Genes RD22 Research Articles

OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues.

An ERF transcription factor OsERF101 is predominantly expressed in rice reproductive tissues and plays an important role in improving rice seed setting rate under drought stress. Drought reduces grain yield due to the cumulative damage effects to plant vegetative and reproductive developmental processes. However, the genes involved in these processes are still not completely understood. In this study, we identified a gene named OsERF101 as an important positive regulator in the adaptive responses to dehydration stress during the reproductive and vegetative stages. This gene encodes a member of APETALA2/Ethylene-Responsive Element Binding Protein (AP2/EREBP) family. OsERF101 was predominantly expressed in flowers, particularly in the tapetum and microspores under normal growth conditions. It was induced by drought, PEG6000 and abscisic acid (ABA) in leaves. During the vegetative stage, OsERF101-overexpression plants were more resistant to osmotic stress caused by PEG6000 compared to the control plants. They also had higher survival and seed setting rates than wild type when subjected to reproductive-stage drought stress. Further physiological analysis revealed that the pollen fertility was improved in the overexpression lines, while the knockout mutant and RNAi lines showed reduced pollen fertility and compromised drought tolerance during the reproductive stage. The increased proline content and peroxidase activity in OsERF101-overexpression plants might contribute to the improved drought-tolerance of plants. In addition, OsERF101-overexpression plants displayed ABA susceptible phenotype, in which the expression levels of ABA-responsive genes RD22, LEA3, and PODs were up-regulated. Taken together, our results indicate that OsERF101 is a gene that regulates dehydration responses during the vegetative and reproductive stages.

Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase gene (CsCSD1) from Cucumis sativus

Superoxide dismutase (SOD) proteins, which are widely present in the plant kingdom, play vital roles in response to abiotic stress. However, the functions of cucumber SOD genes in response to environmental stresses remain poorly understood. In this study, a SOD gene CsCSD1 was identified and functionally characterized from cucumber (Cucumis sativus). The CsCSD1 protein was successfully expressed in E. coli, and its overexpression significantly improved the tolerance of host E. coli cells to salinity stress. Besides, overexpression of CsCSD1 enhanced salinity tolerance during germination and seedling development in transgenic Arabidopsis plants. Further analyses showed that the SOD and CAT (catalase) activities of transgenic plants were significantly higher than those of wild-type (WT) plants under normal growth conditions as well as under NaCl treatment. In addition, the expression of stress-response genes RD22, RD29B and LEA4-5 was significantly elevated in transgenic plants. Our results demonstrate that the CsCSD1 gene functions in defense against salinity stress and may be important for molecular breeding of salt-tolerant plants.

Over-expression of GmHAL3 modulates salt stresses tolerance in transgenic arabidopsis

The halotolerance protein HAL3, also known as SIS2, is a yeast protein that regulates the cell cycle and tolerance to salt stress through inhibition of the Ppz1 type 1 protein phosphatase. Although the roles of HAL3 have been demonstrated during the growth, development, and stress adaptation of Arabidopsis thaliana and Nicotiana tabacum, the function of HAL3 in other plant species, including soybean (Glycine max), has not been elucidated. In this study, GmHAL3a and GmHAL3b were isolated from Glycine max, and their roles were analyzed. GmHAL3a and GmHAL3b transcripts were detected in the roots, stems, leaves and seeds, with higher levels in the roots, and were induced by sodium chloride (NaCl), lithium chloride (LiCl), sorbitol, cold and ABA treatment. Overexpression of GmHAL3a or GmHAL3b in Arabidopsis accelerated the onset of flowering and resulted in more vigorous seed germination and increased tolerance to NaCl, LiCl, and sorbitol stress in seedlings, compared with wild type (WT) and empty vector control (VC) plants. Transgenic Arabidopsis plants accumulated proline and eliminated superoxide radical (O2−) in response to the stress. In addition, transcription levels of the stress-related genes RD22 and P5CS1 were substantially higher in transgenic Arabidopsis than in WT and VC plants. Taken together, the data indicate that GmHAL functions as a positive regulator of the response to salt, lithium cations and sorbitol stress.

Overexpression of the Lotus corniculatus Soloist Gene LcAP2/ERF107 Enhances Tolerance to Salt Stress.

The AP2/ERF play a key role in multiple stress responses in plants. we here report a novel salt stress-related gene, LcAP2/ERF107 that encodes an AP2/ERF protein in Lotus corniculatus cultivar Leo. LcAP2/ERF107 was classified into the soloist subfamiliy based on phylogenetic relationship. The transcription of LcAP2/ERF107 were strongly induced by salt and other phytohormones (ABA, ACC, MeJA). A subcellular localization experiment indicated that LcAP2/ERF107 is a nuclear protein that activates transcription. LcAP2/ERF107 overexpression in Arabidopsis resulted in pleiotropic phenotypes, including higher seed germination rate and transgenic plants with enhanced tolerance to salt stress. Further, under salt tolerance the transgenic lines elevated the relative moisture content; however, the relative electrolyte leakage was lower than in control plants. The expression levels of indicative genes RD22, RD29A, LEA4-5, P5CS1 and P5CS2 were found to be increased in the transgenic plants compared with the WT plants. These results indicated that LcAP2/ERF107 play an important role in the responses of plant to salt stress.

Inspection of the grapevine BURP superfamily highlights an expansion of RD22 genes with distinctive expression features in berry development and ABA-mediated stress responses.

The RESPONSIVE TO DEHYDRATION 22 (RD22) gene is a molecular link between abscisic acid (ABA) signalling and abiotic stress responses. Its expression has been used as a reliable ABA early response marker. In Arabidopsis, the single copy RD22 gene possesses a BURP domain also located at the C-terminus of USP embryonic proteins and the beta subunit of polygalacturonases. In grapevine, a RD22 gene has been identified but putative paralogs are also found in the grape genome, possibly forming a large RD22 family in this species. In this work, we searched for annotations containing BURP domains in the Vitis vinifera genome. Nineteen proteins were defined by a comparative analysis between the two genome predictions and RNA-Seq data. These sequences were compared to other plant BURPs identified in previous genome surveys allowing us to reconceive group classifications based on phylogenetic relationships and protein motif occurrence. We observed a lineage-specific evolution of the RD22 family, with the biggest expansion in grapevine and poplar. In contrast, rice, sorghum and maize presented highly expanded monocot-specific groups. The Vitis RD22 group may have expanded from segmental duplications as most of its members are confined to a region in chromosome 4. The inspection of transcriptomic data revealed variable expression of BURP genes in vegetative and reproductive organs. Many genes were induced in specific tissues or by abiotic and biotic stresses. Three RD22 genes were further studied showing that they responded oppositely to ABA and to stress conditions. Our results show that the inclusion of RNA-Seq data is essential while describing gene families and improving gene annotations. Robust phylogenetic analyses including all BURP members from other sequenced species helped us redefine previous relationships that were erroneously established. This work provides additional evidence for RD22 genes serving as marker genes for different organs or stresses in grapevine.

Physiological responses of transgenic tobacco plants expressing the dehydration-responsive RD22 gene of Vitis vinifera to salt stress

Salinity is one of the most common environmental stresses affecting grapevine productivity. In order to analyze the contribution of the dehydration-responsive RD22 gene of Vitis vinifera L. (VvRD22) to salt tolerance improvement, Agrobacterium-mediated transformation in the tobacco model plant (Nicotiana benthamiana) was carried out and transgenic lines were subjected to in vitro and ex vitro salt treatments for physiological response evaluation. Under in vitro salt stress, the transgenic lines exhibited higher seed germination and growth than the wild type (WT). The ex vitro assays after salt treatment showed better growth and higher chlorophyll content in VvRD22-expressing plants than in the WT. Ion analysis revealed less increase in the levels of sodium (Na+) in leaves and chloride (Cl-) in all organs within the transgenic lines compared to the WT. Interestingly, stabilized calcium (Ca2+) contents were registered in the leaves of transgenic lines. Moreover, the occurrence of an osmotic adjustment based on an overproduction of total soluble sugars was observed within the transgenic lines. These physiological responses suggest that the protective effect of VvRD22 transgenic expression is enabling other physiological mechanisms to function and subsequently contributing to the ability to cope with salt stress. The VvRD22 transgenic expression would be useful in engineering salt stress-tolerant grapevines.

The rice OsDIL gene plays a role in drought tolerance at vegetative and reproductive stages

Drought is one of the critical factors limiting reproductive yields of rice and other crops globally. However, little is known about the molecular mechanism underlying reproductive development under drought stress in rice. To explore the potential gene function for improving rice reproductive development under drought, a drought induced gene, Oryza sativa Drought-Induced LTP (OsDIL) encoding a lipid transfer protein, was identified from our microarray data and selected for further study. OsDIL was primarily expressed in the anther and mainly responsive to abiotic stresses, including drought, cold, NaCl, and stress-related plant hormone abscisic acid (ABA). Compared with wild type, the OsDIL-overexpressing transgenic rice plants were more tolerant to drought stress during vegetative development and showed less severe tapetal defects and fewer defective anther sacs when treated with drought at the reproductive stage. The expression levels of the drought-responsive genes RD22, SODA1, bZIP46 and POD, as well as the ABA synthetic gene ZEP1 were up-regulated in the OsDIL-overexpression lines but the ABA degradation gene ABAOX3 was down-regulated. Moreover, overexpression of OsDIL lessened the down-regulation by drought of anther developmental genes (OsC4, CYP704B2 and OsCP1), providing a mechanism supporting pollen fertility under drought. Overexpression of OsDIL significantly enhanced drought resistance in transgenic rice during reproductive development, while showing no phenotypic changes or yield penalty under normal conditions. Therefore, OsDIL is an excellent candidate gene for genetic improvement of crop yield in adaption to unfavorable environments.

Short-term response of wild grapevines (Vitis vinifera L. ssp. sylvestris) to NaCl salinity exposure: changes of some physiological and molecular characteristics

The physiological and molecular response to salt stress was studied in two wild grapevine (Vitis vinifera L. ssp. sylvestris or Vitis sylvestris) accessions “Khedhayria” and “Houamdia”, previously identified as salt-tolerant and salt-sensitive pair wise. Plants from both accessions were subjected to a progressive salt stress by the use of a nutritional solution containing up to 150 mM NaCl for 2 weeks. Salt stress adversely affected growth and water potential since the first day of exposure to 150 mM NaCl. However, chlorophyll fluorescence parameters were unchanged until 14 days of salt exposure. At that time point the predawn water potential (ΨPD), the non-photochemical quenching of fluorescence (NPQ) and the coefficient of photochemical quenching (qp) were significantly less altered in the tolerant accession. At the molecular level semi-quantitative RT-PCR assays revealed a differential expression of (Vs α-gal/SIP and Vs DHN) genes within these contrasting accessions after exposure to 24 h and 14 days of salt. Comparably, the Vs RD22 gene had increased slightly after only 14 days of treatment in both accessions. These results were the first pieces of information reported on the early and late regulation of salt response genes in wild grapevines. Furthermore, genotype-dependent parameters such as NPQ, qp, mRNA levels of Vs α-gal/SIP and Vs DHN could be used to screen salt-tolerant wild grapevine genotypes.

Gibberellic acid of Arabidopsis regulates the abscisic acid-induced inhibition of stomatal opening in response to light

Gibberellic acid (GA 3) has often known to affect stomatal movement; however, the physiological role of GA 3 in stomatal guard cells has not been defined. In this study, we show that abscisic acid (ABA) is regulated by gibberellic acid via a mechanism opposing that of the ABA-induced inhibition of stomatal opening in response to light in Arabidopsis leaves. GA 3 modulated ABA-induced stomatal closing in response to light in a dose-dependent manner. We found that there did no affect on stomatal opening in response to light in the presence of the gibberellin (GA) biosynthesis inhibitor prohexadione-calcium (BX-112). However, the effects of GA 3 on the ABA-induced inhibition of stomatal opening in response to light were blocked by BX-112. In RT-PCR studies, the expression of the RD22 gene was decreased by the addition of GA 3. The expression of the GA-inducible genes, GASA4 and AtExp1, was reduced by ABA treatment. Our results strongly suggest that GA 3 regulates the ABA-induced inhibition of stomatal opening in response to light.

Identification et caractérisation d'un gène de réponse à la déshydratation « rd22 » chez la vigne ( Vitis vinifera L.)

To identify and isolate genes related to abiotic stresses (salinity and drought) tolerance in grapevine, a candidate gene approach was developed and allowed isolating a full-length cDNA of rd22 gene from the Cabernet Sauvignon variety. The latter, named Vvrd22, is a dehydration-responsive gene that is usually induced by the application of exogenous ABA. Details of the physicochemical parameters and structural properties (molecular mass, secondary structure, conserved domains and motives, putative post-translational modification sites…) of the encoded protein have also been elucidated. The expression study of Vvrd22 was carried out at the berry growth stages and at the level of plant organs and tissues as well as under both drought and salt stresses. The results showed that Vvrd22 is constitutively expressed at a low level in all analyzed tissues. Moreover, salt stress induced Vvrd22 expression, particularly for the tolerant variety (Razegui), contrary to the sensitive one (Syrah), which did not display any expression variation during the stress, which means that Vvrd22 is involved in salt stress response and that its expression level depends on regulatory mechanisms that are efficient only for the tolerant variety. On the other hand, under drought stress, Vvrd22 is induced in an identical manner for both tolerant and sensitive varieties. In addition, stress signal molecules such as ABA (lonely applied or in combination with sucrose) induced Vvrd22 expression, even at a low level. A minimal knowledge about the role and the functionality of this gene is necessary and constitutes a prerequisite condition before starting and including Vvrd22 in any program of improvement of grapevine's abiotic stress tolerance. To cite this article: M. Hanana et al., C. R. Biologies 331 (2008).

Isolation and characterization of a BURP domain‐containing gene BnBDC1 from Brassica napus involved in abiotic and biotic stress

A new gene encoding BURP domain‐containing protein was isolated from oilseed rape (Brassica napus) following drought treatment. The cloned gene, designated as BnBDC1 (GenBank accession number AY293830), contains four major domains: an N‐terminal hydrophobic domain, a short conserved segment, a characteristic repeated region and a C‐terminal BURP domain. BnBDC1 is highly homologous to the dehydration‐responsive RD22 gene from Arabidopsis thaliana, and belongs to the same gene family as BNM2 from B. napus. Northern blot and semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) analyses show that BnBDC1 is constitutively expressed in rape shoots, but is not detectable in the roots, indicating that the expression of BnBDC1 is tissue specific. The expression level of BnBDC1 transcript is upregulated by mannitol, NaCl and abscisic acid (ABA), downregulated by UV irradiation and salicylic acid, and unresponsive to H2O2 and cold treatment. The accumulation of BnBDC1 transcripts generally increases within 2 h after initiation of the promoting stimulus. These results suggest that BnBDC1 has similar functions to those of RD22 of Arabidopsis, and may play an important role in response to osmotic stress and/or plant pathogen infection in B. napus.

Stress memory in plants: a negative regulation of stomatal response and transient induction of rd22 gene to light in abscisic acid-entrained Arabidopsis plants.

All organisms, including plants, perceive environmental stress, and they use this information to modify their behavior or development. Here, we demonstrate that Arabidopsis plants have memory functions related to repeated exposure to stressful concentrations of the phytohormone abscisic acid (ABA), which acts as a chemical signal. Repeated exposure of plants to ABA (40 micro m for 2 h) impaired light-induced stomatal opening or inhibited the response to a light stimulus after ABA-entrainment under both dark/light cycle and continuous light. Moreover, there were transient expressions of the rd22 gene during the same periods under both the growing conditions. Such acquired information in ABA-entrained plants produced a long-term sensitization. When the time of light application was changed, a transient induction of the rd22 gene in plants after ABA-entrainment indicated that these were light-associated responses. These transient effects were also observed in kin1, rab18, and rd29B. The transient expression of AtNCED3, causing the accumulation of endogenous ABA, indicated a possible regulation by ABA-dependent pathways in ABA-entrained plants. An ABA immunoassay supported this hypothesis: ABA-entrained plants showed a transient increase in endogenous ABA level from 220 to 250 pmol g-1 fresh mass at 1-2 h of the training period, whereas ABA-deficient (aba2) mutants did not. Taking into account these results, we propose that plants have the ability to memorize stressful environmental experiences, and discuss the molecular events in ABA-entrained plants.

Molecular cloning and characterization of genes encoding BURP domain-containing protein in the mangrove, Bruguiera gymnorrhiza

We cloned and characterized four independent genes encoding BURP domain-containing proteins from mangrove, Bruguiera gymnorrhiza, two as cDNA (BgBDC2 and BgBDC3) and the other two as genomic DNA (S2 and L3). The putative coding sequences of S2 and L3 were computationally analysed and designated as BgBDC1 and BgBDC4, respectively. These four genes are highly homologous to the dehydration-responsive RD22 gene from Arabidopsis thaliana. Amino acid sequences deduced from the four isolated genes contain characteristic repeated regions, which consist of repeated units whose consensus sequence is similar to that of RD22. The repeated regions, however, differ from each other and from RD22 by the copy number of the repeated unit; x copies in BgBDCX (x=1, 2, 3, and 4), while five copies in RD22. Northern blot analysis probed with BgBDC3 cDNA showed that the amount of RNA hybridized to the probe was retained in the top leaves following 500 mM NaCl treatment, while it distinctly decreased in the lower leaves. Desiccation treatment and exogenous abscisic acid (ABA) treatment also decreased the amount in all leaves. The results indicate that the regulatory mechanisms in B. gymnorrhiza for the expression of RD22 homologues are different from that of RD22.

Isolation and Characterization of a Gene Expressed during Early Embryo Sac Development in Apomictic Guinea Grass ( Panicum maximum)

A novel classification method, based on ovary length, for sampling different developmental stages of embryo sac formation in obligate sexual and facultatively apomictic genotypes of guinea grass (Panicum maximum) has been employed to make a cDNA library and to isolate a stage-specific cDNA clone, probably representing a full-length gene. This A2-134 cDNA, designated ASG-1 (Apomixis Specific Gene), was found to be expressed in flower buds of the apomictic but not of the sexual accession. Furthermore, the gene was also not expressed in buds of apomicts until completion of megasporogenesis but could only be found during a phase characterized by the. appearance of aposporous initial cells (AICs) of the embryo sac. The appearance of these cells is strictly limited to apomictic genotypes. Sequence analysis revealed that the A2-134 cDNA (1,177bp) codes for a protein of 305 amino acids with a molecular mass of 34.2 kDa. The amino acid sequence of A2-134 is related to RD22, a seed-specific and drought-induced gene of Arabidopsis thaliana, to USP, an unknown seed protein precursor of Vicia faba, to a polygalaturonase 1 beta chain precursor (Polygl) of Lycopersicon esculentum and to ADR6, an auxin down-regulated gene of Glycine max. Southern blot analysis showed a different hybridization pattern at the genomic level when A2134 cDNA was hybridized with total DNAs isolated from leaves of sexual as well as apomictic plants, indicating that the gene (or related family members) exists in both types. The cDNA library and the ASG-1 gene should be valuable tools toward understanding the molecular mechanism of apomixis, particularly apospory, and toward the transfer of apomixis to important crops. To our knowledge, A2-134 represents the first case of the isolation and characterization of a cDNA related to apomictic embryo sac development.

Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.

In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) and requires protein biosynthesis for ABA-dependent gene expression. Previous experiments established that a 67-bp DNA fragment of the rd22 promoter is sufficient for dehydration- and ABA-induced gene expression and that this DNA fragment contains two closely located putative recognition sites for the basic helix-loop-helix protein MYC and one putative recognition site for MYB. We have carefully analyzed the 67-bp region of the rd22 promoter in transgenic tobacco plants and found that both the first MYC site and the MYB recognition site function as cis-acting elements in the dehydration-induced expression of the rd22 gene. A cDNA encoding a MYC-related DNA binding protein was isolated by DNA-ligand binding screening, using the 67-bp region as a probe, and designated rd22BP1. The rd22BP1 cDNA encodes a 68-kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors. The rd22BP1 protein binds specifically to the first MYC recognition site in the 67-bp fragment. RNA gel blot analysis revealed that transcription of the rd22BP1 gene is induced by dehydration stress and ABA treatment, and its induction precedes that of rd22. We have reported a drought- and ABA-inducible gene that encodes the MYB-related protein ATMYB2. In a transient transactivation experiment using Arabidopsis leaf protoplasts, we demonstrated that both the rd22BP1 and ATMYB2 proteins activate transcription of the rd22 promoter fused to the beta-glucuronidase reporter gene. These results indicate that both the rd22BP1 (MYC) and ATMYB2 (MYB) proteins function as transcriptional activators in the dehydration- and ABA-inducible expression of the rd22 gene.