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.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

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 previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. 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 plants activated the expression of many of these 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.

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.

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.

Molecular characterization of a cDNA encoding a novel small GTP-binding protein from Arabidopsis thaliana.

A full-length cDNA clone encoding a novel Rab protein AtRab alpha of the monomeric small GTP-binding protein family has been isolated from Arabidopsis thaliana. AtRab alpha has 210 amino acids with a calculated molecular mass of 23.3 kDa. The highest homology was found to Rab1x and Rab1y from Lotus japonicus. Southern blot analysis of genomic DNA indicated that AtRab alpha was encoded by a single copy gene. Northern blot analysis showed that expression of the AtRab alpha mRNA was rich in stems and roots, but poor in leaves and flowers, which is different from the expression pattern of other Arabidopsis Rab genes.

ERD6, a cDNA clone for an early dehydration-induced gene of Arabidopsis, encodes a putative sugar transporter.

Previously, we constructed a cDNA library from Arabidopsis plants that were exposed to dehydration stress for 1 h and obtained the ERD6 clone. Here we report that the ERD6 cDNA consists of 1741 bp and encodes a polypeptide of 496 amino acids having a predicted molecular weight of 54,354. The putative polypeptide of ERD6 is related to those of sugar transporters of bacteria, yeasts, plants and mammals. Hydropathy analysis revealed that ERD6 protein has 12 putative transmembrane domains and a central hydrophilic region. Sequences that are conserved at the ends of the 6th and 12th membrane-spanning domains of sugar transporters are also present in ERD6. These data suggest that ERD6 encodes a sugar transporter. Genomic Southern blots indicate that the ERD6 gene is a member of a multigene family in the Arabidopsis genome. The expression of the ERD6 gene was induced not only by dehydration but also by cold treatment.

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.

Molecular responses to drought and cold stress.

A variety of plant genes are induced by drought and cold stress, and they are thought to be involved in the stress tolerance of the plant. At least five signal transduction pathways control these genes: two are dependent on abscisic acid (ABA), and the others are ABA-independent. A novel cis-acting element involved in one of the ABA-independent signal transduction pathways has been identified. In addition, a number of genes for protein kinases and transcription factors thought to be involved in the stress signal transduction cascades have been shown to be induced by environmental stresses.

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.

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.

Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana.

Among nine cDNA clones (named RD) corresponding to genes that are responsive to dehydration in Arabidopsis thaliana, two clones, RD19 and RD21, were analyzed further. Northern blot analysis revealed that both the RD19 and RD21 mRNAs were not induced by abscisic acid. Neither RD19 nor RD21 mRNA synthesis was responsive to cold or to heat stress. On the other hand, transcription of both the RD19 and RD21 mRNAs was strongly induced under high-salt conditions, which suggests that the genes corresponding to RD19 and RD21 may be induced by changes in the osmotic potential of plant cells. Putative proteins, RD19 and RD21, encoded by two of the RD cDNAs have amino acid (aa) sequences typical of the catalytic sites of cysteine proteinases (CysP). RD21 and RD19 appeared to contain signal peptides that function in protein secretion. RD21 contains an aa sequence similar to that of the C-terminal extension peptide. Phylogenetic tree analysis indicated that the putative RD21 and RD19 proteins are quite different types of CysP. Genomic Southern analysis revealed that each gene family contains at least two members, which do not cross-hybridize. The two genes corresponding to RD19 and RD21 (rd19A and rd21A, respectively) were cloned and their structural analysis revealed the presence of two and four introns, respectively. The numbers and sites of introns differ between the genes, supporting our hypothesis that rd19A and rd21A belong to different subfamilies of genes encoding CysP. The transcription start points were determined by primer extension. Two conserved sequences were found in the promoter regions of the two genes.

Characterization of a gene that encodes a homologue of protein kinase in Arabidopsis thaliana.

Cloning and analysis of a gene that encodes a homologue of protein kinase (PK) from Arabidopsis thaliana is reported. Oligodeoxyribonucleotides (oligos) corresponding to conserved regions in catalytic domains of various PKs were used for polymerase chain reaction (PCR) amplification with genomic DNA from A. thaliana as template, in an attempt to identify genes encoding PK in plants. We obtained several amplified DNA fragments that encoded part of a PK. We screened a genomic DNA library of A. thaliana with these oligos or PCR fragments as probes. Three genomic clones were obtained and one of them, named Atpk7, was sequenced and analyzed. Atpk7 was demonstrated by PCR to contain an intron. The mRNA transcribed from Atpk7 was detected predominantly in root tissue by Northern blot analysis. The transcription start point was determined by primer extension. The deduced amino acid (aa) sequence of the putative product of Atpk7 resembles those of S6 kinases, cyclic nucleotide-dependent PKs and calcium-dependent PKs. From this comparison of aa sequences, the ATPK7 protein is considered to be a member of a novel subfamily of Ser/Thr PKs in plants.

Characterization of two cDNAs (ERD11 and ERD13) for dehydration-inducible genes that encode putative glutathione S-transferases in Arabidopsis thaliana L.

Two cDNA clones, designated ERD11 and ERD13, isolated from a cDNA library from Arabidopsis thaliana L. plants dehydrated for 1 h were sequenced and characterized. These clones encoded polypeptides that were homologous to glutathione S-transferases of tobacco and maize. Genomic Southern hybridization suggested that there are a few additional genes showing high similarity to the ERD11 gene in the Arabidopsis genome. The expression of the genes for ERD11 and ERD13 was induced by dehydration, but was not affected by the application of four plant growth regulators, 2,4-dichlorophenoxyacetic acid 6-benzylaminopurine, abscisic acid, or gibberellic acid.

ATMPKs: a gene family of plant MAP kinases in Arabidopsis thaliana.

We previously reported two cDNAs for MAP kinases (cATMPK1 and cATMPK2) from a dicot plant, Arabidopsis thaliana. We describe here the cloning and characterization of five additional cDNAs encoding novel MAP kinases in Arabidopsis, cATMPK3, cATMPK4, cATMPK5, cATMPK6, and cATMPK7. The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of animal MAP kinases are conserved in all the seven putative ATMPK proteins. Genes for MAP kinases in Arabidopsis constitute a family that contains more than seven members. Sequence analysis suggests that there are at least three subfamilies in the family of Arabidopsis genes for MAP kinases.

Characterization of genes for two-component phosphorelay mediators with a single HPt domain in Arabidopsis thaliana.

Three cDNAs that encode two-component phosphorelay-mediator-like proteins were cloned from Arabidopsis thaliana. Putative proteins (ATHP1-3) contain an HPt (Histidine-containing Phospho transfer)-like domain with a conserved histidine and some invariant residues that are involved in phosphorelay. Growth retardation of YPD1-disrupted yeast cells was reversed with ATHPs, which indicates that ATHPs function as phosphorelay mediators in yeast cells. The ATHP genes are expressed more in roots than in other tissues, similar to the expression of genes for a sensor histidine kinase, ATHK1, and response regulators ATRR1-4. These results suggest that ATHPs function as two-component phosphorelay mediators between sensor histidine kinase and response regulators in Arabidopsis.

Two genes that encode ribosomal-protein S6 kinase homologs are induced by cold or salinity stress in Arabidopsis thaliana.

We have isolated two closely related cDNA clones (cATPK19 and cATPK6) with homology to protein-serine/threonine kinases from Arabidopsis thaliana using the polymerase chain reaction (PCR). The deduced amino acid sequences of the ATPK19 and ATPK6 contain all 11 conserved regions of the catalytic domain of protein kinases, and have homology to p70 ribosomal S6 kinases (52%). ATPK19 and ATPK6 have putative PEST regions in their N- and C-terminal regions, and these regions also contain putative phosphorylation sites that are recognized by casein kinases or proline-directed protein kinases such as cdc2, MAP kinase, and p54 MAP-2 kinase (SAPK). The transcription levels of the ATPK19 and ATPK6 genes rapidly and markedly increased when plants were subjected to cold or high-salt stresses. These observations suggest that ATPK19 and ATPK6 may function in the adaptation of plant cells to cold or high-salt conditions, providing an understanding of the role of protein phosphorylation in plant responses to environmental stresses.

Stress-responsive expression of genes for two-component response regulator-like proteins in Arabidopsis thaliana.

Four cDNAs that encode two-component response regulator-like proteins were cloned from Arabidopsis thaliana. Putative proteins (ATRR1-4) contain a receiver domain with a conserved aspartate residue - a possible phosphorylation site - at the N-terminal half. ATRR2 lacks the C-terminal half; the others contain a C-terminal domain abundant in acidic amino acids or proline residues. ATRR1 and ATRR2 are expressed more in roots than in other tissues and are induced by low temperature, dehydration and high salinity. Levels of ATRR3 and ATRR4 were not affected by stress treatments. These results suggest that ATRRs play distinct physiological roles in Arabidopsis, and that some are involved in stress responses.

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.

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.