu, H., Zhang, H., Wang, X., Song, F. — 2008-04-25

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.

Evolutionary physiology: the extent of C4 and CAM photosynthesis in the genera Anacampseros and Grahamia of the Portulacaceae

Guralnick, L. J., Cline, A., Smith, M., Sage, R. F. — 2008-04-24

The Portulacaceae is one of the few terrestrial plant families known to have both C₄ and Crassulacean acid metabolism (CAM) species. There may be multiple origins of the evolution of CAM within the Portulacaceae but the only clear evidence of C₄ photosynthesis is found in members of the genus Portulaca. In the Portulaca, CAM succulent tissue is overlaid with the C₄ tissue in a unique fashion where both pathways are operating simultaneously. Earlier reports have shown that the clade containing the genera Anacampseros and Grahamia may also contain C₄ photosynthetic species similar to the Portulaca, which would indicate multiple origins of C₄ photosynthesis within the family. The aim of the present study was to ascertain the true photosynthetic nature of these genera. An initial survey of the carbon isotope composition of the Anacampseros ranged from –12.6 to –24.0, indicating very little CAM activity in some species, with other values close to the C₄ range. Anacampseros (=Grahamia) australiana which had been previously identified as a C₄ species had a carbon isotope composition value of –24.0, which is more indicative of a C₃ species with a slight contribution of CAM activity. Other Anacampseros species with C₄-like values have been shown to be CAM plants. The initial isotope analysis of the Grahamia species gave values in the range of –27.1 to –23.6, placing the Grahamia species well towards the C₃ photosynthetic range. Further physiological studies indicated increased night-time CO₂ uptake with imposition of water stress, associated with a large diurnal acid fluctuation and a marked increased phosphoenolpyruvate carboxylase activity. This showed that the Grahamia species are actually facultative CAM plants despite their C₃-like carbon isotope values. The results indicate that the Grahamia and Anacampseros species do not utilize the C₄ photosynthetic pathway. This is the first to identify that the Grahamia species are facultative CAM plants where CAM can be induced by water stress. This work supports earlier physiological work that indicates that this clade containing Anacampseros and Grahamia species comprises predominantly facultative CAM plants. This report suggests there may be only one clade which contains C₄ photosynthetic members with CAM-like characteristics.

On the nature of facultative and constitutive CAM: environmental and developmental control of CAM expression during early growth of Clusia, Kalanchoe, and Opuntia

Winter, K., Garcia, M., Holtum, J. A. M. — 2008-04-24

The capacity to induce crassulacean acid metabolism developmentally (constitutive CAM) and to up-regulate CAM expression in response to drought stress (facultative CAM) was studied in whole shoots of seven species by measuring net CO₂ gas exchange for up to 120 day–night cycles during early growth. In Clusia rosea, CAM was largely induced developmentally. Well-watered seedlings began their life cycle as C₃ plants and developed net dark CO₂ fixation indicative of CAM after the initiation of the fourth leaf pair following the cotyledons. Thereafter, CAM activity increased progressively and drought stress led to only small additional, reversible increases in dark CO₂ fixation. In contrast, CAM expression was overwhelmingly under environmental control in seedlings and mature plants of Clusia pratensis. C₃-type CO₂ exchange was maintained under well-watered conditions, but upon drought stress, CO₂ exchange shifted, in a fully reversible manner, to a CAM-type pattern. Clusia minor showed CO₂ exchange reponses intermediate to those of C. rosea and C. pratensis. Clusia cretosa operated in the C₃ mode at all times. Notably, reversible stress-induced increases of dark CO₂ fixation were also observed during the developmental progression to pronounced CAM in young Kalanchoë daigremontiana and Kalanchoë pinnata, two species considered constitutive CAM species. Drought-induced up-regulation of CAM was even detected in young cladodes of a cactus, Opuntia ficus-indica, an archetypal constitutive CAM species. Evidently, the defining characteristics of constitutive and facultative CAM are shared, to variable degrees, by all species.

Expression of gibberellin 20-oxidase1 (AtGA20ox1) in Arabidopsis seedlings with altered auxin status is regulated at multiple levels

Desgagne-Penix, I., Sponsel, V. M. — 2008-04-24

Bioactive gibberellins (GAs) affect many biological processes including germination, stem growth, transition to flowering, and fruit development. The location, timing, and level of bioactive GA are finely tuned to ensure that optimal growth and development occur. The balance between GA biosynthesis and deactivation is controlled by external factors such as light and by internal factors that include auxin. The role of auxin transport inhibitors (ATIs) and auxins on GA homeostasis in intact light-grown Arabidopsis thaliana (L.) Heynh. seedlings was investigated. Two ATIs, 1-N-naphthylthalamic acid (NPA) and 1-naphthoxyacetic acid (NOA) caused elevated expression of the GA biosynthetic enzyme AtGA20-oxidase1 (AtGA20ox1) in shoot but not in root tissues, and only at certain developmental stages. It was investigated whether enhanced AtGA20ox1 gene expression was a consequence of altered flow through the GA biosynthetic pathway, or was due to impaired GA signalling that can lead to enhanced AtGA20ox1 expression and accumulation of a DELLA protein, Repressor of ga1-3 (RGA). Both ATIs promoted accumulation of GFP-fused RGA in shoots and roots, and this increase was counteracted by the application of GA₄. These results suggest that in ATI-treated seedlings the impediment to DELLA protein degradation may be a deficiency of bioactive GA at sites of GA response. It is proposed that the four different levels of AtGA20ox1 regulation observed here are imposed in a strict hierarchy: spatial (organ-, tissue-, cell-specific) > developmental > metabolic > auxin regulation. Thus results show that, in intact auxin- and auxin transport inhibitor-treated light-grown Arabidopsis seedlings, three other levels of regulation supersede the effects of auxin on AtGA20ox1.

Low temperature effects on leaf physiology and survivorship in the C3 and C4 subspecies of Alloteropsis semialata

Osborne, C. P., Wythe, E. J., Ibrahim, D. G., Gilbert, M. E., Ripley, B. S. — 2008-04-24

The species richness of C₄ grasses is strongly correlated with temperature, with C₄ species dominating subtropical ecosystems and C₃ types predominating in cooler climates. Here, the effects of low temperatures on C₄ and C₃ grasses are compared, controlling for phylogenetic effects by using Alloteropsis semialata, a unique species with C₄ and C₃ subspecies. Controlled environment and common garden experiments tested the hypotheses that: (i) photosynthesis and growth are greater in the C₄ than the C₃ subspecies at high temperatures, but this advantage is reversed below 20 °C; and (ii) chilling-induced photoinhibition and light-mediated freezing injury of leaves occur at higher temperature thresholds in the C₄ than the C₃ plants. Measurements of leaf growth and photosynthesis showed the expected advantages of the C₄ pathway over the C₃ type at high temperatures. These declined with temperature, but were not completely lost until 15 °C, and there was no evidence of a reversal to give a C₃ advantage. Chronic chilling (5–15 °C) or acute freezing events induced a comparable degree of photodamage in illuminated leaves of both subspecies. Similarly, freezing caused high rates of mortality in the unhardened leaves of both subtypes. However, a 2-week chilling treatment prior to these freezing events halved injury in the C₃ but not the C₄ subspecies, suggesting that C₄ leaves lacked the capacity for cold acclimation. These results therefore suggest that C₃ members of this subtropical species may gain an advantage over their C₄ counterparts at low temperatures via protection from freezing injury rather than higher photosynthetic rates.

The peripheral xylem of grapevine (Vitis vinifera) berries. 2. Anatomy and development

Chatelet, D. S., Rost, T. L., Matthews, M. A., Shackel, K. A. — 2008-04-24

It has been hypothesized that the substantial reductions in xylemic water flow occurring at veraison are due to physical disruption (breaking) of the xylem as a result of renewed berry growth. In a companion paper, evidence was presented that the vast majority of xylem tracheary elements remained intact despite the growth of the berry, and it was proposed that existing tracheary elements stretch to accommodate growth and that additional elements may also differentiate after veraison. Measurements of the intergyre distance of tracheary elements in macerated tissue were used to test for stretching, and the numbers of tracheary elements per vascular bundle and of branch points of the peripheral xylem network were analysed to test for continued differentiation from 18 to 120 d after anthesis in Chardonnay berries. The distance between the epidermis and the vasculature increased substantially from pre- to post-veraison, potentially increasing the amount of skin available for analysis of compounds important for winemaking. Tracheary elements continued to differentiate within the existing vascular bundles throughout berry development. Additional vascular bundles also appeared until after veraison, thereby increasing the complexity of the peripheral vascular network. The results also confirmed that tracheary elements stretched by ~20%, but this was not as much as that predicted based on the growth of the vascular diameter (40%). These results complete a comprehensive evaluation of grape berry peripheral xylem during its development and show that tracheary development continues further into berry maturation than previously thought.

The peripheral xylem of grapevine (Vitis vinifera). 1. Structural integrity in post-veraison berries

Chatelet, D. S., Rost, T. L., Shackel, K. A., Matthews, M. A. — 2008-04-24

During the development of many fleshy fruits, water flow becomes progressively more phloemic and less xylemic. In grape (Vitis vinifera L.), the current hypothesis to explain this change is that the tracheary elements of the peripheral xylem break as a result of berry growth, rendering the xylem structurally discontinuous and hence non-functional. Recent work, however, has shown via apoplastic dye movement through the xylem of post-veraison berries that the xylem should remain structurally intact throughout berry development. To corroborate this, peripheral xylem structure in developing Chardonnay berries was investigated via maceration and plastic sectioning. Macerations revealed that, contrary to current belief, the xylem was comprised mostly of vessels with few tracheids. In cross-section, the tracheary elements of the vascular bundles formed almost parallel radial files, with later formed elements toward the epidermis and earlier formed elements toward the centre of the berry. Most tracheary elements remained intact throughout berry maturation, consistent with recent reports of vascular dye movement in post-veraison berries.

Occurrence and forms of Kranz anatomy in photosynthetic organs and characterization of NAD-ME subtype C4 photosynthesis in Blepharis ciliaris (L.) B. L. Burtt (Acanthaceae)

Akhani, H., Ghasemkhani, M., Chuong, S. D. X., Edwards, G. E. — 2008-04-24

Blepharis (Acanthaceae) is an Afroasiatic genus comprising 129 species which occur in arid and semi-arid habitats. This is the only genus in the family which is reported to have some C₄ species. Blepharis ciliaris (L.) B. L. Burtt. is a semi-desert species with distribution in Iran, Oman, and Pakistan. Its form of photosynthesis was investigated by studying different organs. C₄-type carbon isotope composition, the presence of atriplicoid type Kranz anatomy, and compartmentation of starch all indicate performance of C₄ photosynthesis in cotyledons, leaves, and the lamina part of bracts. A continuous layer of distinctive bundle sheath cells (Kranz cells) encircle the vascular bundles in cotyledons and the lateral vascular bundles in leaves. In older leaves, there is extensive development of ground tissue in the midrib and the Kranz tissue becomes interrupted on the abaxial side, and then becomes completely absent in the mature leaf base. Cotyledons have 5–6 layers, and leaves 2–3 layers, of spongy chlorenchyma beneath the veins near the adaxial side of the leaf, indicating bifacial organization of chlorenchyma. As the plant matures, bracts and spines develop and contribute to carbon assimilation through an unusual arrangement of Kranz anatomy which depends on morphology and exposure to light. Stems do not contribute to carbon assimilation, as they lack chlorenchyma tissue and Kranz anatomy. Analysis of C₄ acid decarboxylases by western blot indicates B. ciliaris is an NAD-malic enzyme type C₄ species, which is consistent with the Kranz cells having chloroplasts with well-developed grana and abundant mitochondria.

The influence of epidermal windows on the light environment within the leaves of six succulents

Egbert, K. J., Martin, C. E., Vogelmann, T. C. — 2008-04-23

An omni-directional fibre optic microprobe was used to measure the quantity and quality of light within the leaves of six succulents having epidermal windows, three species having a subterranean growth habit (Haworthia truncata, Lithops olivacea, and Opthalmophyllum longum) and three growing above ground (Peperomia dolabriformis, P. graveolens, and the sprawling vine Senecio rowleyanus). Although light levels at most locations inside the leaves of all species were high, near those incident on the window surfaces, light levels inside the leaves of the two species of Peperomia often greatly exceeded incident light levels, indicating considerable light scattering and focusing by the leaf tissue. The spectral quality of light inside the leaves of all taxa reflected the absorption properties of chlorophyll, with most of the photons in the green wavelengths. Light quality and quantity inside the leaves did not correlate with the growth habit of the plants, the size of the window (as a proportion of the total leaf area), or location inside the leaf, although light levels generally declined and wavelengths increased deeper in the leaves. Application of reflective tape to the windows reduced internal light levels in L. olivacea and S. rowleyanus, although reductions were not always statistically significant. Although light levels throughout the leaves of P. graveolens were substantially and significantly reduced as a result of the application of reflective tape to its windows, the light levels even at the basal chlorenchyma on the abaxial side of the leaf remained high. In all species investigated, the levels of near-infrared radiation inside the leaves were surprisingly high, yet also declined deeper inside the succulent leaves. This near-infrared radiation may add to the heat load of these plants. Furthermore, application of reflective tape to the windows also reduced the amount of near-infrared radiation inside the leaves of the three succulents examined. These results led to a novel, testable hypothesis that may help to explain previous findings that application of reflective tape to the windows of the leaves of these succulents did not effect a reduction in photosynthetic activity.

Is internucleosomal DNA fragmentation an indicator of programmed death in plant cells?

Kuthanova, A., Opatrny, Z., Fischer, L. — 2008-04-23

Specific DNA fragmentation into oligonucleosomal units occurs during programmed cell death (PCD) in both animal and plant cells, usually being regarded as an indicator of its apoptotic character. This internucleosomal DNA fragmentation is demonstrated in tobacco suspension and leaf cells, which were killed immediately by freezing in liquid nitrogen, and homogenization or treatment with Triton X-100. Although these cells could not activate and realize the respective enzymatic processes in a programmed manner, the character of DNA fragmentation was similar to that in the cells undergoing typical gradual PCD induced by 50 µM CdSO₄. This internucleosomal DNA fragmentation was connected with the action of cysteine proteases and the loss of membrane, in particular tonoplast, integrity. The mechanisms of DNase activation in the rapidly killed cells, hypothetical biological relevance, and implications for the classification of cell death are discussed.

Rubisco regulation: a role for inhibitors

2008-04-23

In photosynthesis Rubisco catalyses the assimilation of CO₂ by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.

Rubisco, Rubisco activase, and global climate change

Sage, R. F., Way, D. A., Kubien, D. S. — 2008-04-23

Global warming and the rise in atmospheric CO₂ will increase the operating temperature of leaves in coming decades, often well above the thermal optimum for photosynthesis. Presently, there is controversy over the limiting processes controlling photosynthesis at elevated temperature. Leading models propose that the reduction in photosynthesis at elevated temperature is a function of either declining capacity of electron transport to regenerate RuBP, or reductions in the capacity of Rubisco activase to maintain Rubisco in an active configuration. Identifying which of these processes is the principal limitation at elevated temperature is complicated because each may be regulated in response to a limitation in the other. Biochemical and gas exchange assessments can disentangle these photosynthetic limitations; however, comprehensive assessments are often difficult and, for many species, virtually impossible. It is proposed that measurement of the initial slope of the CO₂ response of photosynthesis (the A/C_i response) can be a useful means to screen for Rubisco activase limitations. This is because a reduction in the Rubisco activation state should be most apparent at low CO₂ when Rubisco capacity is generally limiting. In sweet potato, spinach, and tobacco, the initial slope of the A/C_i response shows no evidence of activase limitations at high temperature, as the slope can be accurately modelled using the kinetic parameters of fully activated Rubisco. In black spruce (Picea mariana), a reduction in the initial slope above 30 °C cannot be explained by the known kinetics of fully activated Rubisco, indicating that activase may be limiting at high temperatures. Because black spruce is the dominant species in the boreal forest of North America, Rubisco activase may be an unusually important factor determining the response of the boreal biome to climate change.

Curiosity and context revisited: crassulacean acid metabolism in the Anthropocene

Osmond, B., Neales, T., Stange, G. — 2008-04-23

Having gained some understanding of the consequences of the CO₂-concentrating mechanisms in crassulacean acid metabolism (CAM) that internalize the photosynthetic environment of the Cretaceous on a daily basis, it may be time to consider potential long-term effects of the planetary CO₂-concentrating mechanism on growth and ecology of these plants in the Anthropocene. This paper emphasizes our limited understanding of the carbohydrate economy of CAM in relation to growth processes and briefly reviews recent studies of the diel cycles of growth in these plants. An inadvertent long-term, regional-scale experiment from the past is revisited in which an Opuntia monoculture grew to occupy >25 million hectares of farmland in central eastern Australia, producing a total biomass of about 1.5 billion tonnes in about 80 years. Although at the time it does not seem to have been recognized that this invasion involved CAM, a botanist from the University of Melbourne, Jean White-Haney emerges as a heroic pioneer in the control of the invader by poison and pioneered its biological control. The Opuntia population was expanding at 10–100 ha h^–1 when it was brought to a halt within a decade by the voracious appetite of Cactoblastis cactorum larvae. It is now known that the female parent moth of this predator detects CAM in O. stricta prior to oviposition by deploying the most sensitive CO₂ detector system yet found in the Lepidoptera. The O. stricta invasion is a dramatic demonstration of the capacity of CAM plants to attain and sustain high biomass; to sequester and retain atmospheric CO₂. In conclusion, experiments are reviewed that show stimulation of CO₂ assimilation, growth, and biomass of CAM plants by elevated atmospheric [CO₂], and the proposition that these plants may have a role in atmospheric CO₂ sequestration is re-examined. This role may be compromised by predators such as Cactoblastis. However the moth CO₂ sensors are adapted to pre-industrial atmospheric [CO₂] and FACE (free-air CO₂ enrichment) experiments show this exquisite system of biological control is also compromised by rising global [CO₂] in the Anthropocene.

Clusia: Holy Grail and enigma

Luttge, U. — 2008-04-23

Clusia is the only genus with bona fide dicotyledonous trees performing Crassulacean acid metabolism (CAM). Clusia minor L. is extraordinarily flexible, being C₃/CAM intermediate and expressing the photosynthetic modes C₃, CAM, CAM-cycling, and CAM-idling. C₃ photosynthesis and CAM can be observed simultaneously in two opposite leaves on a node and possibly even within the same leaf in the interveinal lamina and the major vein tissue, respectively. The relative activity of photosystem II (PSII) indicating photosynthetic energy use, is larger under photorespiratory than under non-photorespiratory conditions due to the particular energy demand of photorespiration. The heterogeneity of PSII over the leaves as visualized by chlorophyll fluorescence imaging in the C₃ mode is larger under non-photorespiratory conditions than under photorespiratory conditions. These observations indicate that photorespiration, presumably by its particular energy demand, synchronizes photosynthetic activity over the leaves. In the CAM mode, the heterogeneity of PSII is more dependent on the transitions between CAM phases. Free-running circadian oscillations of photosynthesis are strongly dampened in both the C₃ and the CAM mode. Photorespiration is under circadian clock control in both the C₃ and the CAM mode. PSII and the heterogeneity of PSII oscillate in phase with CO₂ uptake and photorespiration only under non-photorespiratory conditions. Under photorespiratory conditions, PSII does not oscillate and there is no heterogeneity, again indicating the stabilizing function of photorespiration. Plants acclimatized to perform CAM switch to C₃ photosynthesis during free-running oscillations while subjected to constant illumination.

RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO

2008-04-16

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme in the fixation of CO₂ in the Calvin cycle of plants. Many genome projects have revealed that bacteria, including Bacillus subtilis, possess genes for proteins that are similar to the large subunit of RuBisCO. These RuBisCO homologues are called RuBisCO-like proteins (RLPs) because they are not able to catalyse the carboxylase or the oxygenase reactions that are catalysed by photosynthetic RuBisCO. It has been demonstrated that B. subtilis RLP catalyses the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. The structure of DK-MTP-1-P is very similar to that of ribulose-1,5-bisphosphate (RuBP) and the enolase reaction is a part of the reaction catalysed by photosynthetic RuBisCO. In this review, functional and evolutionary relationships between B. subtilis RLP of the methionine salvage pathway, other RLPs, and photosynthetic RuBisCO are discussed. In addition, the fundamental question, ‘How has RuBisCO evolved?’ is also considered, and evidence is presented that RuBisCOs evolved from RLPs.

Root hydraulic conductivity measured by pressure clamp is substantially affected by internal unstirred layers

Knipfer, T., Steudle, E. — 2008-04-16

Using the root pressure probe in the pressure clamping (PC) mode, the impact of internal unstirred layers (USLs) was quantified for young corn roots, both in experiments and in computer simulations applying the convection/diffusion model of Knipfer et al. In the experiments, water flows (J_Vrs) during PC were analysed in great detail, showing that J_Vrs (and the apparent root hydraulic conductivity) were high during early stages of PC and declined rapidly during the first 80 s of clamping to a steady-state value of 40–30% of the original. The comparison of experimental results with simulations showed that, during PC, internal USLs at the inner surface of the endodermis substantially modify the overall force driving the water. As a consequence, J_Vr and Lp_r were inhibited. Effects of internal USLs were minimized when using the pressure relaxation mode, when internal USLs had not yet developed. Additional stop-clamp experiments and experiments where the endodermis was punctured to reduce the effect of internal USLs verified the existence of internal USLs during PC. Data indicated that the role of pressure propagation along the root xylem for both PC and pressure relaxation modes should be small, as should the effects of filling of the capacities during root pressure probe experiments, which are discussed as an alternative model. The results supported the idea that concentration polarization effects at the endodermis (internal USLs) cause a serious problem whenever relatively large amounts of water (xylem sap) are radially moved across the root, such as during PC or when using the high-pressure flow meter technique.

Catalysis and regulation in Rubisco

Andersson, I. — 2008-04-15

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the incorporation of inorganic CO₂ into the organic molecules of life. Rubisco is extremely inefficient as a catalyst and its carboxylase activity is compromised by numerous side-reactions including oxygenation of its sugar phosphate substrate by atmospheric O₂. The reduction in the catalytic efficiency as a result of these processes has implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Several aspects of Rubisco including its complex biosynthesis and multi-step catalytic reaction are subject to tight control involving light, cellular metabolites, and molecular chaperones. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. These provide a molecular framework for the understanding of these processes at the molecular level.

Leaf succulence determines the interplay between carboxylase systems and light use during Crassulacean acid metabolism in Kalanchoe species

Griffiths, H., Robe, W. E., Girnus, J., Maxwell, K. — 2008-04-11

The photosynthetic physiology of Crassulacean acid metabolism was investigated in two Kalanchoë species with differing leaf succulence. The magnitude of CAM was higher for the more succulent leaves of K. daigremontiana, compared to the less succulent leaves of K. pinnata. High succulence was related to low mesophyll conductance: K. pinnata was able to maximize diurnal carbon gain by the C₃ pathway, whereas increased succulence is associated with a higher commitment to the CAM cycle in K. daigremontiana. The Rubisco specificity factor, , determining selectivity for CO₂ over O₂, was similar for both species (~88), and lower than that of Spinacea (~95), but in contrast to C₄ plants, the Rubisco K_mCO2 (determined independently) was also lower in Kalanchoë spp. than in spinach. Differences in light use were related to the nature of the sink strength in each Phase of CAM, with PEPC activity resulting in low electron transport rates. Decarboxylation was marked by high, non-saturated rates of electron transport, with Rubisco activity and activation state increasing in both species during the course of the light period. The degree of succulence, and extent of CAM activity, was associated with a progressive inhibition of PSII photochemistry and potential Rubisco activity during the night in both species. Rubisco could be ‘woken up’ more rapidly in K. pinnata, whereas 45 min light acclimation was required for full recovery of electron transport and Rubisco activity in K. daigremontiana. Leaf morphology therefore seems to alter the expression of and dependence on CAM, but also the extent of co-regulation of carboxylase networks and light use capacity.

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple)

2008-04-11

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO₂ fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day–night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity

Endo, T., Mihara, Y., Furumoto, T., Matsumura, H., Kai, Y., Izui, K. — 2008-04-11

Introducing a C₄-like pathway into C₃ plants is one of the proposed strategies for the enhancement of photosynthetic productivity. For this purpose it is necessary to provide each component enzyme that exerts strong activity in the targeted C₃ plants. Here, a maize C₄-form phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was engineered for its regulatory and catalytic properties so as to be functional in the cells of C₃ plants. Firstly, amino acid residues Lys-835 and Arg-894 of maize PEPC, which correspond to Lys-773 and Arg-832 of Escherichia coli PEPC, respectively, were replaced by Gly, since they had been shown to be involved in the binding of allosteric inhibitors, malate or aspartate, by our X-ray crystallographic analysis of E. coli PEPC. The resulting mutant enzymes were active but their sensitivities to the inhibitors were greatly diminished. Secondly, a Ser residue (S780) characteristically conserved in all C₄-form PEPC was replaced by Ala conserved in C₃- and root-form PEPCs to decrease the half-maximal concentration (S_0.5) of PEP. The double mutant enzyme (S780A/K835G) showed diminished sensitivity to malate and decreased S_0.5(PEP) with equal maximal catalytic activity (V_m) to the wild-type PEPC, which will be quite useful as a component of the C₄-like pathway to be introduced into C₃ plants.

Malate metabolism in Hoya carnosa mitochondria and its role in photosynthesis during CAM phase III

Hong, H. T. K., Nose, A., Agarie, S., Yoshida, T. — 2008-04-09

This study investigated the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant, Hoya carnosa, in malate metabolism during CAM phase III. The mitochondria showed high malate dehydrogenase (mMDH) and aspartate amino transferase (mAST), and a significant amount of malic enzyme (mME) activities. H. carnosa readily oxidized malate via mME and mMDH in the presence of some cofactors such as thiamine pyrophosphate (TPP), coenzyme A (CoA) or NAD⁺. A high respiration rate of malate oxidation was observed at pH 7.2 with NAD⁺ and glutamate (Glu). Providing AST and Glu simultaneously into the respiratory medium strongly increased the rates of malate oxidation, and this oxidation was gradually inhibited by an inhibitor of -ketoglutarate (-KG) carrier, pyridoxal-5'-phosphate (PLP). The mitochondria readily oxidized aspartate (Asp) or -KG individually with low rates, while they oxidized Asp and -KG simultaneously with high rates, and this simultaneous oxidation was also inhibited by PLP. By measuring the capacity of the mitochondrial shuttle, it was found that the OAA produced via mMDH seemed not to be transported outside the mitochondria, but mAST interconverted OAA and Glu to Asp and -KG, respectively, and exported them out via a malate-aspartate (malate-Asp) shuttle. The data in this research suggest that during phase III of PCK-CAM, H. carnosa mitochondria oxidized malate via both mME and the mMDH systems depending on metabolic requirements. However, malate metabolism by the mMDH system did not operate via a malate-OAA shuttle similarly to Ananas comosus mitochondria, but it operated via a malate-Asp shuttle similarly to Kalanchoë daigremontiana mitochondria.

Structural, biochemical, and physiological characterization of photosynthesis in two C4 subspecies of Tecticornia indica and the C3 species Tecticornia pergranulata (Chenopodiaceae)

2008-04-04

Among dicotyledon families, Chenopodiaceae has the most C₄ species and the greatest diversity in structural forms of C₄. In subfamily Salicornioideae, C₄ photosynthesis has, so far, only been found in the genus Halosarcia which is now included in the broadly circumscribed Tecticornia. Comparative anatomical, cytochemical, and physiological studies on these taxa, which have near-aphyllous photosynthetic shoots, show that T. pergranulata is C₃, and that two subspecies of T. indica (bidens and indica) are C₄ (Kranz-tecticornoid type). In T. pergranulata, the stems have two layers of chlorenchyma cells surrounding the centrally located water storage tissue. The two subspecies of T. indica have Kranz anatomy in reduced leaves and in the fleshy stem cortex. They are NAD-malic enzyme-type C₄ species, with mesophyll chloroplasts having reduced grana, characteristic of this subtype. The Kranz-tecticornoid-type anatomy is unique among C₄ types in the family in having groups of chlorenchymatous cells separated by a network of large colourless cells (which may provide mechanical support or optimize the distribution of radiation in the tissue), and in having peripheral vascular bundles with the phloem side facing the bundle sheath cells. Also, the bundle sheath cells have chloroplasts in a centrifugal position, which is atypical for C₄ dicots. Fluorescence analyses in fresh sections indicate that all non-lignified cell walls have ferulic acid, a cell wall cross-linker. Structural–functional relationships of C₄ photosynthesis in T. indica are discussed. Recent molecular studies show that the C₄ taxa in Tecticornia form a monophyletic group, with incorporation of the Australian endemic genera of Salicornioideae, including Halosarcia, Pachycornia, Sclerostegia, and Tegicornia, into Tecticornia.

C4 photosynthetic isotope exchange in NAD-ME- and NADP-ME-type grasses

Cousins, A. B., Badger, M. R., von Caemmerer, S. — 2008-03-28

Monitoring photosynthetic isotope exchange is an important tool for predicting the influence of plant communities on the global carbon cycle in response to climate change. C₄ grasses play an important role in the global carbon cycle, but their contribution to the isotopic composition of atmospheric CO₂ is not well understood. Instantaneous measurements of ¹³CO₂ (¹³C) and C¹⁸OO (¹⁸O) isotope exchange in five NAD-ME and seven NADP-ME C₄ grasses have been conducted to investigate the difference in photosynthetic CO₂ isotopic fractionation in these subgroups. As previously reported, the isotope composition of the leaf material (¹³C) was depleted in ¹³C in the NAD-ME compared with the NADP-ME grasses. However, ¹³C was not different between subtypes at high light, and, although ¹³C increased at low light, it did so similarly in both subtypes. This suggests that differences in leaf ¹³C between the C₄ subtypes are not caused by photosynthetic isotope fractionation and leaf ¹³C is not a good indicator of bundle sheath leakiness. Additionally, low carbonic anhydrase (CA) in C₄ grasses may influences ¹³C and should be considered when estimating the contribution of C₄ grasses to the global isotopic signature of atmospheric CO₂. It was found that measured ¹⁸O values were lower than those predicted from leaf CA activities and ¹⁸O was similar in all species measured. The ¹⁸O in these C₄ grasses is similar to low ¹⁸O previously measured in C₄ dicots which contain 2.5 times the leaf CA activity, suggesting that leaf CA activity is not a predictor of ¹⁸O in C₄ plants.

The effects of Rubisco activase on C4 photosynthesis and metabolism at high temperature

2008-03-28

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T₁ progeny of transgenic Flaveria bidentis (a C₄ dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C₄ photosynthesis at high temperature. A range of T₁ progeny was screened at 25 °C and 40 °C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6±0.8 µmol m^–2 (447±36 mg m^–2) compared to a Rubisco site content of 14.2±0.8 µmol m^–2. CO₂ assimilation rates and Rubisco carbamylation declined at both 25 °C and 40 °C when the Rubisco activase content dropped below 3 µmol m^–2 (125 mg m^–2), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44±5% at 40 °C compared to 81±2% at 25 °C. When the CO₂ assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C₃ and C₄ metabolites pools. It is concluded that during short-term treatment at 40 °C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C₄ photosynthesis.

Quantum yield variation across the three pathways of photosynthesis: not yet out of the dark

Skillman, J. B. — 2008-03-20

The convergent quantum yield hypothesis (CQY) assumes that thermodynamics and natural selection jointly limit variation in the maximum energetic efficiency of photosynthesis in low light under otherwise specified conditions (e.g. temperature and CO₂ concentration). A literature survey of photosynthetic quantum yield () studies in terrestrial plants from C₃, C₄, and CAM photosynthetic types was conducted to test the CQY hypothesis. Broad variation in values from C₃ plants could partially be explained by accounting for whether the measuring conditions were permissive or restrictive for photorespiration. Assimilatory quotients (AQ), calculated from the CO₂ :O₂ ratios, indicated that 49% and 29% of absorbed light energy was allocated to carbon fixation and photorespiration in C₃ plants, respectively. The unexplained remainder (22%) may represent diversion to various other energy-demanding processes (e.g. starch synthesis, nitrogen assimilation). Individual and cumulative effects of these other processes on photosynthetic efficiency are poorly quantified. In C₄ plants, little variation in values was observed, consistent with the fact that C₄ plants exhibit little photorespiration. As before, AQ values indicate that 22% of absorbed light energy cannot be accounted for by carbon fixation in C₄ plants. Among all three photosynthetic types, the of photosynthesis in CAM plants is the least studied, appears to be highly variable, and may present the greatest challenge to the CQY hypothesis. The high amount of energy diverted to processes other than carbon fixation in C₃ and C₄ plants and the poor characterization of photosynthetic efficiency in CAM plants are significant deficiencies in our otherwise robust understanding of the energetics of terrestrial photoautotrophy.

Co- and post-translational modifications in Rubisco: unanswered questions

Houtz, R. L., Magnani, R., Nayak, N. R., Dirk, L. M. A. — 2008-03-18

Both the large (LS) and small (SS) subunits of Rubisco are subject to a plethora of co- and post-translational modifications. With the exceptions of LS carbamylation and SS transit sequence processing, the remaining modifications, including deformylation, acetylation, methylation, and N-terminal proteolytic processing of the LS, are still biochemically and/or functionally undefined although they are found in nearly all forms of Rubisco from vascular plants. A collection of relatively unique enzymes catalyse these modifications, and several have been characterized in other organisms. Some of the observed modifications in the LS and SS clearly suggest novel changes in enzyme specificity and/or activity, and others have common features with other co- and post-translationally modifying enzymes. With the possible exception of Lys14 methylation in the LS, processing of both the LS and SS of Rubisco is by default an ordered process sequentially leading up to the final forms observed in the holoenzyme. An overview of the nature of structural modifications in the LS and SS of Rubisco is presented, and, where possible, the nature of the enzymes catalysing these modifications (either through similarity with other known enzymes or through direct enzymological characterization) is described. Overall, there are a distinct lack of functional and mechanistic observations for modifications in Rubisco and thus represent many potentially productive avenues for research.

Association of Rubisco activase with chaperonin-60{beta}: a possible mechanism for protecting photosynthesis during heat stress

Salvucci, M. E. — 2008-03-18

Previous studies have shown that inhibition of photosynthesis by moderate heat stress is a consequence of Rubisco deactivation, caused in part by the thermal instability of Rubisco activase. This involvement of Rubisco activase was confirmed in heat stress and recovery experiments using transgenic Arabidopsis plants. Compared with wild-type plants, photosynthesis, the effective quantum yield of photosystem II, and Rubisco activation were less thermotolerant and recovered more slowly in transgenic Arabidopsis plants with reduced levels of Rubisco activase. Immunoblots showed that 65% of the Rubisco activase was recovered in the insoluble fraction after heat stress in leaf extracts of transgenic but not wild-type plants, evidence that deactivation of Rubisco was a consequence of thermal denaturation of Rubisco activase. The transgenic Arabidopsis plants used in this study contained a modified form of Rubisco activase that facilitated affinity purification of Rubisco activase and proteins that potentially interact with Rubisco activase during heat stress. Sequence analysis and immunoblotting identified the β-subunit of chaperonin-60 (cpn60β), the chloroplast GroEL homologue, as a protein that was bound to Rubisco activase from leaf extracts prepared from heat-stressed, but not control plants. Analysis of the proteins by non-denaturing gel electrophoresis showed that cpn60β was associated with Rubisco activase in a high molecular mass complex. Immunoblot analysis established that the apparent association of cpn60β with Rubisco activase was dynamic, increasing with the duration and intensity of the heat stress and decreasing following recovery. Taken together, these data suggest that cpn60β plays a role in acclimating photosynthesis to heat stress, possibly by protecting Rubisco activase from thermal denaturation.

Roles of the bundle sheath cells in leaves of C3 plants

Leegood, R. C. — 2008-03-18

This review considers aspects of the structure and functions of the parenchymatous bundle sheath that surrounds the veins in the leaves of many C₃ plants. It includes a discussion of bundle sheath structure and its related structures (bundle sheath extensions and the paraveinal mesophyll), its relationship to the mestome sheath in some grasses, and its chloroplast content. Its metabolic roles in photosynthesis, carbohydrate synthesis and storage, the import and export of nitrogen and sulphur, and the metabolism of reactive oxygen species are discussed and are compared with the role of the bundle sheath in leaves of C₄ plants. Its role as an interface between the vasculature and the mesophyll is considered in relation to the movement of water and assimilates during leaf development, export of photosynthates, and senescence.

Rubisco gene expression in C4 plants

Patel, M., Berry, J. O. — 2008-03-05

In leaves of most C₄ plants, ribulose 1,5 bisphosphate carboxylase (Rubisco) accumulates only in bundle sheath (bs) cells that surround the vascular centres, and not in mesophyll (mp) cells. It has been shown previously that in the C₄ dicots amaranth and Flaveria bidentis, post-transcriptional control of mRNA translation and stability mediate the C₄ expression patterns of genes encoding the large and small Rubisco subunits (chloroplast rbcL and nuclear RbcS, respectively). Translational control appears to regulate bs cell-specific Rubisco gene expression during early dicot leaf development, while control of mRNA stability appears to mediate bs-specific accumulation of RbcS and rbcL transcripts in mature leaves. Post-transcriptional control is also involved in the regulation of Rubisco gene expression by light, and in response to photosynthetic activity. Transgenic and transient expression studies in F. bidentis provide direct evidence for post-transcriptional control of bs cell-specific RbcS expression, which is mediated by the 5' and 3' untranslated regions (UTRs) of the mRNA. Comparisons of Rubisco gene expression in these dicots and in the monocot maize indicates possible commonalities in the regulation of RbcS and rbcL genes in these divergent C₄ species. Now that the role of post-transcriptional regulation in C₄ gene expression has been established, it is likely that future studies of mRNA–protein interactions will address long-standing questions about the establishment and maintenance of cell type-specificity in these plants. Some of these regulatory mechanisms may have ancestral origins in C₃ species, through modification of pre-existing factors, or by the acquisition of novel C₄ processes.

Large-scale mRNA expression profiling in the common ice plant, Mesembryanthemum crystallinum, performing C3 photosynthesis and Crassulacean acid metabolism (CAM)

Cushman, J. C., Tillett, R. L., Wood, J. A., Branco, J. M., Schlauch, K. A. — 2008-03-03

The common ice plant (Mesembryanthemum crystallinum L.) has emerged as a useful model for molecular genetic studies of Crassulacean acid metabolism (CAM) because CAM can be induced in this species by water deficit or salinity stress. Non-redundant sequence information from expressed sequence tag data was used to fabricate a custom oligonucleotide microarray to compare large-scale mRNA expression patterns in M. crystallinum plants conducting C₃ photosynthesis versus CAM. Samples were collected every 4 h over a 24 h time period at the start of the subjective second day from plants grown under constant light and temperature conditions in order to capture variation in mRNA expression due to salinity stress and circadian clock control. Of 8455 genes, a total of 2343 genes (~28%) showed a significant change as judged by analysis of variance (ANOVA) in steady-state mRNA abundance at one or more time points over the 24 h period. Of these, 858 (10%) and 599 (7%) exhibited a greater than two-fold ratio (TFR) increase or decrease in mRNA abundance, respectively. Functional categorization of these TFR genes revealed that many genes encoding products that function in CAM-related C₄ acid carboxylation/decarboxylation, glycolysis/gluconeogenesis, polysaccharide, polyol, and starch biosynthesis/degradation, protein degradation, transcriptional activation, signalling, stress response, and transport facilitation, and novel, unclassified proteins exhibited stress-induced increases in mRNA abundance. In contrast, salt stress resulted in a significant decrease in transcript abundance for genes encoding photosynthetic functions, protein synthesis, and cellular biogenesis functions. Many genes with CAM-related functions exhibited phase shifts in their putative circadian expression patterns following CAM induction. This report establishes an extensive catalogue of gene expression patterns for future investigations aimed at understanding the complex, transcriptional hierarchies that govern CAM-specific expression patterns. A novel graph-theoretic approach called ‘Max Clique Builder’ is introduced that identifies and organizes sets of coordinately regulated genes, such as those encoding subunits of the vacuolar H⁺-ATPase complex, into tighter functionally related clusters with more similar expression patterns compared with standard hierarchical clustering methods.

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice

2008-03-02

Four enzymes, namely, the maize C₄-specific phosphoenolpyruvate carboxylase (PEPC), the maize C₄-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C₃-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO₂ assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO₂ assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO₂ assimilation, however, was more marked at higher CO₂ concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO₂ inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C₄-like photosynthetic pathway into C₃ plants, are discussed.

My favourite flowering image

Endress, P. K. — 2008-02-27

The heuristic value of drawings in research is emphasized, based on a drawing of flowers of Eupomatia bennettii.

Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships

Tabita, F. R., Satagopan, S., Hanson, T. E., Kreel, N. E., Scott, S. S. — 2008-02-16

There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.

Consequences of C4 photosynthesis for the partitioning of growth: a test using C3 and C4 subspecies of Alloteropsis semialata under nitrogen-limitation

Ripley, B. S., Abraham, T. I., Osborne, C. P. — 2008-02-07

C₄ plants dominate the world's subtropical grasslands, but investigations of their ecology typically focus on climatic variation, ignoring correlated changes in soil nutrient concentration. The hypothesis that higher photosynthetic nitrogen use efficiency (PNUE) in C₄ than in C₃ species allows greater flexibility in the partitioning of growth, especially under nutrient-deficient conditions, is tested here. Our experiment applied three levels of N supply to the subtropical grass Alloteropsis semialata, a unique model system with C₃ and C₄ subspecies. Photosynthesis was significantly higher for the same investment of leaf N in the C₄ than C₃ subspecies, and was unaffected by N treatments. The C₄ plants produced more biomass than the C₃ plants at high N levels, diverting a greater fraction of growth into inflorescences and corms, but less into roots and leaves. However, N-limitation of biomass production caused size-dependent shifts in the partitioning of growth. Root production was higher in small than large plants, and associated with decreasing leaf biomass in the C₃, and inflorescence production in the C₄ plants. Higher PNUE in the C₄ than C₃ subspecies was therefore linked with greater investment in sexual reproduction and storage, and the avoidance of N-limitations on leaf growth, suggesting advantages of the C₄ pathway in disturbed and infertile ecosystems.

The enigmatic contribution of mitochondrial function in photosynthesis

Nunes-Nesi, A., Sulpice, R., Gibon, Y., Fernie, A. R. — 2008-02-05

Considerable cumulative evidence has accrued suggesting a vital role for mitochondrial function in optimizing photosynthesis. Both pharmacological approaches using respiratory inhibitors and reverse genetic approaches have recently underscored the high degree of interconnection between photosynthesis and respiration—the major pathways of energy production which are largely confined to the plastid and mitochondria, respectively. Here recent studies into the nature of these interactions are reviewed, with particular focus on (i) the recently described link between the mitochondrial electron transport chain activity, ascorbate biosynthesis, and photosynthesis; and (ii) the contribution of mitochondrial metabolism to the photorespiratory process. Whilst there is increasing evidence of a role for ascorbate in co-ordinating the rates of respiration and photosynthesis, some data are presented here for plants grown under extreme environmental conditions that suggest that this relationship is not absolute. It thus seems likely that interactions between these compartments are perhaps more numerous and complicated than previously thought. This observation suggests that although the elucidation of the genetic bases of both photorespiration and the Wheeler–Smirnoff pathway of ascorbate biosynthesis has recently been completed, much further research is probably necessary in order to understand fully how energy metabolism is co-ordinated in the illuminated leaf.