Reduced Leaf Water Loss Research Articles

A group III WRKY transcription factor, SlWRKY52, positively regulates drought tolerance in tomato

Drought severely limits crop growth and development, reducing productivity. WRKY transcription factors play crucial roles in regulating diverse aspects of plant growth, development, and responses to stress. Despite this, the functions of tomato WRKYs (SlWRKYs) in response to drought stress remain incompletely understood. Thus, we isolated SlWRKY52, a member of the group III SlWRKYs from the tomato variety ‘Ailsa Craig’, and investigated its role in drought stress responses. SlWRKY52 is an unstable hydrophilic protein chiefly located in the nucleus, which promoter region features cis-acting elements engaged in drought stress response. It was expressed in all tomato tissues and its expression was rapidly induced by drought stress. SlWRKY52 overexpression lines conferred enhanced drought stress resistance in tomato: its involved in abscisic acid (ABA) synthesis, resulted in rapid, ABA-dependent stomatal closure, reduced leaf water loss, lessened photosynthetic damage, higher antioxidant enzyme activity, decreased oxidative damage parameters, increased osmoregulatory substance content, accelerated reactive oxygen species scavenging, and increased expression of regulatory interaction, ABA biosynthesis, and abiotic stress response-related genes. Conversely, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease mutant lines of SlWRKY52 reduced drought stress tolerance. In conclusion, our findings suggest that SlWRKY52 acts as a positive regulator of drought tolerance in tomato and has the potential to be used as a target gene for further molecular breeding.

Suppression of SlDREB3 increases leaf ABA responses and promotes drought tolerance in transgenic tomato plants

Drought susceptibility is a major yield limiting factor in agricultural crops especially in hybrids/varieties that have been bred for high yields. We show that manipulation of the SlDREB3 gene in tomato alters ABA responses and thereby sensitivity of stomatal closure to ABA. SlDREB3 suppression lines show ABA hypersensitivity and rapid stomatal closure in response to ABA while over-expression lines show reduced sensitivity to ABA and open stomata even at high ABA levels with rapid water loss after 10 days of water stress. This is accompanied with high ROS levels and increased membrane damage due to senescence of leaves and drastically reduced survival in drought. The relative water content (RWC) of OEx lines is much reduced even when grown under well-watered conditions. In contrast, suppression lines show greater tolerance to water stress and almost complete survival to 10-day water stress. They show much reduced ROS levels, reduced membrane damage, higher RWC and reduced leaf water loss. These changes are associated with higher expression of ABA signalling pathway genes in suppression lines while these are highly reduced in OEx lines. The studies suggest that control of ABA signalling by SlDREB3 can help in withstanding severe drought.

Exogenous application of coronatine and alginate oligosaccharide to maize seedlings enhanced drought tolerance at seedling and reproductive stages

Plant growth regulators (PGRs) play crucial roles in maize drought tolerance. However, most of the studies are focused on seedling stage under greenhouse conditions, little is known on the effects of grain yields at later growth stage under field environment. It is hypothesized that exogenous application of PGRs can increase maize drought tolerance at different growth stages under field conditions. Here we used alginate oligosaccharide (AOS) and coronatine (COR) to investigate the effects of these two PGRs on maize drought tolerance in a two-year field experiment. The results showed that both COR and AOS treatments reduced leaf water loss and significantly increased the relative leaf water content by 12.6% and 13.0%, thus significantly decreasing superoxide anion accumulation by 49% and 42% in maize seedlings. There was no big difference between these two treatments in the seedling stage. In the subsequent reproductive growth period, although both COR and AOS treatments significantly increased leaf photosynthesis and above-ground growth, including photosynthetic rate by 41% and 33%, relative chlorophyll content (SPAD value) by 8.0% and 6.5%, leaf area by 22% and 15%, plant height by 8.3% and 5.9%, dry matter accumulation by 23% and 17%, respectively, under drought conditions, the effect of COR was superior to the AOS, as evidenced by membership function values of COR treatment were 0.12 and 0.08 larger than that of AOS treatment in 2020 and 2021, respectively. Ultimately, COR and AOS treatments significantly increased the grain yields by 21.7% and 16.2% in 2020, 22.3% and 15.7% in 2021, respectively, under drought conditions. Path analysis showed both PGRs increased final grain yields by increasing thousand-kernel weight (TKW). While the TKW increase with COR treatment was mainly due to the increase of relative chlorophyll content (SPAD value), AOS treatment mainly increased dry matter accumulation under drought conditions.

Wheat TaTIP4;1 Confers Enhanced Tolerance to Drought, Salt and Osmotic Stress in Arabidopsis and Rice.

Tonoplast aquaporins (intrinsic proteins, TIPs) have been indicated to play important roles in plant tolerance to water deficit and salinity. However, the functions of wheat TIPs in response to the stresses are largely unknown. In this study, we observed that transgenic plants overexpressing wheat TaTIP4;1 in Arabidopsis and rice displayed clearly enhanced seed germination and seedling growth under drought, salt and osmotic stress. Compared with wild type plants, Arabidopsis and rice overexpression lines had heightened water contents, reduced leaf water loss, lowered levels of Na+, Na+/K+, H2O2 and malondialdehyde, and improved activities of catalase and/or superoxide dismutase, and increased accumulation of proline under drought, salinity and/or osmotic stresses. Moreover, the expression levels of multiple drought responsive genes clearly elevated upon water dehydration, and the transcription of some salt responsive genes was markedly induced by NaCl treatment in the overexpression lines. Also, the yeast cells containing TaTIP4;1 showed increased tolerance to NaCl and mannitol, and mutation in one of three serines of TaTIP4;1 caused decreased tolerance to the two stresses. These results suggest that TaTIP4;1 serves as an essential positive regulator of seed germination and seedling growth under drought, salt and/or osmotic stress through impacting water relations, ROS balance, the accumulation of Na+ and proline, and stimulating the expression of dozens of stress responsive genes in Arabidopsis and rice. Phosphorylation may modulate the activity of TaTIP4;1.

Drought-Responsive NAC Transcription Factor RcNAC72 Is Recognized by RcABF4, Interacts with RcDREB2A to Enhance Drought Tolerance in Arabidopsis.

RcNAC72, a key transcription factor that may respond to drought stress in Rosa chinensis ‘Old Blush’, was selected in our previous study. In the present study, we found that RcNAC72 is localized in the nucleus and is a transcriptional activator. RcNAC72 expression could be significantly induced by drought, low temperature, salt as well as abscisic acid (ABA) treatment. Analysis of the promoter revealed that multiple abiotic stress and hormone response elements were located in the promoter region. The promoter could respond to drought, low temperature, salt and ABA treatments to activate GUS gene expression. Overexpressing RcNAC72 in Arabidopsis thaliana enhanced sensitivity to ABA and tolerance to drought stress. Silencing of RcNAC72 by virus-induced gene silencing (VIGS) in rose leaves significantly reduced leaf water loss tolerance and leaf extension capacity. Physical interaction of RcNAC72 with RcDREB2A was shown by means of the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. RcABF4 was demonstrated to be able to bind to the promoter of RcNAC72 by means of the yeast one-hybrid (Y1H) assay. These results provide new insights into the regulatory network of RcNAC72 response to drought stress in roses.

MdHB7-like confers drought tolerance and improves water-use efficiency through modulating stomatal density in apple (Malus domestica)

Improving plant drought tolerance and water-use efficiency (WUE) is important for plant survival and crop yield under conditions of water shortage. HD-Zip transcription factors (TFs) have been shown to help plants respond to drought. Here, MdHB7-like (an HD-Zip I TF) was identified as a drought-induced gene in apple plants. Transgenic apple with upregulated expression of MdHB7-like showed enhanced drought tolerance, whereas MdHB7-like RNA interference (RNAi) lines were more sensitive to drought. Further analysis showed that overexpression of MdHB7-like reduced stomatal density, but the stomatal density of MdHB7-like RNAi lines was increased. Transcriptome analysis showed that MdHB7-like overexpressing plants may reduce stomatal density by affecting the expression of stomatal development related genes, such as MdEPFL9.1 and MdEPFL9.2. The decrease in stomatal density reduced leaf water loss, helping to enhance the drought tolerance of overexpression lines. In addition, MdHB7-like overexpression enhanced drought tolerance of apple plants by promoting stomatal closure. Further study showed that the overexpression of MdHB7-like significantly increased WUE under long-term moderate drought conditions, which also related to the reduction of stomatal density. These results suggest that MdHB7-like is a valuable genetic resource for enhancing drought tolerance and increasing the WUE of apple germplasm.

The SlWRKY81 transcription factor inhibits stomatal closure by attenuating nitric oxide accumulation in the guard cells of tomato under drought

The WRKY transcription factors (TFs) play multifaceted roles in plant growth, development, and stress response. Previously, we found that SlWRKY81 negatively regulates tomato tolerance to drought; however, the mechanisms of stomatal regulation in response to drought remain largely unclear. Here, we showed that drought-induced upregulation in the SlWRKY81 transcripts induced photoinhibition and reduced the net photosynthetic rate in tomato leaves. However, silencing SlWRKY81 alleviated those inhibitions and minimized the drought-induced damage. A time-course of water loss showed that SlWRKY81 silencing significantly and consistently reduced leaf water loss, suggesting a role for SlWRKY81 in stomatal movement. Further analysis using light microscopy revealed that SlWRKY81 silencing significantly decreased stomatal aperture and increased the ratio of length to width of stomata under drought. Both biochemical assay and confocal laser scanning microscopy demonstrated that drought-induced upregulation in SlWRKY81 expression inhibited the nitric oxide (NO) accumulation in the guard cells, which was attributed to the simultaneous declines in the activity of nitrate reductase (NR) and NR expression in tomato leaves. The inspection of 3-kb sequences upstream of the predicted transcriptional start site of the NR identified three copies of the core W-box (TTGACC/T) sequence in the promoter region, indicating possible targets of SlWRKY81. Taken together, these data suggest that SlWRKY81 potentially represses NR transcription and thus reduces NO accumulation to attenuate stomatal closure and subsequent drought tolerance. These findings provide an improved understanding of the mechanism of WRKY-induced regulation of stomatal closure, which can be exploited in the future to enhance drought tolerance in crops.

Drought stress modify cuticle of tender tea leaf and mature leaf for transpiration barrier enhancement through common and distinct modes

Cuticle is the major transpiration barrier that restricts non-stomatal water loss and is closely associated with plant drought tolerance. Although multiple efforts have been made, it remains controversial what factors shape up the cuticular transpiration barrier. Previously, we found that the cuticle from the tender tea leaf was mainly constituted by very-long-chain-fatty-acids and their derivatives while alicyclic compounds dominate the mature tea leaf cuticle. The presence of two contrasting cuticle within same branch offered a unique system to investigate this question. In this study, tea seedlings were subjected to water deprivation treatment, cuticle structures and wax compositions from the tender leaf and the mature leaf were extensively measured and compared. We found that cuticle wax coverage, thickness, and osmiophilicity were commonly increased from both leaves. New waxes species were specifically induced by drought; the composition of existing waxes was remodeled; the chain length distributions of alkanes, esters, glycols, and terpenoids were altered in complex manners. Drought treatment significantly reduced leaf water loss rates. Wax biosynthesis-related gene expression analysis revealed dynamic expression patterns dependent on leaf maturity and the severity of drought. These data suggested that drought stress-induced structural and compositional cuticular modifications improve cuticle water barrier property. In addition, we demonstrated that cuticle from the tender leaf and the mature leaf were modified through both common and distinct modes.

A cytosolic NAD+-dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance

BackgroundPlant glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to produce glycerol-3-phosphate (G-3-P), and plays a key role in glycerolipid metabolism as well as stress responses.ResultsIn this study, we report the cloning, enzymatic and physiological characterization of a cytosolic NAD+-dependent GPDH from maize. The prokaryotic expression of ZmGPDH1 in E.coli showed that the enzyme encoded by ZmGPDH1 was capable of catalyzing the reduction of DHAP in the presence of NADH. The functional complementation analysis revealed that ZmGPDH1 was able to restore the production of glycerol-3-phosphate and glycerol in AtGPDHc-deficient mutants. Furthermore, overexpression of ZmGPDH1 remarkably enhanced the tolerance of Arabidopsis to salinity/osmotic stress by enhancing the glycerol production, the antioxidant enzymes activities (SOD, CAT, APX) and by maintaining the cellular redox homeostasis (NADH/NAD+, ASA/DHA, GSH/GSSG). ZmGPDH1 OE Arabidopsis plants also exhibited reduced leaf water loss and stomatal aperture under salt and osmotic stresses. Quantitative real-time RT-PCR analyses revealed that overexpression of ZmGPDH1 promoted the transcripts accumulation of genes involved in cellular redox homeostasis and ROS-scavenging system.ConclusionsTogether, these data suggested that ZmGPDH1 is involved in conferring salinity and osmotic tolerance in Arabidopsis through modulation of glycerol synthesis, stomatal closure, cellular redox and ROS homeostasis.

The Gene Encoding Subunit A of the Vacuolar H+-ATPase From Cotton Plays an Important Role in Conferring Tolerance to Water Deficit.

In plant cells, vacuolar H+-ATPases (V-ATPases) are responsible for deacidification of the cytosol and energisation of the secondary transport processes across the tonoplast. A number of V-ATPase subunit genes have been demonstrated to be involved in the regulation of the plant response to water deficit. However, there are no reports on the role of V-ATPase subunit A (VHA-A) in dehydration tolerance of cotton. In this study, cotton GhVHA-A gene was functionally characterized, especially with regard to its role in dehydration stress tolerance. Expression analysis showed that GhVHA-A was differentially expressed in various cotton organs and was induced by dehydration, low temperature, high salinity, and abscisic acid treatment in leaves. We also report that GhVHA-A improve dehydration tolerance in transgenic tobacco and cotton. Virus-induced gene silencing of GhVHA-A decreased the tolerance of cotton plantlets to dehydration stress. Silencing GhVHA-A decreased chlorophyll content and antioxidant enzyme activities and increased malondialdehyde (MDA) content in cotton under dehydration stress. However, transgenic tobacco expressing GhVHA-A exhibited enhanced dehydration resistance, resulting in reduced leaf water loss, higher average root length, and lower MDA levels under dehydration stress. Meanwhile, overexpression of GhVHA-A in tobacco conferred water deficit tolerance by enhancing osmotic adjustment (proline) and the activities of the antioxidant enzymes superoxide dismutase and peroxidase, thereby enhancing reactive oxygen species detoxification. These results suggest that GhVHA-A plays an important role in conferring resistance to dehydration stress. Our results have identified GhVHA-A as a candidate gene for improving dehydration tolerance in plants.

Overexpression of Nuclear Factor Y subunit StNF-YA7 enhances drought tolerance in potato

Drought, the most devastating abiotic stress, has detrimental effects on crop production. To minimize such adverse effects, it is crucial to improve drought tolerance in crops. Several members of the plant Nuclear Factor Y (NF-Y) gene family are involved in drought tolerance. In this study, we isolated the potato NF-Y subunit A 7 (StNF-YA7) gene from Solanum tuberosum ‘Superior’ and analyzed its relationship to abiotic stress tolerance. StNF-YA7 was induced by drought, salt stress, and abscisic acid (ABA), and a transient expression assay using 35S:StNF-YA7-smGFP showed that StNF-YA7 is localized to the nucleus, as reported for other plant NF-Ys. To examine the involvement of StNF-YA7 in drought tolerance, we generated 35S:StNF-YA7 transgenic potato plants. The transgenic potato plants exhibited enhanced drought tolerance, likely due to reduced leaf water loss. Analysis of the StNF-YA7 promoter (pStNF-YA7) fused to GUS revealed that StNF-YA7 is strongly expressed in vascular tissue. Taken together, these results suggest that enhancing drought tolerance via StNF-YA7 overexpression is associated with reduced leaf water loss, likely through mechanism(s) involving the vascular system.

Wheat bHLH-type transcription factor gene TabHLH1 is crucial in mediating osmotic stresses tolerance through modulating largely the ABA-associated pathway.

Wheat bHLH family gene TabHLH1 is responsive to drought and salt stresses, and it acts as one crucial regulator in mediating tolerance to aforementioned stresses largely through an ABA-associated pathway. Osmotic stresses are adverse factors for plant growth and crop productivity. In this study, we characterized TabHLH1, a gene encoding wheat bHLH-type transcription factor (TF) protein, in mediating plant adaptation to osmotic stresses. TabHLH1 protein contains a conserved basic-helix-loop-helix (bHLH) domain shared by its plant counterparts. Upon PEG-simulated drought stress, salt stress, and exogenous abscisic acid (ABA), the TabHLH1 transcripts in roots and leaves were induced. Under PEG-simulated drought stress and salt stress treatments, the tobacco seedlings with TabHLH1 overexpression exhibited improved growth and osmotic stress-associated traits, showing increased biomass and reduced leaf water loss rate (WLR) relative to wild type (WT). The transgenic lines also possessed promoted stomata closure under drought stress, salt stress, and exogenous ABA and increased proline and soluble sugar contents and reduced hydrogen peroxide (H2O2) amount under osmotic stress conditions, indicating that TabHLH1-mediated osmolyte accumulation and cellular ROS homeostasis contributed to the drought stress and salt stress tolerance. NtPYL12 and NtSAPK2;1, the genes encoding ABA receptor and SnRK2 family kinase, respectively, showed up-regulated expression in lines overexpressing TabHLH1 under osmotic stress and exogenous ABA conditions; overexpression of them conferred plants modified stomata movement, leaf WLR, and growth feature under drought and high salinity, suggesting that these ABA-signaling genes are mediated by wheat TabHLH1 gene and involved in regulating plant responses to simulated drought and salt stresses. Our investigation indicates that the TabHLH1 gene plays critical roles in plant tolerance to osmotic stresses largely through an ABA-dependent pathway.

The Functional and Regulatory Mechanisms of the Thellungiella salsuginea Ascorbate Peroxidase 6 (TsAPX6) in Response to Salinity and Water Deficit Stresses.

Soil salinization is a resource and ecological problem in the world. Thellungiella salsuginea is becoming a new model plant because it resembles its relative species, Arabidopsis thaliana, in small genome and short life cycle. It is highly tolerant to salinity and drought stresses. Ascorbate peroxidase (APX) is an enzyme that clears H2O2 in plants. The function and molecular and regulation mechanisms of APX in T. salsuginea have rarely been reported. In this study, an APX gene, TsApx6, was cloned from T. salsuginea and its responses to abiotic stresses in transgenic Arabidopsis were studied. Under high salinity treatment, the expression of TsApx6 was significantly induced. Under drought treatment, overexpression of TsApx6 increased the survival rate and reduced leaf water loss rate in Arabidopsis. Compared to the wild type plants, high salinity treatment reduced the concentrations of MDA, H2O2 and proline but elevated the activities of APX, GPX, CAT and SOD in the TsApx6-overexpressing plants. Meanwhile, germination rate, cotyledon greening, and root length were improved in the transgenic plants compared to the wild type plants under salt and water deficit conditions. Based on these findings, TsApx6 has an important function in the resistance of plants to certain abiotic stresses. The TsApx6 promoter sequence was obtained using Genome Walking technology. Bioinformatics analysis indicated that it contains some cis-acting elements related to stress response. The treatments of salt, dehydration, and ABA induced the expression of Gus gene under the regulation of the TsApx6 promoter. Mutation analysis showed that the MBS motif present in the TsApx6 promoter might be a key negative regulatory element which has an important effect on the growth and developmental process of plants.

Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana.

MiRNAs play crucial roles in many aspects of plant development and the response to the environment. The miR172 family has been shown to participate in the control of flowering time and the response to abiotic stress. This family regulates the expression of APETALA2 (AP2)-like transcription factors in Arabidopsis. In the present study, soybean (Glycine max L. Merr.) miR172c, a member of the miR172 family, and its target gene were investigated for abiotic stress responses in transgenic Arabidopsis. gma-miR172c was induced by abscisic acid (ABA) treatments and abiotic stresses, including salt and water deficit. 5'-RACE (5'-rapid amplification of cDNA ends) assays indicated that miR172c directed Glyma01g39520 mRNA cleavage in soybeans. Overexpression of gma-miR172c in Arabidopsis resulted in reduced leaf water loss and increased survival rate under stress conditions. Meanwhile, the root length, germination rate, and cotyledon greening of transgenic plants were improved during both high salt and water deficit conditions. In addition, transgenic plants exhibited hypersensitivity to ABA during both the seed germination and post-germination seedling growth stages. Stress-related physiological indicators and the expression of stress/ABA-responsive genes were affected by abiotic treatments. The overexpression of gma-miR172c in Arabidopsis promoted earlier flowering compared with the wild type through modulation of the expression of flowering genes, such as FT and LFY during long days, especially under drought conditions. Glyma01g39520 weakened ABA sensitivity and reduced the tolerance to drought stress in the snz mutant of Arabidopsis by reducing the expression of ABI3 and ABI5. Overall, the present results demonstrate that gma-miR172c confers water deficit and salt tolerance but increased ABA sensitivity by regulating Glyma01g39520, which also accelerates flowering under abiotic stresses.

Over-expression of ApKUP3 enhances potassium nutrition and drought tolerance in transgenic rice

ApKUPs are typical high-affinity potassium (K+) transporters of Alternanthera philoxeroides which are involved in its response to K+ starvation and abiotic stresses. In this study, the overexpression of ApKUP3 gene in rice resulted in enhanced K+ nutrition and drought tolerance of transgenic plants. Compared with wild-type (WT) plants, the transgenic plants showed a better growth performance and a strengthened K+ accumulation under different K+ supplies. The ApKUP3 overexpression in the rice plants also enhanced tolerance to a drought stress, as evidenced by a reduced leaf water loss and an increased total leaf chlorophyll content, stomatal conductance, net photosynthetic rate, and activities of superoxide dismutase, peroxidase, and ascorbate peroxidase (APX). Moreover, the transcription of genes involved in the antioxidation defense system were higher in the transgenic plants than in the WT plants upon the drought stress.

Root-zone CO<sub>2</sub> and root-zone temperature effects on photosynthesis and nitrogen metabolism of aeroponically grown lettuce (Lactuca sativa L.) in the tropics

Effects of elevated root-zone (RZ) CO2 concentration (RZ [CO2]) and RZ temperature (RZT) on photosynthesis, productivity, nitrate (NO3 −), total reduced nitrogen (TRN), total leaf soluble and Rubisco proteins were studied in aeroponically grown lettuce plants in a tropical greenhouse. Three weeks after transplanting, four different RZ [CO2] concentrations (ambient, 360 ppm, and elevated concentrations of 2,000; 10,000; and 50,000 ppm) were imposed on plants at 20°C-RZT or ambient(A)-RZT (24–38°C). Elevated RZ [CO2] resulted in significantly higher light-saturated net photosynthetic rate, but lower light-saturated stomatal conductance. Higher elevated RZ [CO2] also protected plants from both chronic and dynamic photoinhibition (measured by chlorophyll fluorescence Fv/Fm ratio) and reduced leaf water loss. Under each RZ [CO2], all these variables were significantly higher in 20°C-RZT plants than in A-RZT plants. All plants accumulated more biomass at elevated RZ [CO2] than at ambient RZ [CO2]. Greater increases of biomass in roots than in shoots were manifested by lower shoot/root ratios at elevated RZ [CO2]. Although the total biomass was higher at 20°C-RZT, the increase in biomass under elevated RZ [CO2] was greater at A-RZT. Shoot NO3 − and TRN concentrations, total leaf soluble and Rubisco protein concentrations were higher in all elevated RZ [CO2] plants than in plants under ambient RZ [CO2] at both RZTs. Under each RZ [CO2], total leaf soluble and Rubisco protein concentrations were significantly higher at 20°C-RZT than at A-RZT. Our results demonstrated that increased P Nmax and productivity under elevated [CO2] was partially due to the alleviation of midday water loss, both dynamic and chronic photoinhibition as well as higher turnover of Calvin cycle with higher Rubisco proteins.

GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins. In this study, we identified and characterized a gene, GmNFYA3, which encodes the NF-YA subunit of the NF-Y complex in soybeans (Glycine max L.). Real time RT-PCR analysis indicated that GmNFYA3 was induced by abscisic acid (ABA) and abiotic stresses, such as polyethylene glycol, NaCl and cold. Subcellular localization analysis suggested that GmNFYA3 may activate its specific targets in the nucleus. Histochemical β-glucuronidase (GUS) staining revealed that the expression of the GUS gene driven by the GmNFYA3 promoter occurred in various transgenic Arabidopsis tissues. Coexpression in Nicotiana benthamiana and 5' RACE assays indicated that miR169 directs GmNFYA3 mRNA cleavage in vivo. Overexpression of GmNFYA3 resulted in Arabidopsis with reduced leaf water loss and enhanced drought tolerance. In addition, the transgenic Arabidopsis exhibited increased sensitivity to high salinity and exogenous ABA. Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI1, ABI2) and stress-responsive genes, including RD29A and CBF3, were generally higher in GmNFYA3 plants than in wild-type controls under normal conditions. These results suggest that the GmNFYA3 gene functions in positive modulation of drought stress tolerance and has potential applications in molecular breeding to enhance drought tolerance in crops.

The Arabidopsis NFYA5 Transcription Factor Is Regulated Transcriptionally and Posttranscriptionally to Promote Drought Resistance

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:beta-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress-responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

Evidence for the involvement of nitric oxide and reactive oxygen species in osmotic stress tolerance of wheat seedlings: Inverse correlation between leaf abscisic acid accumulation and leaf water loss

Nitric oxide (NO) and reactive oxygen species (ROS) play important roles in both abscisic acid (ABA) signaling and stress-induced ABA accumulation. However, little is known about their physiological roles in the whole plant. In this study, the effects of NO and ROS on leaf water control and the roles of ABA were determined using wheat (Triticum aestivum L.) seedlings. As compared with the control, osmotic stress reduced leaf water loss (LWL) while it increased leaf ABA content. The effects of osmotic stress on LWL and ABA contents were partially reversed by NO scavengers or NO synthase (NOS) inhibitors. Furthermore, sodium nitroprusside (SNP) at concentrations between 0.01 and 10 mM all reduced LWL efficiently and induced ABA accumulation in a dose-dependent manner. When ABA synthesis was inhibited by fluridone or actidione, the effects of SNP on LWL were partially reversed. These results suggest that NO is involved in leaf water maintenance of wheat seedlings under osmotic stress, and one of the possible mechanisms is by stimulating ABA synthesis. The ROS scavengers used in our experiments had no effects on either LWL or ABA accumulation induced by osmotic stress. However, all ROS induced LWL reduction and ABA accumulation significantly. Hydrogen peroxide had the same effects as SNP on LWL and induced ABA accumulation in a dose-dependent manner but had a maximal effect at 1 mM. Fluridone reversed the effects of H2O2 on both LWL reduction and ABA accumulation, while actidione had no effect. These results suggest that ROS are also involved in leaf water maintenance of wheat seedlings by stimulating ABA biosynthesis, but with a different mechanism to that of NO. The ABA-independent mechanism in NO/ROS regulation of leaf water balance is discussed, in relation to our results.

The control of leaf water loss by coconut plants cultured in vitro depends on the type of membrane used for ventilation

SummaryThe physiological responses to vessel ventilation of coconut plants cultured in vitro varied with the type of membrane used for ventilation. Plants cultured in vessels ventilated with one of three types of membranes, polypropylene (PP), polyvinyl chloride (PVC) or Whatman filter paper (W) were compared with plants cultured in conventional sealed (S) vessels. Vessel gas exchange, water loss and medium osmolality were greater in vessels ventilated with W compared with vessels ventilated with PP, PVC or S. Plants cultured in W vessels exhibited lower rates of leaf water loss, narrower stomatal apertures and higher leaf abscisic acid (ABA) concentrations than plants cultured in PP, PVC or S vessels. Plants in PP and PVC vessels showed an intermediate behaviour in all these parameters while plants cultured in S vessels exhibited comparatively high rates of leaf water loss, wide stomatal apertures and low leaf ABA concentrations. The results from the plants cultured in W vessels suggest that dehydration of the vessel medium increased medium osmolality, inducing greater leaf ABA accumulation, which resulted in improved stomatal control and reduced leaf water loss in coconut plants cultured in vitro in the vessels. The use of PP and PVC membranes resulted only in intermediate control of leaf water loss and stomatal functionality compared with S vessels. This occurred despite PP and PVC vessels having similar rates of medium water loss and medium osmolalities compared with S vessels. Because PP and PVC had higher rates of ethylene diffusion, it would appear as if the slight improvement of leaf physiology between plants in PP and PVC vessels and S vessels may be related to differences in ethylene or other gas diffusion rather than differences in medium water loss. In any case, the capacity of plants cultured in vitro to control leaf water loss was related to their leaf ABA concentration. The results of the present work confirm that the use of ventilated systems in the in vitro preacclimatization phase before transplanting results in plants with improved control of water loss that may prove beneficial for survival and performance in the field but care must be taken when selecting the type of ventilation membrane.