Genetic Engineering of Glycine Betaine Biosynthesis Reduces Heat-Enhanced Photoinhibition by Enhancing Antioxidative Defense and Alleviating Lipid Peroxidation in Tomato

Glycine betaine (GB) is a compatible solute that accumulates rapidly to enhance heat tolerance in many plants grown under heat stress. In this study, a BADH gene (betaine aldehyde dehydrogenase) from spinach was introduced into tomato (Lycopersicon esculentum cv. ‘Moneymaker’) via Agrobacterium-mediated transformation. Transgenic tomato lines expressing BADH exhibited higher capabilities for GB accumulation. Chlorophyll fluorescence analysis of wild type (WT) and transgenic plants exposed to heat treatment (42 °C) showed that transgenic plants exhibited higher photosynthetic capacities than WT plants. This finding suggests that GB accumulation increases tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement in D1 protein content, which accelerated the repair of photosystem II (PSII) following heat-enhanced photoinhibition. Significant accumulations of hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^?) were observed in WT plants under heat stress. However, these accumulations were much less for the transgenic plants. An important finding reported herein is that exogenous GB cannot directly reduce the content of reactive oxygen species (ROS). In accordance with a lower relative electrolyte conductivity and malondialdehyde content, the activities of antioxidant enzymes were higher in transgenic lines than in WT plants, indicating that the degree of membrane injury in the transgenic plants was lower compared to the WT plants. These results suggest that GB accumulation in vivo cannot directly eliminate ROS. Rather, higher antioxidant enzyme activities must be maintained to lessen the accumulation of ROS in transgenic plants and to decrease the degree of membrane injury.     

Effects of Shading on the Senescence and Photosynthetic Physiology of the Early-Flowering Rice Mutant FTL10 at Noon

FTL10 is an early-flowering mutant of rice (Oryza sativa L.) with a premature senescent phenotype. Early leaf senescence can cause negative effects on rice yield. Moreover, rice leaves are damaged under high-light conditions, which promote rice senescence. Artificial shading can reduce the amount of light absorbed by rice leaves. The aim of this study was to investigate the effects of shading at noon (11:30–14:00) on the senescence and yield of FTL10. The results showed that shading improved the total antioxidant capacity of rice leaves, reduced the accumulation of reactive oxygen species (ROS) and reduced the expression of genes related to senescence. In the shaded group, the degradation rate of chlorophyll and Rubisco proteins, which are related to photosynthesis, was relatively slow, and the photosynthetic rate was relatively high. Compared with those under the natural growth conditions, the proportion of photosynthetic electron allocated to photorespiration in the shaded group rice leaves was lower, and the proportion allocated to carbon fixation was higher. The yield data showed that the single-spike weight and yield per plant of rice significantly increased after shading. Therefore, our research shows that shading at noon could delay FTL10 senescence and increase yields.

     

Plasma membrane-generated ROS and their possible contribution to leaf cell growth of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) MSC16 mitochondrial mutant

Reactive oxygen species (ROS) generally regarded as harmful products of oxygenic metabolism causing oxidative stress and cell damage are also important for control and regulation of biological processes. ROS can be generated by various enzymatic activities and removed by an array of ROS-scavenging molecules in the cell. In plants, the generation of ROS initiated by the plasma membrane NADPH oxidase can be used for controlled polymer breakdown leading to cell wall loosening during extension growth. The mosaic (MSC16) mitochondrial mutant of cucumber (Cucumis sativus L.) has marked phenotypic changes, including a slower growth rate which partially may result from disturbed leaf carbon and energy metabolism and ROS/antioxidants equilibrium. Cytochemical localization of H₂O₂ in leaf cells showed lower total level of H₂O₂ particularly in the apoplast of MSC16 leaf cells as compared to WT. The activity of plasma membrane NADPH oxidase (EC 1.6.3.1) was about 30% lower in plasmalemma vesicles isolated from MSC16 leaf tissue as compared to WT. The total foliar ascorbate pool (reduced and oxidized) was about 35% higher in MSC16 compared to WT leaves due to an increased content of the oxidized form. About 3% of the whole-leaf ascorbate was localized in the apoplast but in MSC16 it was considerably more reduced. We conclude that the lower apoplastic ROS content caused by decreased activity of plasma membrane NADPH oxidase and lower amounts of H₂O₂ in the apoplast may also contribute to altered growth of the MSC16 cucumber mutant.     

Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant

The Arabidopsis thaliana ascorbate-deficient vtc-1 mutant has only 30% ascorbate contents of the wild type (WT). This ascorbate-deficient mutant was used here to study the physiological roles of ascorbate under salt stress in vivo. Salt stress resulted in a more significant decrease in CO2 assimilatory capacity in the vtc-1 mutant than in the WT. Photosystem II function in the Arabidopsis vtc-1 mutant also showed an increased sensitivity to salt stress. Oxidative stress, indicated by the hydrogen peroxide content, increased more dramatically in the vtc-1 mutant than in the WT under salt stress. To clarify the reason for the increased oxidative stress in the vtc-1 mutant, the contents of small antioxidant compounds and the activities of several antioxidant enzymes in the ascorbate-glutathione cycle were measured. Despite an elevated glutathione pool in the vtc-1 mutant, the ascorbate contents and the reduced form of ascorbate decreased very rapidly under salt stress. These results showed that the activities of MDAR and DHAR were lower in the vtc-1 mutant than in the WT under salt stress. Thus, low intrinsic ascorbate and an impaired ascorbate-glutathione cycle in the vtc-1 mutant under salt stress probably induced a dramatic decrease in the reduced form of ascorbate, which resulted in both enhanced ROS contents and decreased NPQ in the vtc-1 mutant.     

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP⁺-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.     

The antioxidative defense system is involved in the delayed senescence in a wheat mutant tasg1

Wheat, which is the most important food crop worldwide, is a cereal that presents considerable potential for increased yield. A new wheat (Triticum aestivum L.) mutant tasg1 with delayed leaf senescence was constructed using ethyl methane sulfonate as a mutagen. Natural senescence in tasg1 was distinctly delayed in the field, as indicated by the slower progression of chlorophyll degradation, net photosynthetic rate than its wild type. Further, the malondialdehyde and the hydrogen peroxide content was lower and antioxidative enzyme activity higher in tasg1 than those in its wild type during both natural senescence and methyl viologen-induced oxidative stress. The results suggest that tasg1 is a functional stay-green wheat mutant with the Type B (in which senescence initiates on schedule, but progresses at a rate lower than that in the respective WTs) or Type A (in which senescence initiates late but proceeds at a normal rate) and B combination and that the competence of the antioxidant defense system is one of the most important mechanisms underlying the expression of the stay-green phenotype.     

ATHKl基因调节拟南芥渗透胁迫信号转导过程

以拟南芥ATHKl基因T-DNA插入所产生的缺失突变体和野生型ws(wassilewskija)生态型为材料,分析了它们在生理和基因表达方面的差异.结果表明突变体的离体叶片失水率明显大于野生型;在30%PEG-6000胁迫后,野生型和ATHKJ突变体的细胞膜离子外渗率比胁迫前分别增加了50%和80%.PEG胁迫48 h时突变体的萎蔫程度明显大于野生型ws.以上结果说明ATHKl突变体的抗渗透胁迫能力低于野生型,即ATHKl基因参与了拟南芥适应逆境的调节反应.利用DDRT-PCR技术研究二者在PEG胁迫36h后的基因表达差异,分离到9个在野生型中被PEG诱导表达而在突变体中未被诱导的参与逆境应答的基因片段,其中包括MAPKKKl8和丝氨酸/苏氨酸蛋白激酶基因,即ATHKJ基因失活引起下游基因响应渗透胁迫的能力减弱,进一步说明ATHKJ基因参与拟南芥适应逆境的调节反应,并且ATHKl可能在逆境信号转导组分MAPK的上游起作用,很可能是植物体中的渗透感受器.     

Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation

Phosphatidic acid (PA) level increases during various stress conditions. However, the physiological roles of this lipid in stress response remain largely unknown. In this study, we report that PA induced leaf cell death and elevated the levels of reactive oxygen species (ROS) in the whole leaf and single cells. To further elucidate the mechanism of PA-induced cell death, we then examined whether Rho-related small G protein (ROP) 2, which enhanced ROS production in an in vitro assay, is involved in PA-induced ROS production and cell death. In response to PA, transgenic leaves of Arabidopsis expressing a constitutively active rop2 mutant exhibited earlier cell death and higher levels of ROS than wild type (WT), whereas those expressing a dominant-negative rop2 mutant exhibited later cell death and lower ROS. However, in the absence of exogenous PA, no spontaneous cell death or elevated ROS was observed in constitutively active rop2 plants, suggesting that the activation of ROP GTPase alone is insufficient to activate the ROP-mediated ROS generation pathway. These results suggest that PA modulates an additional factor required for the active ROP-mediated ROS generation pathway. Therefore, PA may be an important regulator of ROP-regulated ROS generation and the cell death process during various stress and defense responses of plants.     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H₂O₂. The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenouslyapplied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H₂O₂. Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.     

Physiological and molecular adaptations to drought in Andean potato genotypes

The drought stress tolerance of two Solanum tuberosum subsp. andigena landraces, one hybrid (adgxtbr) and Atlantic (S. tuberosum subsp. tuberosum) has been evaluated. Photosynthesis in the Andigena landraces during prolonged drought was maintained significantly longer than in the Tuberosum (Atlantic) line. Among the Andigena landraces, 'Sullu' (SUL) was more drought resistant than 'Negra Ojosa' (NOJ). Microarray analysis and metabolite data from leaf samples taken at the point of maximum stress suggested higher mitochondrial metabolic activity in SUL than in NOJ. A greater induction of chloroplast-localized antioxidant and chaperone genes in SUL compared with NOJ was evident. ABA-responsive TFs were more induced in NOJ compared with SUL, including WRKY1, mediating a response in SA signalling that may give rise to increased ROS. NOJ may be experiencing higher ROS levels than SUL. Metabolite profiles of NOJ were characterized by compounds indicative of stress, for example, proline, trehalose, and GABA, which accumulated to a higher degree than in SUL. The differences between the Andigena lines were not explained by protective roles of compatible solutes; hexoses and complex sugars were similar in both landraces. Instead, lower levels of ROS accumulation, greater mitochondrial activity and active chloroplast defences contributed to a lower stress load in SUL than in NOJ during drought.     

A novel stress-associated protein SbSAP14 from Sorghum bicolor confers tolerance to salt stress in transgenic rice

We have characterized a member of the stress-associated protein (SAP) gene family from Sorghum bicolor (SbSAP14) with A20 and AN1 zinc-finger domains. Expression profiling revealed that SbSAP14 is specifically induced in response to dehydration, salt, and oxidative stress as well as abscisic acid treatment. During the early stage of salt stress, overexpression of SbSAP14 was able to prevent yellowing and withering of the leaf tip of rice plants. Measurements of malondialdehyde, ion leakage, and chlorophyll content demonstrated that transgenic rice had an enhanced tolerance to oxidative damage caused by salt stress. Under prolonged salt stress, transgenic rice plants had a higher seed germination rate and higher percentage seedling survival than wild-type (WT) plants. Importantly, in vivo and in situ assays revealed that the accumulation of reactive oxygen species in transgenic rice plants was significantly lower than that in WT plants. Among the six antioxidant genes tested, APX2, CatB, CatC, and SodA1 showed a higher expression level in transgenic rice than in WT rice. Based on these results, we propose that SbSAP14 may play a key role in antioxidant defense systems and possibly be involved in the induction of antioxidant genes in plants, suggesting a possible mechanism of the SAP gene family in stress defense response.     

An inactivating mutation in the vacuolar arginine exporter gene Vae results in culture degeneration in the fungus Metarhizium robertsii

Culture degeneration usually results in great commercial losses in the economically important filamentous fungi, but the genetic causes of the degeneration remain elusive. In the fungus Metarhizium robertsii, we found that deletion of the vacuolar arginine exporter gene Vae caused culture degeneration. Compared to the WT strain, the mutant showed increased apoptosis, reactive oxygen species (ROS) level and mitochondrial membrane potential collapse, reduced conidial yield and abnormal lipid droplet formation. The extent of the degeneration in the mutant gradually increased over the successive subculturing, which eventually became irreversible; compared to the third subculture of the mutant, the seventh subculture showed a lower conidial yield and pathogenicity to insects, stronger apoptosis, higher ROS level and a smaller number of conidial lipid droplets. Incorporation of the genomic clone of Vae could not restore the WT phenotypes in the seventh subculture, but could in the third one. Loss-of-function in Vae resulted in vacuolar arginine accumulation and reduction in the cytosolic arginine. This downregulated the expression of the regulator CAG9 of G protein signalling pathway, which accounted for most of the phenotypic changes associated with the degeneration of the mutant. We identified a deleterious mutation that causes culture degeneration in a filamentous fugus.