ABA Stress Research Articles

Molecular cloning and expression of AmCDPK from mangrove Avicennia marina under elevated temperature.

Considered as an essential calcium sensor, the calcium-dependent protein kinase (CDPK) family plays a critical part in terrestrial plants' responses to both biotic and abiotic stresses. In the study, Avicennia marina was proved to have better heat tolerance than other species. A CDPK gene was cloned from mangrove species A. marina using RACE-PCR and designated as AmCDPK. By predicting and analyzing its properties, structures and expression patterns, we found that the amino acid sequence, containing a kinase domain and four EF-hand Ca2+-binding sites, shared high identity with Handroanthus impetiginosus and Sesamum indicum. Quantitative real-time PCR data analysis suggested that AmCDPK demonstrated significant up-regulation under heat stress. It is likely that AmCDPK is a versatile gene involved in various stresses, including dehydration, cold, light, defense and ABA stress responses by analyzing cis-elements. It is the first time that CDPKs from mangroves have been cloned and our results brought evidence to the effect of AmCDPK on heat stress, which is particularly important under the background of global warming.

The Maize AP2/EREBP Transcription Factor ZmEREB160 Enhances Drought Tolerance in Arabidopsis

Drought is a main factor affecting plant growth and crop production. APETALA2/Ethylene Responsive Element Binding Protein (AP2/EREBP) transcription factors (TFs) are involved in the response to abiotic stress and their functions in ABA signaling and the regulation of drought response have been intensively studied. However, AP2/EREBP TFs have been limited in the maize. The objective of our study was to identify the function of the novel maize AP2/EREBP gene ZmEREB160. Expression levels analysis in maize revealed that ZmEREB160 is significantly induced by PEG6000, NaCl and ABA treatment. ZmEREB160 localized to the nucleus when transiently expressed in Arabidopsis leaf protoplasts. ZmEREB160 activated the reporter gene and exhibited transcriptional activation activity in yeast cells. When overexpression of ZmEREB160 in Arabidopsis significantly enhanced tolerance to osmotic and ABA stress, overexpressed seedlings were longer roots under ABA and mannitol treatments compared with wild type. In addition, overexpression of ZmEREB160 Arabidopsis seedlings was found to elevate survival rate compared with wild type plants under drought stress. During the drought treatment, qRT-PCR assays showed that the expression levels of ABA/drought stress-related genes, ABI2, ABI5, COR15, DREB2A and RD29B were up-regulated in ZmEREB160 transgenic plants, transgenic plants accumulated more proline content than wild type plants. These results indicate that ZmEREB160 functions in response to drought and ABA stresses, and participates in ABA signaling pathway and may enhance drought tolerance.

DEAR4, a Member of DREB/CBF Family, Positively Regulates Leaf Senescence and Response to Multiple Stressors in Arabidopsis thaliana.

Leaf senescence is a programmed developmental process regulated by various endogenous and exogenous factors. Here we report the characterization of the senescence-regulating role of DEAR4 (AT4G36900) from the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) family in Arabidopsis. The expression of DEAR4 is associated with leaf senescence and can be induced by ABA, JA, darkness, drought and salt stress. Transgenic plants over-expressing DEAR4 showed a dramatically enhanced leaf senescence phenotype under normal and dark conditions while the dear4 knock-down mutant displayed delayed senescence. DEAR4 over-expressing plants showed decreased seed germination rate under ABA and salt stress conditions as well as decreased drought tolerance, indicating that DEAR4 was involved in both senescence and stress response processes. Furthermore, we found that DEAR4 protein displayed transcriptional repressor activities in yeast cells. DEAR4 could directly repress the expression of a subset of COLD-REGULATED (COR) and RESPONSIVE TO DEHYDRATION (RD) genes which have been shown to be involved in leaf longevity and stress response. Also we found that DERA4 could induce the production of Reactive oxygen species (ROS), the common signal of senescence and stress responses, which gives us the clue that DEAR4 may play an integrative role in senescence and stress response via regulating ROS production.

Molecular cloning and functional analysis of a cold-regulated gene Wcor18 in wheat

To explore the function of COR gene in wheat, Wcor18 was cloned from wheat cultivar Jing 841. Expression level of Wcor18 increased with vernalization time prolongation. The full length of Wcor18 gene is 498 bp and encodes 165 amino acids. The predicted Wcor18 protein is a type of unstable hydrophilic protein and is prone to be hydrolyzed and to be oxidized. Gene structure analysis and phylogenetic tree analysis showed Wcor18 gene contains two exons and coding proteins are quite conservative among the donor ancestors of common wheat. Wcor18 can quickly respond to low temperature, high salt, drought, and ABA stresses at wheat seedling stage. The highly expression of Wcor18 gene under the − 15 °C and the rapid response of Wcor18 gene under several stress conditions indicated that the gene may play many roles in stress response of wheat.

Genome-wide identification and expression analysis of NADPH oxidase genes in response to ABA and abiotic stresses, and in fibre formation in Gossypium.

Plasma membrane NADPH oxidases, also named respiratory burst oxidase homologues (Rbohs), play pivotal roles in many aspects of growth and development, as well as in responses to hormone signalings and various biotic and abiotic stresses. Although Rbohs family members have been identified in several plants, little is known about Rbohs in Gossypium. In this report, we characterized 13, 13, 26 and 19 Rbohs in G. arboretum, G. raimondii, G. hirsutum and G. barbadense, respectively. These Rbohs were conservative in physical properties, structures of genes and motifs. The expansion and evolution of the Rbohs dominantly depended on segmental duplication, and were under the purifying selection. Transcription analyses showed that GhRbohs were expressed in various tissues, and most GhRbohs were highly expressed in flowers. Moreover, different GhRbohs had very diverse expression patterns in response to ABA, high salinity, osmotic stress and heat stress. Some GhRbohs were preferentially and specifically expressed during ovule growth and fiber formation. These results suggest that GhRbohs may serve highly differential roles in mediating ABA signaling, in acclimation to environmental stimuli, and in fiber growth and development. Our findings are valuable for further elucidating the functions and regulation mechanisms of the Rbohs in adaptation to diverse stresses, and in growth and development in Gossypium.

Functional Analysis of <i>Arabidopsis thaliana</i> Galactinol Synthase <i>AtGolS2</i> in Response to Abiotic Stress

Soil salt-alkalization is one of the adverse factors limiting crop yields. Identification of key salt-alkaline tolerant genes is of great significance for molecular breeding of stress-resistant crops. In this study, a T-DNA insertion Arabidopsis mutant atgols2 showing higher sensitivity to bicarbonate salt-alkaline stress was screened out against NaHCO 3 treatment. Further bioinformatic analysis revealed that the AtGolS2 gene encoded a galactinol synthase, which is a member of the glycosyltransferase family A superfamily. We predicted the protein interaction network of AtGolS2 via SMART online analysis, and found that these AtGolS2 interacting proteins were related to lipid metabolism, galactose biosynthesis and raffinose biosynthesis, and participated in abiotic stress responses. By using the online expression data, we showed that AtGolS2 expression responded to salt, osmotic, drought and ABA stress. PCR amplification by using the three primers method verified the homozygous T-DNA insertion in atgols2. Phenotypic assays further uncovered that atgols2 mutant was more sensitive to high salt, osmotic and ABA stresses than the wild type Arabidopsis. Taken together, results in this study revealed the positive function of AtGolS2 in bicarbonate salt-alkaline, high salt, osmotic and ABA stresses, which will facilitate further research regarding the function and molecular mechanism of the GolS family genes in stress responses.&nbsp

ABA signaling is negatively regulated by GbWRKY1 through JAZ1 and ABI1 to affect salt and drought tolerance.

GbWRKY1 can function as a negative regulator of ABA signaling via JAZ1 and ABI1, with effects on salt and drought tolerance. WRKY transcription factors play important roles in plant development and stress responses. GbWRKY1 was initially identified as a defense-related gene in cotton and negatively regulates the response to fungal pathogens by activating the expression of JAZ1. Here, we characterized the role of GbWRKY1, an orthologue of the Arabidopsis gene AtWRKY75, in abiotic stress (salt and drought) and established novel connection between JAZ1 and ABA signaling in Arabidopsis. GbWRKY1 is nucleus localized and its expression is significantly induced by treatment with ABA and osmotic stresses NaCl and PEG. Increased levels of expression of GbWRKY1 in transgenic Arabidopsis enhance sensitivity to salt and drought as revealed by seed germination tests and soil stress experiments. Similarly, GbWRKY1 overexpression cotton plants also display increased sensitivity to PEG treatment and drought. Expression analysis shows that the induction of two ABA responsive genes, RAB18 and RD29A by NaCl, mannitol, and ABA treatment is significantly impaired in GbWRKY1 overexpression Arabidopsis lines. GbWRKY1 overexpression Arabidopsis displays a strong ABA-insensitive phenotype at both germination and early stages of seedling development. Further genetic evidence suggested that the ABA-insensitive phenotype of GbWRKY1 overexpression Arabidopsis was dependent on JAZ1, and overexpression of JAZ1 also displayed an ABA-insensitive phenotype. In addition, yeast two hybrid and bimolecular fluorescence complementation assays showed that JAZ1 directly interacts with ABI1, a key negative regulator of ABA signaling. We, therefore, demonstrate that GbWRKY1 acts as a negative regulator of ABA signaling, through an interaction network involving JAZ1 and ABI1, to regulate salt and drought tolerance.

Foxtail millet (Setaria italica (L.) P. Beauv) CIPKs are responsive to ABA and abiotic stresses.

CBL-interacting protein kinases (CIPKs) have been shown to regulate a variety of environmental stress-related signalling pathways in plants. Foxtail millet (Setaria italica (L.) P. Beauv) is known worldwide as a relatively stress-tolerant C4 crop species. Although the foxtail millet genome sequence has been released, little is known about the functions of CIPKs in foxtail millet. Therefore, a systematic genome-wide analysis of CIPK genes in foxtail millet was performed. In total, 35 CIPK members were identified in foxtail millet and divided into four subgroups (I to IV) on the basis of their phylogenetic relationships. Phylogenetic and gene structure analyses clearly divided all SiCIPKs into intron-poor and intron-rich clades. Cis-element analysis subsequently indicated that these SiCIPKs may be involved in responses to abiotic stimuli, hormones, and light signalling during plant growth and development, and stress-induced expression profile analysis revealed that all the SiCIPKs are involved in various stress signalling pathways. These results suggest that the CIPK genes in foxtail millet exhibit the basic characteristics of CIPK family members and play important roles in response to abiotic stresses. The results of this study will contribute to future functional characterization of abiotic stress responses mediated by CIPKs in foxtail millet.

Genome-wide identification and functional analysis of the WDR protein family in potato.

WD-repeat (WDR) proteins are highly abundant and participate in a seemingly wide range of interactions and cellular functions acting as scaffolding molecules. However, WDR identification in potato has not been conducted so far. In this study, we demonstrated the presence of at least 168 WDR genes in potato (Solanum tuberosum L.) which can be subdivided into five discrete clusters (Cluster I-V) and 10 classes inferred from their phylogenetic features of the constituent genes and the distribution of domains. These genes are distributed on all 12 chromosomes, of which chromosome 3 carries the most genes with 26 StWDRs. The expression of potato WDR genes showed tissue specificity with a high expression in carpels, callus and roots, and the expression patterns were obviously different among different genes. Transcript profiling of 168 StWDR genes revealed the particular tissues in which the 168 StWDR are expressed, and displayed a high expression in carpels, callus and roots. Most StWDRs were modulated by salt, ABA and Verticillium dahliae stresses, of which StWD092 was found to be highly expressed under all the three stresses. These outcomes revealed the intricate crosstalk between WDRs and other regulatory networks in the event of adverse milieu.

Molecular cloning and functional characterization of a sweetpotato chloroplast IbDHAR3 gene in response to abiotic stress

Dehydroascorbate reductase (DHAR) plays a critical role in the regeneration of ascorbic acid (AsA), and widely involved in plant tolerance to biotic and abiotic stresses. In this study, the IbDHAR3 gene was cloned from sweetpotato cultivar Xushu 18 by RT-PCR. The full-length of this gene was 813 bp which encodes 270 amino acids. The IbDHAR3 protein contained two conserved domains of glutathione S-transferase (GST) and GST-C-DHAR, and one chloroplast transit peptide with 52 amino acids length. Transient expression in tobacco leaf epidermal cells indicated that IbDHAR3 protein is subcellular localized to chloroplast. The qRT-PCR results revealed that the relative expression level of IbDHAR3 in leaves is much higher than that in other tissues, and could be up-regulated by ABA, drought, salinity, and high-temperature stresses. The seed germination rate and root elongation were increased in contrast to wild type under mannitol and NaCl stresses in T3 transgenic Arabidopsis overexpressing IbDHAR3 gene. The soil drought experiments showed that the overexpression of IbDHAR3 gene in Arabidopsis reduced the malondialdehyde (MDA) and the H2O2 content, enhanced the level of AtGR gene expression, superoxide dismutase (SOD), ascorbate peroxidase (APX), DHAR activity, and the AsA content. Therefore, overexpression of IbDHAR3 gene could enhance the ability of scavenging reactive oxygen species such as H2O2 by promoting AsA-glutathione cycle and related antioxidant enzymes system, thereby contributing to increased stress tolerance in Arabidopsis.

Screening of abiotic stress-responsive cotton genes using a cotton full-length cDNA overexpressing Arabidopsis library.

Cotton (Gossypium hirsutum L.) is a major crop and the main source of natural fiber worldwide. Because various abiotic and biotic stresses strongly influence cotton fiber yield and quality, improved stress resistance of this crop plant is urgently needed. In this study, we used Gateway technology to construct a normalized full-length cDNA overexpressing (FOX) library from upland cotton cultivar ZM12 under various stress conditions. The library was transformed into Arabidopsis to produce a cotton-FOX-Arabidopsis library. Screening of this library yielded 6,830 transgenic Arabidopsis lines, of which 757 were selected for sequencing to ultimately obtain 659 cotton ESTs. GO and KEGG analyses mapped most of the cotton ESTs to plant biological process, cellular component, and molecular function categories. Next, 156 potential stress-responsive cotton genes were identified from the cotton-FOX-Arabidopsis library under drought, salt, ABA, and other stress conditions. Four stress-related genes identified from the library, designated as GhCAS, GhAPX, GhSDH, and GhPOD, were cloned from cotton complementary DNA, and their expression patterns under stress were analyzed. Phenotypic experiments indicated that overexpression of these cotton genes in Arabidopsis affected the response to abiotic stress. The method developed in this study lays a foundation for high-throughput cloning and rapid identification of cotton functional genes.

Genome-wide analysis of sulfotransferase genes and their responses to abiotic stresses in Chinese cabbage (Brassica rapa L.).

Sulfotransferases (SOTs; EC 2.8.2.-), which are widespread from prokaryotes to eukaryotes, constitute a multi-protein family that plays crucial roles in plant growth, development and stress adaptation. However, this family has not been systemically investigated in Brassica rapa. Here, a genome-wide systemic analysis of SOT genes in B. rapa subsp. pekinensis, a globally cultivated vegetable, were conducted. We identified 56 SOT genes from the whole B. rapa genome using Arabidopsis SOT sequences as queries and classified them into nine groups, rather than the eight groups of previous research. 56 B. rapa SOT genes (BraSOTs) were distributed on all 10 chromosomes except for chromosome 5. Of these, 27 BraSOTs were distributed in seven clusters on five chromosomes (ChrA01, ChrA02, Chr03, ChrA07, and Chr09). Among the BraSOT proteins, 48 had only one SOT_1 domain and 6 had two, while 2 had one SOT_3 domain. Additionally, 47 BraSOT proteins contained only known SOT domains. The remaining nine proteins, five in group-VIII and two in group-IX, contained additional transmembrane domains. Specific motif regions I and IV for 3′-phosphoadenosine 5′-phosphosulfate binding were found in 41 BraSOT proteins. Introns were present in only 18 BraSOT genes, and all seven BraSOT genes in groups VIII and IX had more than three introns. To identify crucial SOTs mediating the response to abiotic stress in B. rapa, expression changes in 56 BraSOT genes were determined by quantitative RT-PCR after drought, salinity, and ABA treatments, and some BraSOT genes were associated with NaCl, drought and ABA stress, e.g. Bra017370, Bra009300, Bra027880.

Genome-wide characterization of the AP2/ERF gene family in radish (Raphanus sativus L.): Unveiling evolution and patterns in response to abiotic stresses

Main conclusionAmong 247 RsAP2/ERF identified, the majority of the 21 representatives were preferably expressed under drought and heat while suppressed under heavy metals, indicating their potential roles in abiotic stress responses and tolerance.APETALA2/Ethylene-Responsive factor (AP2/ERF) transcription factor (TF) is one of the largest gene families in plants that play a fundamental role in growth and development as well as biotic and/or abiotic stresses responses. Although AP2/ERFs have been extensively characterized in many plant species, little is known about this family in radish, which is an important root vegetable with various medicinal properties. The available genome provides valuable opportunity to identify and characterize the global information on AP2/ERF TFs in radish. In this study, a total of 247 ERF family genes were identified from the radish genome, and sequence alignment and phylogenetic analyses classified the AP2/ERF superfamily into five groups (AP2, ERF, DREB, RAV and soloist). Motif analysis showed that other than AP2/ERF domains, other conserved regions were selectively distributed among different clades in the phylogenetic tree. Chromosome location analysis showed that tandem duplication may result in the expansion of RsAP2/ERF gene family. The RT-qPCR analysis confirmed that a proportion of AP2/ERF genes were preferably expressed under drought and heat stresses, whereas they were suppressed under the ABA and heavy metal stresses. These results provided valuable information for further evolutionary and functional characterization of RsAP2/ERF genes, and contributed to genetic improvement of stress tolerances in radish and other root vegetable crops.

Novel Maize NAC Transcriptional Repressor ZmNAC071 Confers Enhanced Sensitivity to ABA and Osmotic Stress by Downregulating Stress-Responsive Genes in Transgenic Arabidopsis.

NAC TFs play crucial roles in response to abiotic stresses in plants. Here, ZmNAC071 was identified as a nuclear located transcriptional repressor. Overexpression of ZmNAC071 in Arabidopsis enhanced sensitivity of transgenic plants to ABA and osmotic stress. The expression levels of SODs, PODs, P5CSs,and AtMYB61 were inhibited by ZmNAC071, which results in reduced ROS scavenging and proline content, increased ROS level, and water loss. Besides, the expression levels of some ABA or abiotic stress-related genes, like ABIs, RD29A, DREBs, and LEAs were also significantly inhibited by ZmNAC071. Yeast one-hybrid assay demonstrated that ZmNAC071 specifically bound to the cis-acting elements containing CGT[G/A] core sequences in the promoter of stress-related genes, suggesting that ZmNAC071 may participate in the regulation of transcription of these genes through recognizing the core sequences CGT[G/A]. These results will facilitate further studies concerning the cis-elements and downstream genes targeted by ZmNAC071 in maize.

PGPR-induced OsASR6 improves plant growth and yield by altering root auxin sensitivity and the xylem structure in transgenic Arabidopsis thaliana

Plant-growth-promoting rhizobacteria (PGPR) improve plant growth by altering the root architecture, although the mechanisms underlying this alteration have yet to be unravelled. Through microarray analysis of PGPR-treated rice roots, a large number of differentially regulated genes were identified. Ectopic expression of one of these genes, OsASR6 (ABA STRESS RIPENING6), had a remarkable effect on plant growth in Arabidopsis. Transgenic lines over-expressing OsASR6 had larger leaves, taller inflorescence bolts and greater numbers of siliques and seeds. The most prominent effect was observed in root growth, with the root biomass increasing four-fold compared with the shoot biomass increase of 1.7-fold. Transgenic OsASR6 over-expressing plants showed higher conductance, transpiration and photosynthesis rates, leading to an ˜30% higher seed yield compared with the control. Interestingly, OsASR6 expression led to alterations in the xylem structure, an increase in the xylem vessel size and altered lignification, which correlated with higher conductance. OsASR6 is activated by auxin and, in turn, increases auxin responses and root auxin sensitivity, as observed by the increased expression of auxin-responsive genes, such as SAUR32 and PINOID, and the key auxin transcription factor, ARF5. Collectively, these phenomena led to an increased root density. The effects of OsASR6 expression largely mimic the beneficial effects of PGPRs in rice, indicating that OsASR6 activation may be a key factor governing PGPR-mediated changes in rice. OsASR6 is a potential candidate for the manipulation of rice for improved productivity.

The MATH-BTB BPM3 and BPM5 subunits of Cullin3-RING E3 ubiquitin ligases target PP2CA and other clade A PP2Cs for degradation

Early abscisic acid signaling involves degradation of clade A protein phosphatases type 2C (PP2Cs) as a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. At later steps, ABA induces up-regulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. It has been demonstrated that ABA induces the degradation of existing ABI1 and PP2CA through the PUB12/13 and RGLG1/5 E3 ligases, respectively. However, other unidentified E3 ligases are predicted to regulate protein stability of clade A PP2Cs as well. In this work, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric cullin3 (CUL3)-RING-based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2, and HAB1. BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity, whereas bpm3 bpm5 plants show increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Using biochemical and genetic assays, we demonstrated that ubiquitination of PP2CA depends on BPM function. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C coreceptor levels to regulate early ABA signaling as well as the later desensitizing-resetting steps.

Transcription factor WRKY30 mediates resistance to Cucumber mosaic virus in Arabidopsis

WRKY transcription factors are key regulators in regulating abiotic or biotic stress response in Arabidopsis. Previous studies showed that WRKY30 expression was induced by oxidative stress treatment, fungal elicitor, SA and ABA. However, functions of WRKY30 on viral defense are not well studied. Here, we found that Arabidopsis WRKY DNA binding protein 30 (WRKY30) plays essential roles in regulating Cucumber mosaic virus (CMV) resistance. The expression of WRKY30 was induced by CMV infection and wrky30 mutant displayed more susceptibility (including higher oxidative damages, induced reactive oxygen species synthesis and more PSII photochemistry compromise), while WRKY30 overexpression plants (WRKY30OX) exhibited more resistance to CMV infection. Moreover BRs-induced CMV tolerance is partly dependent on WRKY30. And WRKY30 expression increased after BL treatment. All these demonstrated that WRKY30 works as a positive regulator in plant CMV resistance process.

LlDREB1G, a novel DREB subfamily gene from Lilium longiflorum, can enhance transgenic Arabidopsis tolerance to multiple abiotic stresses

In plants, most DREB1s/CBFs proteins with a single AP2/ERF domain participate in responses to abiotic stresses. Here, we cloned a DREB subfamily gene from Lilium longiflorum (designated as LlDREB1G). Sequence analyses revealed that it is composed of 219 amino acids, and contains an AP2/ERF domain, a putative nuclear localization signal (NLS), a DSAW/R and LWSY motif. LlDREB1G, which was localized to the nucleus, showed strongly induced expression under low temperature, and was also stimulated by mechanical agitation, heat shock, ABA and H2O2 stresses. Moreover, the LlDREB1G gene had an obvious circadian rhythm and organ specificity in expression. The LlDREB1G transcription factor was functional in the heterologous system (Arabidopsis). Transgenic Arabidopsis plants with higher transcription of LlDREB1G gene exhibited increased acquired thermotolerance and resistance to freezing and dehydration stresses. Besides, the transgenic seeds were insensitive to exogenous ABA treatment than those of the wild type (WT) in germination experiments. Compared with the WT seedlings, the seedlings of transgenic Arabidopsis overexpressing LlDREB1G showed strongly upregulated expression of stress-related genes after cold, drought or acclimation to heat shock treatments. Furthermore, overexpression of LlDREB1G increased the proline levels and survival rates, but decreased the electrolyte leakage and H2O2 accumulation in the transgenic plants after freezing, dehydration and high temperature treatments. These results imply that LlDREB1G gene may be involved in the responses to multiple abiotic stresses in an ABA-dependent and -independent manner. LlDREB1G gene, a DREB subfamily gene from Lilium longiflorum, may be involved in the responses to multiple abiotic stresses in an ABA-dependent and -independent manner.

Development of Agrobacterium-mediated transient expression system in Caragana intermedia and characterization of CiDREB1C in stress response

BackgroundThe Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia.ResultsIn this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment.ConclusionsThe optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.

Regulation of Antioxidant Enzymes and Invertases by Hydrogen Peroxide and Nitric Oxide Under ABA and Water-Deficit Stress in Wheat

The present investigation reports the activities of different antioxidant enzymes and invertases (acid, alkaline and cell wall bound) in shoots and roots of 5.5-day-old seedlings of wheat cultivars PBW 644 (drought tolerant and ABA higher sensitive) and PBW 343 (drought susceptible and ABA lesser sensitive) under ABA and water-deficit stress in the presence of H2O2/nitric oxide (NO) donors as well as scavengers. Drought stress was produced by 20% PEG 6000. Sodium nitroprusside as NO donor and dimethylthiourea and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) as H2O2 and NO scavengers, respectively, were used. Roots of PBW 644 increased activities of antioxidant enzymes and invertases (acid and alkaline) and sucrose content, while PBW 343 decreased these under PEG. This was related to the presence of endogenous H2O2/NO regulation of these parameters under PEG in roots of PBW 644 but deficient in roots of PBW 343 but improved under exogenous H2O2/NO for some parameters. However, compared to PEG, roots of PBW 343 showed endogenous H2O2/NO regulation of many these parameters under ABA. Under ABA, endogenous H2O2/NO regulation was comparable between cultivars. This study showed that H2O2/NO signalling plays an important role in drought tolerance by upregulating antioxidant enzymes and invertases under stress. ABA sensitivity level and ABA levels present in cells may help to produce an effective endogenous H2O2/NO signalling.