Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A:AtDREB1A for the improvement of drought tolerance.

We evaluated the molecular, anatomical and physiological properties of a soybean line transformed to improve drought tolerance with an rd29A:AtDREB1A construct. This construct expressed dehydration- responsive element binding protein DREB1A from the stress-inducible rd29A promoter. The greenhouse growth test included four randomized blocks of soybean plants, with each treatment performed in triplicate. Seeds from the non-transformed soybean cultivar BR16 and from the genetically modified soybean P58 line (T(2) generation) were grown at 15% gravimetric humidity for 31 days. To induce water deficit, the humidity was reduced to 5% gravimetric humidity (moderate stress) for 29 days and then to 2.5% gravimetric humidity (severe stress). AtDREB1A gene expression was higher in the genetically modified P58 plants during water deficit, demonstrating transgene stability in T(2) generations and induction of the rd29A promoter. Drought-response genes, including GmPI-PLC, GmSTP, GmGRP, and GmLEA14, were highly expressed in plants submitted to severe stress. Genetically modified plants had higher stomatal conductance and consequently higher photosynthetic and transpiration rates. In addition, they had more chlorophyll. Overexpression of AtDREB1A may contribute to a decrease in leaf thickness; however, a thicker abaxial epidermis was observed. Overexpression of AtDREB1A in soybean appears to enhance drought tolerance.

Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions.

Water deficit is the major abiotic constraint affecting crop productivity in peanut (Arachis hypogaea L.). Water use efficiency under drought conditions is thought to be one of the most promising traits to improve and stabilize crop yields under intermittent water deficit. A transcription factor DREB1A from Arabidopsis thaliana, driven by the stress inducible promoter from the rd29A gene, was introduced in a drought-sensitive peanut cultivar JL 24 through Agrobacterium tumefaciens-mediated gene transfer. The stress inducible expression of DREB1A in these transgenic plants did not result in growth retardation or visible phenotypic alterations. T3 progeny of fourteen transgenic events were exposed to progressive soil drying in pot culture. The soil moisture threshold where their transpiration rate begins to decline relative to control well-watered (WW) plants and the number of days needed to deplete the soil water was used to rank the genotypes using the average linkage cluster analysis. Five diverse events were selected from the different clusters and further tested. All the selected transgenic events were able to maintain a transpiration rate equivalent to the WW control in soils dry enough to reduce transpiration rate in wild type JL 24. All transgenic events except one achieved higher transpiration efficiency (TE) under WW conditions and this appeared to be explained by a lower stomatal conductance. Under water limiting conditions, one of the selected transgenic events showed 40% higher TE than the untransformed control.

Plant histidine kinases: an emerging picture of two-component signal transduction in hormone and environmental responses.

In the Arabidopsis thaliana genome, 11 genes encode bacterial-type two-component histidine kinases. Genetic and biochemical analyses indicate that five two-component histidine kinase-like proteins (ETR1, ETR2, EIN4, ERS1, and ERS2) function as ethylene receptors. A hybrid histidine kinase, CRE1 (also known as AHK4), acts as a cytokinin receptor, and a set of response regulators may be involved in cytokinin signal transduction. In addition to CRE1, histidine kinases CKI1 and CKI2 are likely to play important roles in cytokinin signaling. A database search of the entire Arabidopsis genome sequence has identified two additional homologs of CRE1. Arabidopsis seems to employ a hybrid histidine kinase, ATHK1, as an osmosensor. Plants widely use two-component systems in the detection of, and signal transduction by, the growth regulators ethylene and cytokinin, as well as in their responses to environmental stimuli.

Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis.

Abscisic acid (ABA), a plant hormone, is involved in responses to environmental stresses such as drought and high salinity, and is required for stress tolerance. ABA is synthesized de novo in response to dehydration. 9-cis-epoxycarotenoid dioxygenase (NCED) is thought to be a key enzyme in ABA biosynthesis. Here we demonstrate that the expression of an NCED gene of Arabidopsis, AtNCED3, is induced by drought stress and controls the level of endogenous ABA under drought-stressed conditions. Overexpression of AtNCED3 in transgenic Arabidopsis caused an increase in endogenous ABA level, and promoted transcription of drought- and ABA-inducible genes. Plants overexpressing AtNCED3 showed a reduction in transpiration rate from leaves and an improvement in drought tolerance. By contrast, antisense suppression and disruption of AtNCED3 gave a drought-sensitive phenotype. These results indicate that the expression of AtNCED3 plays a key role in ABA biosynthesis under drought-stressed conditions in Arabidopsis. We improved drought tolerance by gene manipulation of AtNCED3 causing the accumulation of endogenous ABA.

Characterization of four extensin genes in Arabidopsis thaliana by differential gene expression under stress and non-stress conditions.

From Arabidopsis thaliana we isolated four different cDNAs that encode extensins, a family of cell-wall hydroxyproline-rich glycoproteins (HRGPs). Putative proteins (AtExt2-5) contained one open reading frame and characteristic Ser-(Pro)4 sequences organized in a high-order repetitive motif. AtExt2-5 genes were strongly expressed during rehydration after dehydration. They were also expressed after treatment with various amino acids. In particular, AtExt3 and five mRNAs were abundantly accumulated after treatment with L-Ser, Hyp, and L-Pro, which are major components of extensin proteins. The AtExt transcripts were strongly expressed in root tissues of both unbolted and bolted plants. The transcripts of AtExt2, 3, and 5 were also detected in the lower stem and flower buds, and that of AtExt4 was detected in bolted flowers. Therefore, we suggest that these four AtExt genes are novel extensin genes in A. thaliana, because the expression of atExt1, which has already been isolated from A. thaliana, was different from these.

Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor.

Plant productivity is greatly affected by environmental stresses such as drought, salt loading and freezing. We reported that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses in Arabidopsis. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic Arabidopsis plants activated the expression of many of theses stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress-inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter. As the DRE-related regulatory element is not limited to Arabidopsis the DREB1A cDNA and the rd29A promoter may be useful for improving the stress tolerance of agriculturally important crops by gene transfer.

Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture.

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.

Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray.

Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, cold-treated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a cDNA microarray using approximately 1300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs for drought- and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes that have not been reported as drought- or cold-inducible genes previously. Twelve stress-inducible genes were identified as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microarray analyses, the six genes were identified as novel drought- and cold-inducible genes that are controlled by DREB1A. Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify potential cis-acting DNA elements by combining the expression data with the genomic sequence data.

Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions.

The induction of the dehydration-responsive Arabidopsis gene, rd29B, is mediated mainly by abscisic acid (ABA). Promoter analysis of rd29B indicated that two ABA-responsive elements (ABREs) are required for the dehydration-responsive expression of rd29B as cis-acting elements. Three cDNAs encoding basic leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using the yeast one-hybrid system and were designated AREB1, AREB2, and AREB3 (ABA-responsive element binding protein). Transcription of the AREB1 and AREB2 genes is up-regulated by drought, NaCl, and ABA treatment in vegetative tissues. In a transient transactivation experiment using Arabidopsis leaf protoplasts, both the AREB1 and AREB2 proteins activated transcription of a reporter gene driven by ABRE. AREB1 and AREB2 required ABA for their activation, because their transactivation activities were repressed in aba2 and abi1 mutants and enhanced in an era1 mutant. Activation of AREBs by ABA was suppressed by protein kinase inhibitors. These results suggest that both AREB1 and AREB2 function as transcriptional activators in the ABA-inducible expression of rd29B, and further that ABA-dependent posttranscriptional activation of AREB1 and AREB2, probably by phosphorylation, is necessary for their maximum activation by ABA. Using cultured Arabidopsis cells, we demonstrated that a specific ABA-activated protein kinase of 42-kDa phosphorylated conserved N-terminal regions in the AREB proteins.

An Arabidopsis gene encoding a Ca2+-binding protein is induced by abscisic acid during dehydration.

An Arabidopsis thaliana RD20 cDNA, which was isolated as one of drought-inducible genes, encodes a putative protein with a conserved EF-hand Ca2+-binding domain. The recombinant RD20 protein was shown to bind Ca2+. The transcription of RD20 gene was induced not only by drought but also by ABA and high salinity.

Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system.

We have so far cloned a cDNA encoding a hybrid-type histidine kinase (ATHK1), three cDNAs encoding phosphorelay intermediates (ATHP1-3), and four cDNAs encoding response regulators (ATRR1-4) from Arabidopsis thaliana. To determine which molecules constitute a His to Asp phosphorelay pathway, we examined protein-protein interactions between them using a pairwise yeast two-hybrid analysis, as an initial step. We detected a specific interaction between ATHK1 and ATHP1. We further examined protein-protein interactions between ATHP1-3 and other histidine kinases. We detected interactions between ETR1 and all ATHPs, and between CKI1 and ATHP1 or ATHP2. Interestingly, ERS1 could not interact with any ATHPs. We also examined protein-protein interactions between ATHP1-3 and ATRR1-4. The results indicated that ATHP2 could interact with ATRR4, and that ATHP3 could interact with ATRR1 or ATRR4. However, ATHP1 could not interact with any ATRRs. On the basis of these results, we discuss the possible phosphorelay networks in an Arabidopsis two-component system.

Molecular cloning and characterization of a cDNA encoding proline transporter in rice.

A cDNA encoding a proline (Pro) transporter (ProT) was isolated and characterized from a cDNA library prepared from 14-d-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the rice ProT protein (OsProT) had 68.8% homology to the ProT protein 1 from Arabidopsis thaliana and 59.6% homology to that from Lycopersicon esculentum. Northern blot analysis revealed that the gene for OsProT (OsProT) was expressed in all organs examined, comparatively strongly in leaf sheath and stem. Salt treatment did not induce expression of OsProT but strongly induced expression of the gene for delta1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in Pro biosynthesis. Southern blot analysis revealed that OsProT has a gene family. OsProT specifically transported L-Pro in a transport assay using Xenopus laevis oocytes.

A novel Arabidopsis thaliana dynamin-like protein containing the pleckstrin homology domain.

A full-length cDNA encoding a novel type of plant dynamin-like protein, ADL3, was isolated from Arabidopsis thaliana. ADL3 is a high molecular weight GTPase whose GTP-binding domain shows a low homology to those of other plant dynamin-like proteins. ADL3 contains the pleckstrin homology domain as is in mammalian dynamins, although other plant dynamin-like proteins reported lack this domain. The ADL3 gene was expressed weakly in various tissues, except for siliques with high level expression, which is distinct from the case for other plant dynamin-like protein genes. Taken together, it is predicted that the mode of activation of ADL3 is different from those of other plant homologues.

A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea.

Four cDNA clones named CPRD (cowpea responsive to dehydration) corresponding to genes that are responsive to dehydration were isolated using differential screening of a cDNA library prepared from 10-h dehydrated drought-tolerant cowpea (Vigna unguiculata) plants. One of the cDNA clones has a homology to 9-cis-epoxycarotenoid dioxygenase (named VuNCED1), which is supposed to be involved in abscisic acid (ABA) biosynthesis. The GST (glutathione S-transferase)-fused protein indicates a 9-cis-epoxycarotenoid dioxygenase activity, which catalyzes the cleavage of 9-cis-epoxycarotenoid. The N-terminal region of the VuNCED1 protein directed the fused sGFP (synthetic green-fluorescent protein) into the plastids of the protoplasts, indicating that the N-terminal sequence acts as a transit peptide. Both the accumulation of ABA and expression of VuNCED1 were strongly induced by drought stress in the 8-d-old cowpea plant, whereas drought stress did not trigger the expression of VuABA1 (accession no. AB030295) gene that encodes zeaxanthin epoxidase. These results indicate that the VuNCED1 cDNA encodes a 9-cis-epoxycarotenoid dioxygenase and that its product has a key role in the synthesis of ABA under drought stress.

Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways.

Recently, a major transcription system that controls abscisic-acid-independent gene expression in response to dehydration and low temperature has been identified. The system includes the DRE/CRT (dehydration-responsive element/C-repeat) cis-acting element and its DNA-binding protein, DREB/CBF (DRE-binding protein/C-repeat binding factor), which has an AP2 domain. DREB/CBF contains two subclasses, DREB1/CBF and DREB2, which are induced by cold and dehydration, respectively, and control the expression of various genes involved in stress tolerance. Recent studies are providing evidence of differences between dehydration-signaling and cold-stress-signaling cascades, and of cross-talk between them.

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression.

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5'-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The beta-glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.

Two-component systems in plant signal transduction.

In plants, two-component systems play important roles in signal transduction in response to environmental stimuli and growth regulators. Genetic and biochemical analyses indicate that sensory hybrid-type histidine kinases, ETR1 and its homologs, function as ethylene receptors and negative regulators in ethylene signaling. Two other hybrid-type histidine kinases, CKI1 and ATHK1, are implicated in cytokinin signaling and osmosensing processes, respectively. A data base search of Arabidopsis ESTs and genome sequences has identified many homologous genes encoding two-component regulators. We discuss the possible origins and functions of these two-component systems in plants.

Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana.

Synthesis, degradation, and transport of proline (Pro) are thought to cooperatively control its endogenous levels in higher plants in response to environmental conditions. To evaluate the function of Pro degradation in the regulation of the levels of Pro and to elucidate roles of Pro in stress tolerance, we generated antisense transgenic Arabidopsis plants with an AtProDH cDNA encoding proline dehydrogenase (ProDH), which catalyzes Pro degradation. Several transgenic lines accumulated Pro at higher levels than wild-type plants, providing evidence for a key role of ProDH in Pro degradation in Arabidopsis. These antisense transgenics were more tolerant to freezing and high salinity than wild-type plants, showing a positive correlation between Pro accumulation and stress tolerance in plants.

Evaluation of a cDNA scanning method concerning the fidelity and efficiency of cDNA selection using the YAC CIC3B1-S region of Arabidopsis thaliana chromosome 5.

We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated. We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region. In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones. Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein. We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region. We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning.