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1.  Brassinosteroids are involved in response of cucumber (Cucumis sativus) to iron deficiency 
Annals of Botany  2012;110(3):681-688.
Background and Aims
Brassinosteroids (BR) are a class of plant polyhydroxysteroids with diverse functions in plant growth and development. However, there is little information on the role of BRs played in the response to nutrient deficiency.
To evaluate the role of BR in the response of plants to iron (Fe) deficiency, the effect of 24-epibrassinolide (EBR) on ferric reductase (FRO) activity, acidification of the rhizosphere and Fe content in cucumber (Cucumis sativus) seedlings under Fe-deficient (1 µm FeEDTA) and Fe-sufficient (50 µm FeEDTA) conditions were investigated.
Key Results
There was a significant increase in FRO activity upon exposure of cucumber seedlings to an Fe-deficient medium, and the Fe deficiency-induced increase in FRO activity was substantially suppressed by EBR. In contrast, application of EBR to Fe-sufficient seedlings stimulated FRO activity. Ethylene production evoked by Fe deficiency was suppressed by EBR, while EBR induced ethylene production from Fe-sufficient seedlings. Fe contents in shoots were reduced by treatment with EBR, while Fe contents in roots were markedly increased under both Fe-deficient and Fe-sufficient conditions. The reductions in Fe contents of shoots were independent of chlorophyll (CHL) contents under Fe-sufficient conditions, but they were positively correlated with CHL contents under Fe-deficient conditions. At the transcriptional level, transcripts encoding FRO (CsFRO1) and Fe transporter (CsIRT1) were increased upon exposure to the Fe-deficient medium, and the increases in transcripts were reversed by EBR.
The results demonstrate that BRs are likely to play a negative role in regulating Fe-deficiency-induced FRO, expressions of CsFRO1 and CsIRT1, as well as Fe translocation from roots to shoots.
PMCID: PMC3400454  PMID: 22684685
Brassinosteroids; iron deficiency; cucumber; Cucumis sativus; ferric reductase activity; Fe translocation
2.  Role of brassinosteroids in alleviation of phenanthrene–cadmium co-contamination-induced photosynthetic inhibition and oxidative stress in tomato 
Journal of Experimental Botany  2012;64(1):199-213.
Heavy metal pollution often occurs together with organic contaminants. Brassinosteroids (BRs) induce plant tolerance to several abiotic stresses, including phenanthrene (PHE) and cadmium (Cd) stress. However, the role of BRs in PHE+Cd co-contamination-induced stress amelioration is unknown. Here, the interactive effects of PHE, Cd, and 24-epibrassinolide (EBR; a biologically active BR) were investigated in tomato plants. The application of Cd (100 µM) alone was more phytotoxic than PHE applied alone (100 µM); however, their combined application resulted in slightly improved photosynthetic activity and pigment content compared with Cd alone after a 40 d exposure. Accumulation of reactive oxygen species and membrane lipid peroxidation were induced by PHE and/or Cd; however, the differences in effect were insignificant between Cd and PHE+Cd. The foliar application of EBR (0.1 µM) to PHE- and/or Cd-stressed plants alleviated photosynthetic inhibition and oxidative stress by causing enhancement of the activity of the enzymes and related transcript levels of the antioxidant system, secondary metabolism, and the xenobiotic detoxification system. Additionally, PHE and/or Cd residues were significantly decreased in both the leaves and roots after application of EBR, more specifically in PHE+Cd-stressed plants when treated with EBR, indicating a possible improvement in detoxification of these pollutants. The findings thus suggest a potential interaction of EBR and PHE for Cd stress alleviation. These results advocate a positive role for EBR in reducing pollutant residues for food safety and also strengthening phytoremediation.
PMCID: PMC3528031  PMID: 23201830
Brassinosteroids; food safety; heavy metal; photosynthesis; phytoremediation; polycyclic aromatic hydrocarbons (PAHs)
3.  Physiological and biochemical effect of 24-epibrassinoslide on cold tolerance in maize seedlings 
Germination and early seedling growth are important for establishment of maize because maize is chilling sensitive crop and low temperature during early period of growth can be detrimental to subsequent crop growth and productivity. Therefore, it is important to protect maize seedling from cold stress. A study was conducted on induced cold tolerance by 24-epibrassinoslide (EBR) at the Indian Agricultural Research Institute, New Delhi, India. Maize seedlings were raised in green house condition (25/18 °C day-night temperatures). Ten days old seedlings were treated with EBR (0.0, 0.01, 0.1, 1.0 and 10 μM) and then divided into two sets, one set was kept in greenhouse (25/18 °C day-night temperatures) and another was transferred to net house (cold stress). Data on various morpho-physiological traits was recorded after 7, 14 and 21 days of treatment. Exogenous application of 1.0 μM EBR had significant effect on growth and morpho-physiological traits under both conditions. The maize seedlings treated with EBR were more tolerant to cold stress than the untreated one. Significant increase in plant height, dry matter accumulation, chlorophyll content, total soluble proteins and starch contents was observed under both conditions, however, the results were more pronounced under cold stress. 1.0 μ M concentration being the most effective under both conditions. Maintenance of high tissue water content, reduced membrane injury index, increased total chlorophyll, soluble sugar and protein content were taken as the possible indicators of EBR induced chilling tolerance.
PMCID: PMC3550514  PMID: 23814437
24-epibrassinoslide; Cold tolerance; Maize; Glycine betaine; Total chlorophyll; Membrane injury index
4.  A role of brassinosteroids in early fruit development in cucumber 
Journal of Experimental Botany  2008;59(9):2299-2308.
Brassinosteroids (BRs) are essential for many biological processes in plants, however, little is known about their roles in early fruit development. To address this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes were examined in two cucumber cultivars that differ in parthenocarpic capacity. The application of EBR induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set and, subsequently, fruit growth in Jinchun No. 2, a cultivar with natural parthenocarpic capacity, and this inhibitory effect could be rescued by the application of EBR. RT-PCR analysis showed both pollination and EBR induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. cDNA sequences for CsCycD3;1 and CsCycD3;2 were isolated through PCR amplification. Both CsCycD3;1 and CsCycD3;2 transcripts were up-regulated by EBR treatment and pollination but strongly repressed by Brz treatment. Meanwhile, BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.
PMCID: PMC2423651  PMID: 18515830
Brassinosteroids; cell division; Cucumis sativus; cyclin; flow cytometry; parthenocarpy
5.  Identification of MicroRNA 395a in 24-Epibrassinolide-Regulated Root Growth of Arabidopsis thaliana Using MicroRNA Arrays 
Brassinosteroids (BRs) are endogenous plant hormones and are essential for normal plant growth and development. MicroRNAs (miRNAs) of Arabidopsis thaliana are involved in mediating cell proliferation in leaves, stress tolerance, and root development. The specifics of BR mechanisms involving miRNAs are unknown. Using customized miRNA array analysis, we identified miRNAs from A. thaliana ecotype Columbia (Col-0) regulated by 24-epibrassinolide (EBR, a highly active BR). We found that miR395a was significantly up-regulated by EBR treatment and validated its expression under these conditions. miR395a was over expressed in leaf veins and root tissues in EBR-treated miR395a promoter::GUS plants. We integrated bioinformatics methods and publicly available DNA microarray data to predict potential targets of miR395a. GUN5—a multifunctional protein involved in plant metabolic functions such as chlorophyll synthesis and the abscisic acid (ABA) pathway—was identified as a possible target. ABI4 and ABI5, both genes positively regulated by ABA, were down-regulated by EBR treatment. In summary, our results suggest that EBR regulates seedling development and root growth of A. thaliana through miR395a by suppressing GUN5 expression and its downstream signal transduction.
PMCID: PMC3742243  PMID: 23839095
brassinosteroids; miR395a; root growth; Arabidopsis thaliana; microRNA array
6.  Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways 
BMC Plant Biology  2010;10:151.
Brassinosteroids (BRs) play crucial roles in plant development and also promote tolerance to a range of abiotic stresses. Although much has been learned about their roles in plant development, the mechanisms by which BRs control plant stress responses and regulate stress-responsive gene expression are not fully known. Since BR interacts with other plant hormones, it is likely that the stress tolerance conferring ability of BR lies in part in its interactions with other stress hormones.
Using a collection of Arabidopsis mutants that are either deficient in or insensitive to abscisic acid (ABA), ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), we studied the effects of 24-epibrassinloide (EBR) on basic thermotolerance and salt tolerance of these mutants. The positive impact of EBR on thermotolerance in proportion to wild type was evident in all mutants studied, with the exception of the SA-insensitive npr1-1 mutant. EBR could rescue the ET-insensitive ein2 mutant from its hypersensitivity to salt stress-induced inhibition of seed germination, but remained ineffective in increasing the survival of eto1-1 (ET-overproducer) and npr1-1 seedlings on salt. The positive effect of EBR was significantly greater in the ABA-deficient aba1-1 mutant as compared to wild type, indicating that ABA masks BR effects in plant stress responses. Treatment with EBR increased expression of various hormone marker genes in both wild type and mutant seedlings, although to different levels.
These results together indicate that the redox-sensitive protein NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1), a master regulator of SA-mediated defense genes, is likely a critical component of EBR-mediated increase in thermotolerance and salt tolerance, but it is not required for EBR-mediated induction of PR-1 (PATHOGENESIS-RELATED1) gene expression; that BR exerts anti-stress effects independently as well as through interactions with other hormones; that ABA inhibits BR effects during stress; and that BR shares transcriptional targets with other hormones.
PMCID: PMC3095295  PMID: 20642851
7.  Changes in Photosynthetic Rates and Gene Expression of Leaves during a Source–Sink Perturbation in Sugarcane 
Annals of Botany  2007;101(1):89-102.
Background and Aims
In crops other than sugarcane there is good evidence that the size and activity of carbon sinks influence source activity via sugar-related regulation of the enzymes of photosynthesis, an effect that is partly mediated through coarse regulation of gene expression.
In the current study, leaf shading treatments were used to perturb the source–sink balance in 12-month-old Saccharum spp. hybrid ‘N19’ (N19) by restricting source activity to a single mature leaf. Changes in leaf photosynthetic gas exchange variables and leaf and culm sugar concentrations were subsequently measured over a 14 d period. In addition, the changes in leaf gene response to the source–sink perturbation were measured by reverse northern hybridization analysis of an array of 128 expressed sequence tags (ESTs) related to photosynthetic and carbohydrate metabolism.
Key Results
Sucrose concentrations in immature culm tissue declined significantly over the duration of the shading treatment, while a 57 and 88% increase in the assimilation rate (A) and electron transport rate (ETR), respectively, was observed in the source leaf. Several genes (27) in the leaf displayed a >2-fold change in expression level, including the upregulation of several genes associated with C4 photosynthesis, mitochondrial metabolism and sugar transport. Changes in gene expression levels of several genes, including Rubisco (EC 4·1·1·39) and hexokinase (HXK; EC 2·7·1·1), correlated with changes in photosynthesis and tissue sugar concentrations that occurred subsequent to the source–sink perturbation.
These results are consistent with the notion that sink demand may limit source activity through a kinase-mediated sugar signalling mechanism that correlates to a decrease in source hexose concentrations, which, in turn, correlate with increased expression of genes involved in photosynthesis and metabolite transport. The signal feedback system reporting sink sufficiency and regulating source activity may be a potentially valuable target for future genetic manipulation to increase sugarcane sucrose yield.
PMCID: PMC2701831  PMID: 17942591
C4 metabolism; array; hexose; photosynthesis; sucrose; Saccharum spp.; source–sink; gene expression
8.  Interaction of Brassinosteroids and Polyamines Enhances Copper Stress Tolerance in Raphanus Sativus  
Journal of Experimental Botany  2012;63(15):5659-5675.
Brassinosteroids (BRs) and polyamines (PAs) regulate various responses to abiotic stress, but their involvement in the regulation of copper (Cu) homeostasis in plants exposed to toxic levels of Cu is poorly understood. This study provides an analysis of the effects of exogenously applied BRs and PAs on radish (Raphanus sativus) plants exposed to toxic concentrations of Cu. The interaction of 24-epibrassinolide (EBR, an active BR) and spermidine (Spd, an active PA) on gene expression and the physiology of radish plants resulted in enhanced tolerance to Cu stress. Results indicated that the combined application of EBR and Spd modulated the expression of genes encoding PA enzymes and genes that impact the metabolism of indole-3-acetic acid (IAA) and abscisic acid (ABA) resulting in enhanced Cu stress tolerance. Altered expression of genes implicated in Cu homeostasis appeared to be the main effect of EBR and Spd leading to Cu stress alleviation in radish. Ion leakage, in vivo imaging of H2O2, comet assay, and improved tolerance of Cu-sensitive yeast strains provided further evidence for the ability of EBR and Spd to improve Cu tolerance significantly. The study indicates that co-application of EBR and Spd is an effective approach for Cu detoxification and the maintenance of Cu homeostasis in plants. Therefore, the use of these compounds in agricultural production systems should be explored.
PMCID: PMC3444278  PMID: 22915739
Abscisic acid; brassinosteroids; comet assay; copper transporters; Cu homeostasis; Cu-sensitive yeast; indole-3-acetic acid; oxidative stress; polyamines
9.  Brassinosteroids-Induced Systemic Stress Tolerance was Associated with Increased Transcripts of Several Defence-Related Genes in the Phloem in Cucumis sativus 
PLoS ONE  2013;8(6):e66582.
Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.
PMCID: PMC3686678  PMID: 23840504
10.  Source/sink interactions underpin crop yield: the case for trehalose 6-phosphate/SnRK1 in improvement of wheat 
Considerable interest has been evoked by the analysis of the regulatory pathway in carbohydrate metabolism and cell growth involving the non-reducing disaccharide trehalose (TRE). TRE is at small concentrations in mesophytes such as Arabidopsis thaliana and Triticum aestivum, excluding a role in osmoregulation once suggested for it. Studies of TRE metabolism, and genetic modification of it, have shown a very wide and more important role of the pathway in regulation of many processes in development, growth, and photosynthesis. It has now been established that rather than TRE, it is trehalose 6-phosphate (T6P) which has such profound effects. T6P is the intermediary in TRE synthesis formed from glucose-6-phosphate and UDP-glucose, derived from sucrose, by the action of trehalose phosphate synthase. The concentration of T6P is determined both by the rate of synthesis, which depends on the sucrose concentration, and also by the rate of breakdown by trehalose-6-phosphate phosphatase which produces TRE. Changing T6P concentrations by genetically modifying the enzymes of synthesis and breakdown has altered photosynthesis, sugar metabolism, growth, and development which affect responses to, and recovery from, environmental factors. Many of the effects of T6P on metabolism and growth occur via the interaction of T6P with the SnRK1 protein kinase system. T6P inhibits the activity of SnRK1, which de-represses genes encoding proteins involved in anabolism. Consequently, a large concentration of sucrose increases T6P and thereby inhibits SnRK1, so stimulating growth of cells and their metabolic activity. The T6P/SnRK1 mechanism offers an important new view of how the distribution of assimilates to organs, such as developing grains in cereal plants, is achieved. This review briefly summarizes the factors determining, and limiting, yield of wheat (particularly mass/grain which is highly conserved) and considers how T6P/SnRK1 might function to determine grain yield and might be altered to increase them. Increasing the potential rate of filling and mass/grain are ways in which total crop yield could be increased with good husbandry which maintains crop assimilation Cereal yields globally are not increasing, despite the greater production required to meet human demand. Careful targeting of T6P is showing much promise for optimization of source/sink for yield improvement and offers yet further possibilities for increasing sink demand and grain size in wheat.
PMCID: PMC4142875  PMID: 25202319
source/sink; wheat; grain yield; yield components; food security; sucrose; trehalose 6-phosphate; SnRK1
11.  Role of Evidence-Based Reviews in Surgery in teaching critical appraisal skills and in journal clubs 
Canadian Journal of Surgery  2013;56(4):E98-E102.
Evidence-Based Reviews in Surgery (EBRS) is a program developed to teach critical appraisal skills to general surgeons and residents. The purpose of this study was to assess the use of EBRS by general surgery residents across Canada and to assess residents’ opinions regarding EBRS and journal clubs.
We surveyed postgraduate year 2–5 residents from 15 general surgery programs. Data are presented as percentages and means.
A total of 231 residents (58%, mean 56% per program, range 0%–100%) responded: 172 (75%) residents indicated that they know about EBRS and that it is used in their programs. More than 75% of residents who use EBRS agreed or strongly agreed that the EBRS clinical and methodological articles and reviews are relevant. Only 55 residents (24%) indicated that they used EBRS online. Most residents (198 [86%]) attend journal clubs. The most common format is a mandatory meeting held at a special time every month with faculty members with epidemiological and clinical expertise. Residents stated that EBRS articles were used exclusively (13%) or in conjunction with other articles (57%) in their journal clubs. Most respondents (176 of 193 [91%]) stated that journal clubs are very or somewhat valuable to their education.
The EBRS program is widely used among general surgery residents across Canada. Although most residents who use EBRS rate it highly, a large proportion are unaware of EBRS online features. Thus, future efforts to increase awareness of EBRS online features and increase its accessibility are required.
PMCID: PMC3728260  PMID: 23883511
12.  Phenotypic landscape inference reveals multiple evolutionary paths to C4 photosynthesis 
eLife  2013;2:e00961.
C4 photosynthesis has independently evolved from the ancestral C3 pathway in at least 60 plant lineages, but, as with other complex traits, how it evolved is unclear. Here we show that the polyphyletic appearance of C4 photosynthesis is associated with diverse and flexible evolutionary paths that group into four major trajectories. We conducted a meta-analysis of 18 lineages containing species that use C3, C4, or intermediate C3–C4 forms of photosynthesis to parameterise a 16-dimensional phenotypic landscape. We then developed and experimentally verified a novel Bayesian approach based on a hidden Markov model that predicts how the C4 phenotype evolved. The alternative evolutionary histories underlying the appearance of C4 photosynthesis were determined by ancestral lineage and initial phenotypic alterations unrelated to photosynthesis. We conclude that the order of C4 trait acquisition is flexible and driven by non-photosynthetic drivers. This flexibility will have facilitated the convergent evolution of this complex trait.
eLife digest
Plants rely on carbon for their growth and survival: in a process called photosynthesis, they use energy from sunlight to convert carbon dioxide and water into carbohydrates and oxygen gas. The chemical reactions that make up photosynthesis are powered by a chain of enzymes, and plants must ensure that these enzymes—which are in the leaves of the plant—are supplied with enough carbon dioxide and water. Carbon dioxide from the atmosphere enters plants through pores in their leaves, but water must be carried up the plant from the roots.
The type of photosynthesis used by about 90% of flowering plant species—including tomatoes and rice—is called C3 photosynthesis. The first step in this process begins with an enzyme called RuBisCO, which reacts with carbon dioxide and a substance called RuBP to form molecules that contain three carbon atoms (hence the name C3 photosynthesis).
In a hot climate, however, a plant can lose a lot of water through the pores in its leaves: closing these pores allows the plant to retain water, but this also reduces the supply of carbon dioxide. Under these circumstances this causes problems because RuBisCO uses oxygen to break down RuBP, instead of creating sugars, when carbon dioxide is not readily available. To prevent this process, which wastes a lot of energy and resources, some plants—including maize, sugar cane and many other agricultural staples—have evolved an alternative process called C4 photosynthesis. Although it is more complex than C3 photosynthesis, and required many changes to be made to the structure of leaves, C4 photosynthesis has evolved on more than 60 different occasions.
In C4 plants, the mesophyll—the region that is associated with the capture of carbon dioxide by RuBisCO in C3 plants—contains high levels of an alternative enzyme called PEPC that converts carbon dioxide molecules into an acid that contains four carbon atoms. To avoid carbon dioxide being captured by both enzymes, C4 plants evolved to relocate RuBisCO from the mesophyll to a second set of cells in an airtight structure known as the bundle sheath. The four-carbon acids produced by PEPC diffuse to the cells in the bundle sheath, where they are broken down into carbon dioxide molecules, and photosynthesis then proceeds as normal. This process allows photosynthesis to continue when the level of carbon dioxide in the leave is low because the plant has closed its pores to retain water.
Since C4 plants grow faster than C3 plants, and also require less water, plant biologists would like to introduce certain C4 traits into C3 crop plants. To help with this process, Williams, Johnston et al. have used computational methods to explore how C4 photosynthesis evolved from ancestral C3 plants. This involved investigating the prevalence of 16 traits that are common to C4 plants in a total of 73 species that undergo C3 or C4 photosynthesis (including 37 species that possess characteristics of both C3 and C4).
Williams, Johnston et al. then went on to produce a new mathematical model that represents evolutionary processes as pathways across a multi-dimensional “landscape”. The model shows that traits can be acquired in various orders, and that C4 photosynthesis evolved through a number of independent pathways. Some traits that evolved early in the transitions to C4 photosynthesis influenced how evolution proceeded, providing “foundations” upon which further changes evolved.
Interestingly, the structure of the leaf itself appeared to change before any of the photosynthetic enzymes changed. This led Williams, Johnston et al. to conclude that climate change—in particular, the declines in carbon dioxide levels that occurred in prehistoric times—was probably not responsible for the original evolution of C4 photosynthesis. Nevertheless, these results could help with efforts to adapt important C3 crop plants to on-going changes in our climate.
PMCID: PMC3786385  PMID: 24082995
convergent evolution; C4 photosynthesis; Bayesian model; Other
13.  Harvest index, a parameter conditioning responsiveness of wheat plants to elevated CO2  
Journal of Experimental Botany  2013;64(7):1879-1892.
The expansion of the world’s population requires the development of high production agriculture. For this purpose, it is essential to identify target points conditioning crop responsiveness to predicted [CO2]. The aim of this study was to determine the relevance of ear sink strength in leaf protein and metabolomic profiles and its implications in photosynthetic activity and yield of durum wheat plants exposed to elevated [CO2]. For this purpose, a genotype with high harvest index (HI) (Triticum durum var. Sula) and another with low HI (Triticum durum var. Blanqueta) were exposed to elevated [CO2] (700 µmol mol–1 versus 400 µmol mol–1 CO2) in CO2 greenhouses. The obtained data highlighted that elevated [CO2] only increased plant growth in the genotype with the largest HI; Sula. Gas exchange analyses revealed that although exposure to 700 µmol mol–1 depleted Rubisco content, Sula was capable of increasing the light-saturated rate of CO2 assimilation (Asat) whereas, in Blanqueta, the carbohydrate imbalance induced the down-regulation of Asat. The specific depletion of Rubisco in both genotypes under elevated [CO2], together with the enhancement of other proteins in the Calvin cycle, revealed that there was a redistribution of N from Rubisco towards RuBP regeneration. Moreover, the down-regulation of N, NO3 –, amino acid, and organic acid content, together with the depletion of proteins involved in amino acid synthesis that was detected in Blanqueta grown at 700 µmol mol–1 CO2, revealed that inhibition of N assimilation was involved in the carbohydrate imbalance and consequently with the down-regulation of photosynthesis and growth in these plants.
PMCID: PMC3638836  PMID: 23564953
2D proteomic; Acclimation; C/N; CO2; Harvest index; Wheat.
14.  The Enterococcus faecium Enterococcal Biofilm Regulator, EbrB, Regulates the esp Operon and Is Implicated in Biofilm Formation and Intestinal Colonization 
PLoS ONE  2013;8(5):e65224.
Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162_2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization.
PMCID: PMC3669262  PMID: 23741484
15.  Effects of manganese-excess on CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport of leaves, and antioxidant systems of leaves and roots in Citrus grandis seedlings 
BMC Plant Biology  2010;10:42.
Very little is known about the effects of manganese (Mn)-excess on citrus photosynthesis and antioxidant systems. Seedlings of sour pummelo (Citrus grandis) were irrigated for 17 weeks with nutrient solution containing 2 μM (control) or 500 μM (excess) MnSO4. The objective of this study were to understand the mechanisms by which Mn-excess leads to a decrease in CO2 assimilation and to test the hypothesis that Mn-induced changes in antioxidant systems differ between roots and leaves.
Mn-excess decreased CO2 assimilation and stomatal conductance, increased intercellular CO2 concentration, but did not affect chlorophyll (Chl) level. Both initial and total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity in Mn-excess leaves decreased to a lesser extent than CO2 assimilation. Contents of glucose, fructose, starch and total nonstructural carbohydrates did not differ between Mn-excess leaves and controls, while sucrose content was higher in the former. Chl a fluorescence (OJIP) transients from Mn-excess leaves showed increased O-step and decreased P-step, accompanied by positive L- and K-bands. Mn-excess decreased maximum quantum yield of primary photochemistry (Fv/Fm) and total performance index (PItot,abs), but increased relative variable fluorescence at I-steps (VI) and energy dissipation. On a protein basis, Mn-excess leaves displayed higher activities of monodehydroascorbate reductase (MDAR), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) and contents of antioxidants, similar ascorbate peroxidase (APX) activities and lower dehydroascorbate reductase (DHAR) activities; while Mn-excess roots had similar or lower activities of antioxidant enzymes and contents of antioxidants. Mn-excess did not affect malondialdehyde (MDA) content of roots and leaves.
Mn-excess impaired the whole photosynthetic electron transport chain from the donor side of photosystem II (PSII) up to the reduction of end acceptors of photosystem I (PSI), thus limiting the production of reducing equivalents, and hence the rate of CO2 assimilation. Both the energy dissipation and the antioxidant systems were enhanced in Mn-excess leaves, while the antioxidant systems in Mn-excess roots were not up-regulated, but still remained high activity. The antioxidant systems in Mn-excess roots and leaves provided sufficient protection to them against oxidative damage.
PMCID: PMC2848762  PMID: 20205939
16.  Chloroplastic thioredoxin-f and thioredoxin-m1/4 play important roles in brassinosteroids-induced changes in CO2 assimilation and cellular redox homeostasis in tomato 
Journal of Experimental Botany  2014;65(15):4335-4347.
Virus-induced gene silencing (VIGS) was used in this study to characterize the role of thioredoxin-f and thioredoxin-m1/4 in brassinosteroid-induced changes in CO2 assimilation and cellular redox homeostasis in tomato.
Chloroplast thioredoxins (TRXs) and glutathione function as redox messengers in the regulation of photosynthesis. In this work, the roles of chloroplast TRXs in brassinosteroids (BRs)-induced changes in cellular redox homeostasis and CO2 assimilation were studied in the leaves of tomato plants. BRs-deficient d ^im plants showed decreased transcripts of TRX-f, TRX-m2, TRX-m1/4, and TRX-x, while exogenous BRs significantly induced CO2 assimilation and the expression of TRX-f, TRX-m2, TRX-m1/4, and TRX-x. Virus-induced gene silencing (VIGS) of the chloroplast TRX-f, TRX-m2, TRX-m1/4, and TRX-y genes individually increased membrane lipid peroxidation and accumulation of 2-Cys peroxiredoxin dimers, and decreased the activities of the ascorbate–glutathione cycle enzymes and the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in the leaves. Furthermore, partial silencing of TRX-f, TRX-m2, TRX-m1/4, and TRX-y resulted in decreased expression of genes involved in the Benson–Calvin cycle and decreased activity of the associated enzymes. Importantly, the BRs-induced increase in CO2 assimilation and the increased expression and activities of antioxidant- and photosynthesis-related genes and enzymes were compromised in the partially TRX-f- and TRX-m1/4-silenced plants. All of these results suggest that TRX-f and TRX-m1/4 are involved in the BRs-induced changes in CO2 assimilation and cellular redox homeostasis in tomato.
PMCID: PMC4112637  PMID: 24847092
Antioxidant; Benson–Calvin cycle; chloroplast; 2-Cys peroxiredoxin; glutathione; photosynthesis.
17.  Grafting Helps Improve Photosynthesis and Carbohydrate Metabolism in Leaves of Muskmelon 
The most important quality for muskmelon (Cucumis melo L.) is their sweetness which is closely related to the soluble sugars content. Leaves are the main photosynthetic organs in plants and thus the source of sugar accumulation in fruits since sugars are translocated from leaves to fruits. The effects of grafting muskmelon on two different inter-specific (Cucurbita maxima×C. moschata) rootstocks was investigated with respect to photosynthesis and carbohydrate metabolism. Grafting Zhongmi1 muskmelon on RibenStrong (GR) or Shengzhen1 (GS) rootstocks increased chlorophyll a, chlorophyll b and chlorophyll a+b content and the leaf area in middle and late developmental stages of the plant compared to the ungrafted Zhongmi1 check (CK). Grafting enhanced the net photosynthesis rate, the stomatal conductance, concentration of intercellular CO2 and transpiration rate. Grafting influenced carbohydrates contents by changing carbohydrate metabolic enzymes activities which was observed as an increase in acid invertase and neutral invertase activity in the functional leaves during the early and middle developmental stages compared to CK. Grafting improved sucrose phosphate synthase and stachyose synthase activities in middle and late developmental stages, thus translocation of sugars (such as sucrose, raffinose and stachyose) in GR and GS leaves were significantly enhanced. However, compared with CK, translocation of more sugars in grafted plants did not exert feedback inhibition on photosynthesis. Our results indicate that grafting muskmelon on inter-specific rootstocks enhances photosynthesis and translocation of sugars in muskmelon leaves.
PMCID: PMC3204406  PMID: 22043173
Chlorophyll content; Photosynthesis; Raffinose family oligosaccharides (RFOs); Carbohydrate metabolism; Grafting; Muskmelon
18.  CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply 
BMC Plant Biology  2009;9:43.
Although the effects of P deficiency on tea (Camellia sinensis (L.) O. Kuntze) growth, P uptake and utilization as well as leaf gas exchange and Chl a fluorescence have been investigated, very little is known about the effects of P deficiency on photosynthetic electron transport, photosynthetic enzymes and carbohydrates of tea leaves. In this study, own-rooted 10-month-old tea trees were supplied three times weekly for 17 weeks with 500 mL of nutrient solution at a P concentration of 0, 40, 80, 160, 400 or 1000 μM. This objective of this study was to determine how P deficiency affects CO2 assimilation, Rubisco, carbohydrates and photosynthetic electron transport in tea leaves to understand the mechanism by which P deficiency leads to a decrease in CO2 assimilation.
Both root and shoot dry weight increased as P supply increased from 0 to 160 μM, then remained unchanged. P-deficient leaves from 0 to 80 μM P-treated trees showed decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentration. Both initial and total Rubisco activity, contents of Chl and total soluble protein in P-deficient leaves decreased to a lesser extent than CO2 assimilation. Contents of sucrose and starch were decreased in P-deficient leaves, whereas contents of glucose and fructose did not change significantly except for a significant increase in the lowest P leaves. OJIP transients from P-deficient leaves displayed a rise at the O-step and a depression at the P-step, accompanied by two new steps at about 150 μs (L-step) and at about 300 μs (K-step). RC/CSo, TRo/ABS (or Fv/Fm), ETo/ABS, REo/ABS, maximum amplitude of IP phase, PIabs and PItot, abs were decreased in P-deficient leaves, while VJ, VI and dissipated energy were increased.
P deficiency decreased photosynthetic electron transport capacity by impairing the whole electron transport chain from the PSII donor side up to the PSI, thus decreasing ATP content which limits RuBP regeneration, and hence, the rate of CO2 assimilation. Energy dissipation is enhanced to protect P-deficient leaves from photo-oxidative damage in high light.
PMCID: PMC2685392  PMID: 19379526
19.  The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness 
Journal of Experimental Botany  2014;65(20):5959-5973.
Extended darkness induces a transient increase in sugars and trehalose pathway gene expression.
Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed.
PMCID: PMC4203130  PMID: 25271261
Maize; shade stress,; trehalose-6-phosphate; trehalose gene family; diurnal cycle,; quantitative RT-PCR.
20.  Over-expressing the C3 photosynthesis cycle enzyme Sedoheptulose-1-7 Bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO2 fumigation (FACE) 
BMC Plant Biology  2011;11:123.
Biochemical models predict that photosynthesis in C3 plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme Rubisco to carboxylate RuBP (Vc,max), or the regeneration of RuBP via electron transport (J). At current atmospheric [CO2] levels Rubisco is not saturated; consequently, elevating [CO2] increases the velocity of carboxylation and inhibits the competing oxygenation reaction which is also catalyzed by Rubisco. In the future, leaf photosynthesis (A) should be increasingly limited by RuBP regeneration, as [CO2] is predicted to exceed 550 ppm by 2050. The C3 cycle enzyme sedoheptulose-1,7 bisphosphatase (SBPase, EC has been shown to exert strong metabolic control over RuBP regeneration at light saturation.
We tested the hypothesis that tobacco transformed to overexpressing SBPase will exhibit greater stimulation of A than wild type (WT) tobacco when grown under field conditions at elevated [CO2] (585 ppm) under fully open air fumigation. Growth under elevated [CO2] stimulated instantaneous A and the diurnal photosynthetic integral (A') more in transformants than WT. There was evidence of photosynthetic acclimation to elevated [CO2] via downregulation of Vc,max in both WT and transformants. Nevertheless, greater carbon assimilation and electron transport rates (J and Jmax) for transformants led to greater yield increases than WT at elevated [CO2] compared to ambient grown plants.
These results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of C3 crops grown at [CO2] expected by the middle of the 21st century.
PMCID: PMC3185276  PMID: 21884586
climate change; photosynthetic carbon reduction cycle; C3 plants; RuBP regeneration; electron transport; improving photosynthesis
21.  Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2? 
Journal of Experimental Botany  2011;62(11):3957-3969.
Wheat plants (Triticum durum Desf., cv. Regallo) were grown in the field to study the effects of contrasting [CO2] conditions (700 versus 370 μmol mol−1) on growth, photosynthetic performance, and C management during the post-anthesis period. The aim was to test whether a restricted capacity of sink organs to utilize photosynthates drives a loss of photosynthetic capacity in elevated CO2. The ambient 13C/12C isotopic composition (δ13C) of air CO2 was changed from –10.2‰ in ambient [CO2] to –23.6‰ under elevated [CO2] between the 7th and the 14th days after anthesis in order to study C assimilation and partitioning between leaves and ears. Elevated [CO2] had no significant effect on biomass production and grain filling, and caused an accumulation of C compounds in leaves. This was accompanied by up-regulation of phosphoglycerate mutase and ATP synthase protein content, together with down-regulation of adenosine diphosphate glucose pyrophosphatase protein. Growth in elevated [CO2] negatively affected Rubisco and Rubisco activase protein content and induced photosynthetic down-regulation. CO2 enrichment caused a specific decrease in Rubisco content, together with decreases in the amino acid and total N content of leaves. The C labelling revealed that in flag leaves, part of the C fixed during grain filling was stored as starch and structural C compounds whereas the rest of the labelled C (mainly in the form of soluble sugars) was completely respired 48 h after the end of labelling. Although labelled C was not detected in the δ13C of ear total organic matter and respired CO2, soluble sugar δ13C revealed that a small amount of labelled C reached the ear. The 12CO2 labelling suggests that during the beginning of post-anthesis the ear did not contribute towards overcoming flag leaf carbohydrate accumulation, and this had a consequent effect on protein expression and photosynthetic acclimation.
PMCID: PMC3134354  PMID: 21511906
C management; elevated CO2; photosynthetic acclimation; proteomic characterization; Rubisco; stable isotopes
22.  A Rice Plastidial Nucleotide Sugar Epimerase Is Involved in Galactolipid Biosynthesis and Improves Photosynthetic Efficiency 
PLoS Genetics  2011;7(7):e1002196.
Photosynthesis is the final determinator for crop yield. To gain insight into genes controlling photosynthetic capacity, we selected from our large T-DNA mutant population a rice stunted growth mutant with decreased carbon assimilate and yield production named photoassimilate defective1 (phd1). Molecular and biochemical analyses revealed that PHD1 encodes a novel chloroplast-localized UDP-glucose epimerase (UGE), which is conserved in the plant kingdom. The chloroplast localization of PHD1 was confirmed by immunoblots, immunocytochemistry, and UGE activity in isolated chloroplasts, which was approximately 50% lower in the phd1-1 mutant than in the wild type. In addition, the amounts of UDP-glucose and UDP-galactose substrates in chloroplasts were significantly higher and lower, respectively, indicating that PHD1 was responsible for a major part of UGE activity in plastids. The relative amount of monogalactosyldiacylglycerol (MGDG), a major chloroplast membrane galactolipid, was decreased in the mutant, while the digalactosyldiacylglycerol (DGDG) amount was not significantly altered, suggesting that PHD1 participates mainly in UDP-galactose supply for MGDG biosynthesis in chloroplasts. The phd1 mutant showed decreased chlorophyll content, photosynthetic activity, and altered chloroplast ultrastructure, suggesting that a correct amount of galactoglycerolipids and the ratio of glycolipids versus phospholipids are necessary for proper chloroplast function. Downregulated expression of starch biosynthesis genes and upregulated expression of sucrose cleavage genes might be a result of reduced photosynthetic activity and account for the decreased starch and sucrose levels seen in phd1 leaves. PHD1 overexpression increased photosynthetic efficiency, biomass, and grain production, suggesting that PHD1 plays an important role in supplying sufficient galactolipids to thylakoid membranes for proper chloroplast biogenesis and photosynthetic activity. These findings will be useful for improving crop yields and for bioenergy crop engineering.
Author Summary
Photosynthesis is carried out in chloroplast, a plant-specific organelle. Photosynthetic membranes in chloroplasts contain high levels of glycolipids, and UDP-galactose is a dominating donor for glycolipid biosynthesis. Although glycolipid assembly of photosynthetic membranes has been characterized at the genetic and enzymatic level, the mechanism of substrate supply of UDP-galactose for the glycolipid biosynthetic pathway remains obscure. By genetic screening of rice mutants that are impaired in photosynthetic capacity and carbon assimilation, we identified PHD1 as a novel nucleotide sugar epimerase involved in a process of glycolipid biosynthesis and participating in photosynthetic membrane biogenesis. PHD1 was preferentially expressed in green and meristem tissues, and the PHD1 protein was targeted to chloroplasts. We revealed that UDP-galactose for glycolipid biosynthesis catalyzed by the new enzyme was generated inside chloroplasts, and the reduced amounts of glycolipids in the mutant led to decreased chlorophyll content and photosynthetic activity. Overexpression of this gene lead to growth acceleration, enhanced photosynthetic efficiency, and finally improved biomass and grain yield in rice. These results suggest that PHD1 has significant economic implications in both traditional crop improvement and bioenergy crop production.
PMCID: PMC3145628  PMID: 21829379
23.  Antagonistic actions of boron against inhibitory effects of aluminum toxicity on growth, CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, and photosynthetic electron transport probed by the JIP-test, of Citrus grandis seedlings 
BMC Plant Biology  2009;9:102.
Little information is available on the amelioration of boron (B) on aluminum (Al)-induced photosynthesis inhibition. Sour pummelo (Citrus grandis) seedlings were irrigated for 18 weeks with nutrient solution containing 4 B levels (2.5, 10, 25 and 50 μM H3BO3) × 2 Al levels (0 and 1.2 mM AlCl3·6H2O). The objectives of this study were to determine how B alleviates Al-induced growth inhibition and to test the hypothesis that Al-induced photosynthesis inhibition can be alleviated by B via preventing Al from getting into shoots.
B had little effect on plant growth, root, stem and leaf Al, leaf chlorophyll (Chl), CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), Chl a fluorescence (OJIP) transient and related parameters without Al stress except that root, stem and leaf B increased with increasing B supply and that 50 μM B decreased slightly root dry weight. Al-treated roots, stems and leaves displayed a higher or similar B. B did not affect root Al under Al stress, but decreased stem and leaf Al level. Shoot growth is more sensitive to Al stress than root growth, CO2 assimilation, Chl, Rubisco, OJIP transient and most related parameters. Al-treated leaves showed decreased CO2 assimilation, but increased or similar intercellular CO2 concentration. Both initial and total Rubisco activity in Al-treated leaves decreased to a lesser extent than CO2 assimilation. Al decreased maximum quantum yield of primary photochemistry and total performance index, but increased minimum fluorescence, K-band, relative variable fluorescence at J- and I-steps. B could alleviate Al-induced increase or decrease for all these parameters. Generally speaking, the order of B effectiveness was 25 μM > 10 μM ≥ 50 μM (excess B) > 2.5 μM.
We propose that Al-induced photosynthesis inhibition was mainly caused by impaired photosynthetic electron transport chain, which may be associated with growth inhibition. B-induced amelioration of root inhibition was probably caused by B-induced changes in Al speciation and/or sub-cellular compartmentation. However, B-induced amelioration of shoot and photosynthesis inhibition and photoinhibitory damage occurring at both donor and acceptor sides of photosystem II could be due to less Al accumulation in shoots.
PMCID: PMC2731759  PMID: 19646270
24.  Evidence-Based Research in Complementary and Alternative Medicine II: The Process of Evidence-Based Research 
It is a common practice in contemporary medicine to follow stringently the scientific method in the process of validating efficacy and effectiveness of new or improved modes of treatment intervention. It follows that these complementary or alternative interventions must be validated by stringent research before they can be reliably integrated into Western medicine. The next decades will witness an increasing number of evidence-based research directed at establishing the best available evidence in complementary and alternative medicine (CAM). This second paper in this lecture series examines the process of evidence-based research (EBR) in the context of CAM. We outline the fundamental principles, process and relevance of EBR, and its implication to CAM. We underscore areas of future development in EBR. We note that the main problem of applying EBR to CAM at present has to do with the fact that the contribution of EBR can be significant only to the extent to which studies used in the process of EBR are of good quality. All too often CAM research is not of sufficient quality to warrant the generation of a consensus statement. EBR, nevertheless, can contribute to CAM by identifying current weaknesses of CAM research. We present a revised instrument to assess quality of the literature.
PMCID: PMC1375240  PMID: 16550218
evidence-based research; systematic review; consolidated standards of randomized trials; Markov model; complementary and alternative medicine
25.  Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.) 
Journal of Experimental Botany  2010;62(1):111-123.
Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (Nase) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and Nase activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by Nase activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with Nase inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψs). At the nodule level, drought had an inhibitory effect on Nase activity. This decrease in Nase activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation in the nodule could also be associated with an osmoregulatory response to drought and might function as a protective agent against ROS. In droughted nodules, the decrease in N2 fixation was caused by an increase in oxygen resistance that was induced in the nodule. This was a mechanism to avoid oxidative damage associated with reduced respiration activity and the consequent increase in oxygen content. This study highlighted that even though drought had a direct effect on leaves, the deleterious effects of drought on nodules also conditioned leaf responsiveness.
PMCID: PMC2993905  PMID: 20797998
Drought; Medicago sativa; N2 fixation; N remobilization; oxidative stress; photosynthesis; proteome; Rubisco

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