Influence of intergenotypic competition on multigenerational persistence of abiotic stress resistance transgenes in populations of Arabidopsis thaliana.

Reducing crop losses due to abiotic stresses is a major target of agricultural biotechnology that will increase with climate change and global population growth. Concerns, however, have been raised about potential ecological impacts if transgenes become established in wild populations and cause increased competitiveness of weedy or invasive species. Potential risks will be a function of transgene movement, population sizes, and fitness effects on the recipient population. While key components influencing gene flow have been extensively investigated, there have been few studies on factors subsequent to transgene movement that can influence persistence and competitiveness. Here, we performed multiyear, multigenerational, assessment to examine fitness effects and persistence of three mechanistically different abiotic stress tolerance genes: C-repeat binding factor 3/drought responsive element binding factor 1a (CBF3/DREB1a); Salt overly sensitive 1 (SOS1); and Mannose-6-phosphate reductase (M6PR). Transgenic Arabidopsis thaliana overexpressing these genes were grown in pure populations and in competition with wild-type (WT) parents for six generations spanning a range of field environment conditions. Growth, development, biomass, seed production, and transgene frequency were measured at each generation. Seed planted for each generation was obtained from the previous generation as would occur during establishment of a new genotype in the environment. The three transgenes exhibited different fitness effects and followed different establishment trajectories. In comparison with pure populations, CBF3 lines exhibited reduced dry weight, seed yield, and viable seed yield, relative to WT background. In contrast, overexpression of SOS1 and M6PR did not significantly impact productivity measures in pure populations. In competition with WT, negative fitness effects were magnified. Transgene frequencies were significantly reduced for CBF3 and SOS1 while frequencies of M6PR appeared to be subject to genetic drift. These studies demonstrate the importance of fitness effects and intergenotype competition in influencing persistence of transgenes conferring complex traits.

Post photosynthetic carbon partitioning to sugar alcohols and consequences for plant growth.

The occurrence of sugar alcohols is ubiquitous among plants. Physiochemical properties of sugar alcohols suggest numerous primary and secondary functions in plant tissues and are often well documented. In addition to functions arising from physiochemical properties, the synthesis of sugar alcohols may have significant influence over photosynthetic, respiratory, and developmental processes owing to their function as a large sink for photosynthates. Sink strength is demonstrated by the high concentrations of sugar alcohols found in plant tissues and their ability to be readily transported. The plant scale distribution and physiochemical function of these compounds renders them strong candidates for functioning as stress metabolites. Despite this, several aspects of sugar alcohol biosynthesis and function are poorly characterised namely: 1) the quantitative characterisation of carbon flux into the sugar alcohol pool; 2) the molecular control governing sugar alcohol biosynthesis on a quantitative basis; 3) the role of sugar alcohols in plant growth and ecology; and 4) consequences of sugar alcohol synthesis for yield production and yield quality. We highlight the need to adopt new approaches to investigating sugar alcohol biosynthesis using modern technologies in gene expression, metabolic flux analysis and agronomy. Combined, these approaches will elucidate the impact of sugar alcohol biosynthesis on growth, stress tolerance, yield and yield quality.

Transcriptional variation in response to salt stress in commonly used Arabidopsis thaliana accessions.

Transcriptional variation is increasingly recognized as a component of genetic diversity and environmental adaptation. It can also provide insights into stress responsive determinants and underlying adaptive mechanisms. Prior studies showed phenotypic differences in response to salinity stress for two widely used Arabidopsis thaliana accessions, Wassilewskija-2 (Ws) and Columbia-0 (Col). This study examined changes in global gene expression in relation to differences in response to salt stress among Ws, Col, and the glabrous mutant of Col [Col(gl)]. Transcripts most highly affected by accession and salt stress were related to abiotic or biotic stress responses. Approximately 60% of salt-induced changes in Ws overlapped with changes in Col, suggesting common salt stress responses. However, a markedly greater number of genes was altered in the highly salt sensitive Col, likely reflecting both adaptive responses and salt injury. The Col(gl) transcriptome was least affected by salt. Many salt-responsive transcripts observed in Col were altered in Col(gl) prior to salt stress, indicating that even without salt, the gl1-1 mutation induced a suite of stress responsive genes. Regardless of salt stress, there were greater transcriptomic differences between Col and Col(gl) than between Col and Ws. The transcript expression differences between [Ws vs. Col] and [Col(gl) vs. Col] formed largely non-overlapping sets. Thus, although Ws, Col and Col(gl) are commonly and sometimes interchangeably used, here they displayed distinct responses. Collectively, their observed expression differences likely reflect a combination of adaptive traits, response to injury, or phenotypic buffering of mutational effects.

Transcriptomic and physiological variations of three Arabidopsis ecotypes in response to salt stress.

Salt stress is one of the major abiotic stresses in agriculture worldwide. Analysis of natural genetic variation in Arabidopsis is an effective approach to characterize candidate salt responsive genes. Differences in salt tolerance of three Arabidopsis ecotypes were compared in this study based on their responses to salt treatments at two developmental stages: seed germination and later growth. The Sha ecotype had higher germination rates, longer roots and less accumulation of superoxide radical and hydrogen peroxide than the Ler and Col ecotypes after short term salt treatment. With long term salt treatment, Sha exhibited higher survival rates and lower electrolyte leakage. Transcriptome analysis revealed that many genes involved in cell wall, photosynthesis, and redox were mainly down-regulated by salinity effects, while transposable element genes, microRNA and biotic stress related genes were significantly changed in comparisons of Sha vs. Ler and Sha vs. Col. Several pathways involved in tricarboxylic acid cycle, hormone metabolism and development, and the Gene Ontology terms involved in response to stress and defense response were enriched after salt treatment, and between Sha and other two ecotypes. Collectively, these results suggest that the Sha ecotype is preconditioned to withstand abiotic stress. Further studies about detailed gene function are needed. These comparative transcriptomic and analytical results also provide insight into the complexity of salt stress tolerance mechanisms.

Resveratrols in grape berry skins and leaves in vitis germplasm.

BACKGROUND: Resveratrol is an important stilbene that benefits human health. However, it is only distributed in a few species including grape and is very expensive. At present, grape has been an important source resveratrol. However, the details are scarce on resveratrol distribution in different Vitis species or cultivars. METHODOLOGY/PRINCIPAL FINDING: The composition and content of resveratrols were investigated by HPLC for assessing genotypic variation in berry skins and leaves of 75 grape cultivars, belonging to 3 species and 7 interspecific hybrids. Trans-resveratrol, cis-piceid and trans-piceid were detected in berry skins and leaves, but cis-resveratrol was not. Resveratrol content largely varied with genetic background as well as usage. In most cultivars, total resveratrol including the above three compounds was higher in berry skins than leaves. In berry skins of most cultivars and leaves of almost all cultivars, cis-piceid was the most abundant resveratrol; trans-resveratrol and trans-piceid were minor components. Some specific cultivars were found with extremely high levels of trans-resveratrol, cis- piceid, trans-piceid or total resveratrols in berry skins or leaves. In skins and leaves, rootstock cultivars had a higher content of total resveratrols, and the cultivated European type cultivars and their hybrids with V. labrusca had relatively low totals. There were no significant correlations of the amounts of total resveratrols or any individual resveratrol between berry skins and leaves. All 75 cultivars can be divided into four groups based on the composition of resveratrols and their concentration by principal component analysis. CONCLUSION: Resveratrol content of grape berries and leaves varied largely with their genetic background and usage. Rootstock cultivars had a higher content of total resveratrols than the other germplasm. Total resveratrols were lower in leaves than berry skins in most cultivars. Cis-piceid was the most abundant resveratrol in most cultivars, and trans-res and trans-pd were minor components.

Comparison of salt stress resistance genes in transgenic Arabidopsis thaliana indicates that extent of transcriptomic change may not predict secondary phenotypic or fitness effects.

Engineered abiotic stress resistance is an important target for increasing agricultural productivity. There are concerns, however, regarding possible ecological impacts of transgenic crops. In contrast to the first wave of transgenic crops, many abiotic stress resistance genes can initiate complex downstream changes. Transcriptome profiling has been suggested as a comprehensive non-targeted approach to examine the secondary effects. We compared phenotypic and transcriptomic effects of constitutive expression of genes intended to confer salt stress tolerance by three different mechanisms: a transcription factor, CBF3/DREB1a; a metabolic gene, M6PR, for mannitol biosynthesis; and the Na⁺/H⁺ antiporter, SOS1. Transgenic CBF3, M6PR and SOS1 Arabidopsis thaliana were grown together in the growth chamber, greenhouse and field. In the absence of salt, M6PR and SOS1 lines performed comparably with wild type; CBF3 lines exhibited dwarfing as reported previously. All three transgenes conferred fitness advantage when subjected to 100 mm NaCl in the growth chamber. CBF3 and M6PR affected transcription of numerous abiotic stress-related genes as measured by Affymetrix microarray analysis. M6PR additionally modified expression of biotic stress and oxidative stress genes. Transcriptional effects of SOS1 in the absence of salt were smaller and primarily limited to redox-related genes. The extent of transcriptome change, however, did not correlate with the effects on growth and reproduction. Thus, the magnitude of global transcriptome differences may not predict phenotypic differences upon which environment and selection act to influence fitness. These observations have implications for interpretation of transcriptome analyses in the context of risk assessment and emphasize the importance of evaluation within a phenotypic context.

Photosynthetic responses to heat treatments at different temperatures and following recovery in grapevine (Vitis amurensis L.) leaves.

BACKGROUND: The electron transport chain, Rubisco and stomatal conductance are important in photosynthesis. Little is known about their combined responses to heat treatment at different temperatures and following recovery in grapevines (Vitis spp.) which are often grown in climates with high temperatures. METHODOLOGY/FINDINGS: The electron transport function of photosystem II, the activation state of Rubisco and the influence of stomatal behavior were investigated in grapevine leaves during heat treatments and following recovery. High temperature treatments included 35, 40 and 45°C, with 25°C as the control and recovery temperature. Heat treatment at 35°C did not significantly (P>0.05) inhibit net photosynthetic rate (P(n)). However, with treatments at 40 and 45°C, P(n) was decreased, accompanied by an increase in substomatal CO(2) concentration (C(i)), decreases in stomatal conductance (g(s)) and the activation state of Rubisco, and inhibition of the donor side and the reaction center of PSII. The acceptor side of PSII was inhibited at 45°C but not at 40°C. When grape leaves recovered following heat treatment, P(n), g(s) and the activation state of Rubisco also increased, and the donor side and the reaction center of PSII recovered. The increase in P(n) during the recovery period following the second 45°C stress was slower than that following the 40°C stress, and these increases corresponded to the donor side of PSII and the activation state of Rubisco. CONCLUSIONS: Heat treatment at 35°C did not significantly (P>0.05) influence photosynthesis. The decrease of P(n) in grape leaves exposed to more severe heat stress (40 or 45°C) was mainly attributed to three factors: the activation state of Rubisco, the donor side and the reaction center of PSII. However, the increase of P(n) in grape leaves following heat stress was also associated with a stomatal response. The acceptor side of PSII in grape leaves was responsive but less sensitive to heat stress.

Global gene expression analysis of transgenic, mannitol-producing, and salt-tolerant Arabidopsis thaliana indicates widespread changes in abiotic and biotic stress-related genes.

Mannitol is a putative osmoprotectant contributing to salt tolerance in several species. Arabidopsis plants transformed with the mannose-6-phosphate reductase (M6PR) gene from celery were dramatically more salt tolerant (at 100 mM NaCl) as exhibited by reduced salt injury, less inhibition of vegetative growth, and increased seed production relative to the wild type (WT). When treated with 200 mM NaCl, transformants produced no seeds, but did bolt, and exhibited less chlorosis/necrosis and greater survival and dry weights than the WT. Without salt there were no M6PR effects on growth or phenotype, but expression levels of 2272 genes were altered. Many fewer differences (1039) were observed between M6PR and WT plants in the presence of salt, suggesting that M6PR pre-conditioned the plants to stress. Previous work suggested that mannitol is an osmoprotectant, but mannitol levels are invariably quite low, perhaps inadequate for osmoprotectant effects. In this study, transcriptome analysis reveals that the M6PR transgene activated the downstream abscisic acid (ABA) pathway by up-regulation of ABA receptor genes (PYL4, PYL5, and PYL6) and down-regulation of protein phosphatase 2C genes (ABI1 and ABI2). In the M6PR transgenic lines there were also increases in transcripts related to redox and cell wall-strengthening pathways. These data indicate that mannitol-enhanced stress tolerance is due at least in part to increased expression of a variety of stress-inducible genes.

Salicylic acid alleviates decreases in photosynthesis under heat stress and accelerates recovery in grapevine leaves.

BACKGROUND: Although the effect of salicylic acid (SA) on photosynthesis of plants including grapevines has been investigated, very little is yet known about the effects of SA on carbon assimilation and several components of PSII electron transport (donor side, reaction center and acceptor side). In this study, the impact of SA pretreatment on photosynthesis was evaluated in the leaves of young grapevines before heat stress (25 degrees C), during heat stress (43 degrees C for 5 h), and through the following recovery period (25 degrees C). Photosynthetic measures included gas exchange parameters, PSII electron transport, energy dissipation, and Rubisco activation state. The levels of heat shock proteins (HSPs) in the chloroplast were also investigated. RESULTS: SA did not significantly (P < 0.05) influence the net photosynthesis rate (Pn) of leaves before heat stress. But, SA did alleviate declines in Pn and Rubisco activation state, and did not alter negative changes in PSII parameters (donor side, acceptor side and reaction center QA) under heat stress. Following heat treatment, the recovery of Pn in SA-treated leaves was accelerated compared with the control (H2O-treated) leaves, and, donor and acceptor parameters of PSII in SA-treated leaves recovered to normal levels more rapidly than in the controls. Rubisco, however, was not significantly (P < 0.05) influenced by SA. Before heat stress, SA did not affect level of HSP 21, but the HSP21 immune signal increased in both SA-treated and control leaves during heat stress. During the recovery period, HSP21 levels remained high through the end of the experiment in the SA-treated leaves, but decreased in controls. CONCLUSION: SA pretreatment alleviated the heat stress induced decrease in Pn mainly through maintaining higher Rubisco activation state, and it accelerated the recovery of Pn mainly through effects on PSII function. These effects of SA may be related in part to enhanced levels of HSP21.

A novel mannose-based selection system for plant transformation using celery mannose-6-phosphate reductase gene.

To investigate its potential application as a selectable marker for plant transformation, the mannitol producing, celery mannose-6-phosphate reductase gene (M6PR) was transformed into Arabidopsis and tobacco using Agrobacterium tumefaciens-mediated transformation. Mannose-tolerance assays in transgenic materials revealed that the M6PR can act as a selectable marker gene in either a positive or a negative selection mode depending on the plant species. For mannose sensitive species, such as Arabidopsis, expression of M6PR enhanced mannose tolerance and provided a positive selection for transgenic seeds. On medium containing 2 g/L mannose, transgenic seeds germinated, whereas wild type (WT) seeds did not. For mannose-tolerant species, expression of M6PR increased mannose sensitivity in tobacco and enabled a negative selection for transgenic leaves and seeds. Mannose at 30 g/L blanched leaf explants from all 29 transgenic tobacco events with M6PR. In contrast, 30 g/L mannose did not inhibit shoot regeneration from leaf explants of WT or transgenic plants with either an antisense M6PR or a plasmid control. Similarly, mannose at 30 g/L inhibited seed germination of transgenic tobacco seeds with M6PR but not that of WT or transgenic tobacco with either the antisense M6PR or the plasmid control. Northern blot confirmed transcripts of the M6PR in transgenic tobacco, and accumulation of mannitol verified activity of the M6PR in tobacco leaves. Either positive or negative selection using the celery M6PR is versatile for plant transformation. Additionally, the celery M6PR is a potential target gene for improving salt-tolerance in plants due to mannitol accumulation.

Heat acclimation induced acquired heat tolerance and cross adaptation in different grape cultivars: relationships to photosynthetic energy partitioning.

Several mechanisms on acquired heat tolerance and cross adaptation have been proposed; however, relationships to photosynthetic energy partitioning remain unknown. The effects of heat pretreatment on cold and heat tolerance in grapevine leaves of two cultivars ('Jingxiu', cold sensitive; 'Beta', cold tolerant) were evident in changes in the antioxidant system, lipid peroxidation, net photosynthesis rate and also in chlorophyll fluorescence according : Y(II) + Y(NPQ) + Y(NO) = 1, where Y(II) is the effective PSII quantum yield; Y(NPQ) is regulated energy dissipation as a protective mechanism; and Y(NO) is non-regulated energy dissipation as a damaging mechanism. Heat pretreatment enhanced heat tolerance in the two cultivars, which was associated with less energy partitioned in non-regulated energy dissipation, less lipid peroxidation and higher antioxidant enzyme (catalase, ascorbate peroxidase and guaiacol peroxidase) activities compared with control plants under heat stress. Heat pretreatment also induced cold tolerance in 'Jingxiu' and 'Beta' leaves. This cross adaptation seemed to be attributable in part to less non-regulated energy dissipation in pretreated 'Jingxiu' and 'Beta' than the controls under cold stress. The evidence that lipid peroxidation was less and antioxidant enzyme activities were higher in pretreated plants under cold stress further corroborated the results from energy partitioning.

Response of mannitol-producing Arabidopsis thaliana to abiotic stress.

In celery, mannitol is a primary photosynthetic product that is associated with celery's exceptional salt tolerance. Arabidopsis plants transformed with celery's mannose-6-phosphate reductase (M6PR) gene produce mannitol and grow normally in the absence of stress. Daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant and specific leaf weight) over a 12-day period showed less effect of salt (100 mm NaCl) on the M2 transformant than wild type (WT). Following a 12-day treatment of WT, M2 and M5 plants with 100 or 200 mm NaCl the total shoot fresh weight, leaf number, and leaf area were significantly greater in transformants than in WT plants. The efficiency of use of energy for photochemistry by PSII was measured daily under growth conditions. In WT plants treated with 100 mm NaCl, the PSII yield begin decreasing after 6 days with a 50% loss in yield after 12 days, indicating a severe loss in PSII efficiency; whereas, there was no effect on the transformants. Under atmospheric levels of CO2, growth with 200 mm NaCl caused an increase in the substomatal levels of CO2 in WT plants but not in transformants. It also caused a marked decrease in carboxylation efficiency under limiting levels of CO2 in WT compared with transformants. When stress was imposed and growth reduced by withholding water for 12 days, which resulted in a similar decrease in relative water content to salt-treated plants, there were no differences among the genotypes in PSII yields or growth. The results suggest mannitol, which is known to be a compatible solute and antioxidant, protects photosynthesis against salt-related damage to chloroplasts.

The effects of UV-B radiation on photosynthesis in relation to Photosystem II photochemistry, thermal dissipation and antioxidant defenses in winter wheat (Triticum aestivum L.) seedlings at different growth temperatures.

To study the UV-B effect on photosynthesis in winter wheat at different day/night temperatures, biologically effective UV-B radiation at 4.2 (LUVB) and 10.3 (HUVB) kJ m-2 d-1 was provided on the seedlings at 25/20°C or 10/5°C. UV-B radiation inhibited net photosynthesis rate (Pn) by enhanced intensity and decreased temperature without change of intercellular CO2 concentrations (Ci). Decreased maximal quantum yield of Photosystem II (Fv/Fm) and increased minimum fluorescence (Fo) were observed in HUVB at both temperatures and LUVB at 10/5°C. HUVB increased total pool size (VAZ) of xanthophyll cycle pigments, but decreased the de-epoxidation state (DEPS) of these pigments at both temperatures, while LUVB only decreased DEPS at 10/5°C. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and the redox states of ascorbate and glutathione (AsA/DAsA and GSH/GSSG) were enhanced at 25/20°C, while there were increased SOD and CAT, unaltered APX activities and AsA/DHA, as well as decreased GR activity and GSH/GSSG in LUVB and HUVB at 10/5°C. UV-B radiation resulted in higher H2O2 and thiobarbituric acid reactive substance (TBARS) concentrations at 10/5°C than 25/20°C. It appears that low temperature alone did not influence photosynthesis but aggravated UV-B induced photoinhibition, which was associated with PSII photochemistry rather than stomatal limitation. Xanthophyll cycle pigments failed to provide photoprotection through thermal dissipation. The antioxidant system was up-regulated in LUVB and HUVB at 25/20°C, but was impaired at 10/5°C. Low temperature intensified UV-B induced photoinhibition and damage by weakening the antioxidant system.

Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues.

The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g. almond [Prunus dulcis (P. Mill.) D.A. Webber], apple [Malus pumila P. Mill.], cherry [Prunus spp.], peach [Prunus persica L. Batsch], and pear [Pyrus communis]). To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol. Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae). Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (K(m) sorbitol of 0.81 mM for PcSOT1 and 0.64 mM for PcSOT2). Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development. PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest. In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves. In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves. Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits. Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues. Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity.