Yoshu Yoshiba

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.

A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis.

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 is caused 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 early responsive to dehydration stress), isolated from plants dehydrated for 1 hr, revealed that it encodes a protein with identity to products of the yeast PUT1 (for proline utilization) 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 ERD5 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 ERD5 is localized in the mitochondrial fraction and accumulates in response to proline in cultured cells. Fusion genes for ERD5 and PUT1 complemented a put1 mutant of yeast, allowing put1 to grow with proline as the source of nitrogen. These results suggest that ERD5 encodes a precursor of proline dehydrogenase (oxidase), which is regulated at the level of mRNA accumulation in both dehydrated and rehydrated plants.

Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.

A cDNA for Δ1-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.

Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development.

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.

Water Stress-Induced Genes inArabidopsis thaliana

Plants respond to conditions of severe environmental changes or stresses, such as dehydration, low temperature, high-salinity with a number of physiological and developmental changes. We have isolated more than thirty genes that respond to dehydration stresses at transcriptional level. In the present study, we will report the expression and function of differentArabidopsis genes involved in the kinase cascade and in the proline biosynthesis. MAP kinases play key roles in the signalling pathway activated by growth factors and in stress response in animals. The mRNA levels of three protein kinases, ATMEKK1 (MAP kinase kinase kinase), ATMPK3 (MAP kinase) and ATPK19 (ribosomal S6 kinase), increased markedly and simultaneously in response to touch, cold and salinity stresses. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the post-translational level but also at the transcriptional level in plants. It is also suggested that MAP kinase cascades in plants may function in transducing signals under environmental stresses. Proline is thought to be a compatible osmolyte like glycine betaine, which plays a role in counteracting the effects of osmotic stress in plants. We have isolated two cDNAs fromArabidopsis (cAtPSCS andcAtPSCR) encoding P5CS (Δ1-pyrroline-5-carboxylate synthetase) and P5CR (Δ1-pyrroline-5-carboxylate reductase) which play key roles in the biosynthesis of proline in plants under water stress. Northern blot analysis revealed that theAtP5CS gene was induced by dehydration, high salt and treatment with ABA. Moreover, a simultaneous accumulation of proline was observed as a result of these treatments ofArabidopsis plants. By contrast, theAtPSCR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that theAtP5CS gene plays a principal role in the biosynthesis of proline inArabidopsis under osmotic stress.

Ethylene Production and Growth Accerelation in the Seedlings of Dwarf and Semidwarf Mutants Derived from a Cultivar of Rice (Oryza sativa L.).

The rate of seedling growth of the semidwarf mutant cultivar Reimei and the dwarf mutant line, Fukei 71 were found to be much faster than their original cultivar Fujiminori. Such rapid early growth of the dwarf and semidwarf mutants was significantly inhibited by the treatment with the ethylene synthesis inhibitor, aminoethoxyvinylglycine (AVG) . For example, with 25μM of AVG treatment the elongation was inhibited by 36% and 41% during the growth of Reimei and Fukei 71 seedlings, respectively, as compared with 13% for that of Fujiminori. Gas chromatography showed that ethylene production in Reimei and Fukei 71 were about 2.5 and 7.1-fold higher than that of Fujiminori, respectively. It was suggested that the stimulation of the growth in dwarf and semidwarf mutants might be derived from the increased ethylene production.