The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper–like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper–like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants.

Members of the NAC gene family encode plant-specific transcription factors and are widely distributed in plant species. The OsNAC6 gene is one of many NAC genes in rice and has high similarity to genes in the ATAF subfamily. Here we show that OsNAC6 is induced by cold, salt, drought and abscisic acid (ABA). We found that OsNAC6 is also induced by wounding. The response of OsNAC6 to wounding is very rapid and strong. OsNAC6 was also induced by jasmonic acid (JA), a plant hormone that activates defense responses against herbivores and pathogens. Our results imply that OsNAC6, besides having a role in plant adaptation to abiotic stresses, also integrates signals derived from both abiotic and biotic stresses.

Petunias (Petunia hybrida cv. 'Mitchell') accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by Delta(1)-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57-1.01% of the total amino acids in the transgenic plants, or 1.5-2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.

To clarify the mechanisms of stress tolerance in rice and to search for rice genes associated with these mechanisms, we analyzed genes induced by a high salinity treatment using the PCR-subtractive hybridization method (PCR-subtraction). Seedlings of the salt-tolerant rice cultivar Dee-geo-woo-gen (DGWG) were either treated with 250 m M NaCl for 5 h or left untreated, and PCR-subtraction was then performed using the untreated ( control) plants as a driver and the NaCl-treated plants as a tester. We obtained 384 clones of tester-specific cDNAs as salt-inducible candidates. Northern analysis performed with the cDNA fragments showed that 65 clones had been induced by the NaCl treatment. Sequence analysis and database searching indicated that these clones have homology to proteins functional for detoxification, stress response, and signal transduction in plants. Of these clones, 22% coded for unknown proteins and 12% gave no hits. We selected eight clones from each functional category and analyzed their expression pattern in DGWG. For temporal analysis, seedlings were treated with H2O or 250 m M NaCl for 0, 0.5, 1, 2, 5, 10 or 24 h. Different patterns of transcript regulation were found. For the analysis of expression in response to various types of stress and abscisic acid (ABA) treatments, seedlings were treated for 5 h or 10 h with H2O, dehydration, cold ( 4 degrees C), heat ( 40 degrees C), mannitol, ABA, or wounding. All clones were strongly up-regulated by osmotic stress ( dehydration and mannitol) and the ABA treatment.

Arginine decarboxylase (ADC) catalyzes the first step of polyamine (PA) biosynthesis to produce putrescine (Put) from arginine (Arg). One of the 2 Arabidopsis ADC genes, AtADC2, is induced in response to salt stress causing the accumulation of free Put. To analyze the roles of stress-inducible AtADC2 gene and endogenous Put in stress tolerance, we isolated a Ds insertion mutant of AtADC2 gene (adc2-1) and characterized its phenotypes under salt stress. In the adc2-1 mutant, free Put content was reduced to about 25% of that in the control plants and did not increase under salt stress. Furthermore, the adc2-1 mutant was more sensitive to salt stress than the control plants. The stress sensitivity of adc2-1 was recovered by the addition of exogenous Put. These results indicate that endogenous Put plays an important role in salt tolerance in Arabidopsis. AtADC2 is a key gene for the production of Put under not only salinity conditions, but also normal conditions.

To characterize the genes for enzymes involved in the biosynthesis of polyamines (PAs), their expression profiles were investigated and the levels of PAs in Arabidopsis thaliana quantified. In the Arabidopsis genome, eight genes involved in PAs biosynthesis were identified and the expression profiles of these genes were analysed, not only under abiotic stress to determine whether they were stress-inducible, constitutive, or stress-repressible, but also in various organs to show their tissue specificity. AtADC2 and AtSPMS mRNAs, encoding arginine decarboxylase and spermine synthase, clearly increased in response to NaCl and dehydration and abscisic acid treatments. Stress-inducible accumulation of AtADC2 mRNA correlated with putrescine (Put) accumulation under NaCl and dehydration treatments. In a cold condition, AtSAMDC2 mRNA increased significantly. AtADC2 and AtSAMDC2 mRNA were expressed in sexual organs such as flowers, buds and immature siliques. PAs also accumulated in sexual organs. These results suggest that the transcripts of eight genes involved in PA biosynthesis show different profiles of expression not only in response to environmental stress but also during plant development.

Proline is one of the most common compatible osmolytes in water-stressed plants. The accumulation of proline in dehydrated plants is caused both by the activation of proline biosynthesis and by the inactivation of proline degradation; a decrease in the level of accumulated proline in rehydrated plants iscaused both by the inhibition of proline biosynthesis and by the activation of proline degradation. The proline biosynthetic pathway has been well characterized, but the degradation of proline is poorly understood. Sequence analysis of an Arabidopsis cDNA clone, ERD5 (for garly [esponsive to dehydration stress), isolated from plants dehydrated for 1 hr, revealed that i t encodes a protein with identity to products of the yeast PU7Y (for eroline Uilization) gene (23.6% over 364 amino acids) and the Drosophila sluggish-A gene (34.5% over 255 amino acids). Their gene products are precursors of proline oxidases (dehydrogenase) (EC 1.5.99.8), which are the first enzymes involved in the conversion of proline to glutamic acid. Proline oxidase is localized in mitochondria. RNA gel blot analysis demonstrated that transcripts of the ERDS gene were undetectable when plants had been dehydrated for 10 hr, but large amounts of the transcript accumulated when plants subsequently were rehydrated. Elevated levels of the transcript were also found in plants that had been incubated in a medium that contained proline. Immunologically, we showed that the product of ERDS is localized i n the mitochondrial fraction and accumulates in response to proline in cultured cells. Fusion genes for ERDS and PU7Y complemented a put7 mutant of yeast, allowing putl to grow with proline as the source of nitrogen. These results suggest that ERDS encodes a precursor of proline dehydrogenase (oxidase), which is regulated at the level of mRNA accumulation in both dehydrated and rehydrated plants.

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.

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.

Δ1-Pyrroline-5-carboxylate synthetase 1 (P5CS1) is the rate-limiting enzyme in the biosynthesis of proline by Arabidopsis thaliana. Results of Northern analysis using aba1, abi1, and abi3 mutants of A. thaliana suggest that the expression of the P5CS1 gene under water stress is induced via abscisic acid (ABA)-biosynthesis-dependent and -independent pathways. Expression via ABA biosynthesis does not require protein synthesis. Analysis using transgenic A. thaliana containing a P5CS1 promoter/GUS fused gene indicated that the P5CS1 gene of A. thaliana is expressed in the whole plant under dehydration and in reproductive organs and tissues (flower buds and surrounding parts, pollen and pistils, and young siliques in the early stage of seed formation) under unstressed conditions. Cis-acting elements involved in dehydration-responsive gene expression are shown to be located in a 117-bp region between positions −621 and −504 upstream from the transcriptional initiation site.

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild-type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L-Pro but not by D-Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.