OSM1/SYP61: A Syntaxin Protein in Arabidopsis Controls Abscisic Acid–Mediated and Non-Abscisic Acid–Mediated Responses to Abiotic Stress

Abstract

To identify the genetic loci that control salt tolerance in higher plants, a large-scale screen was conducted with a bialaphos marker-based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line, osm1 (for osmotic stress-sensitive mutant), exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root-bending assay. The osm1 mutant displayed a more branched root pattern with or without stress and was hypersensitive to inhibition by Na ؉ , K ؉ , and Li ؉ but not Cs ؉ . Plants of the osm1 mutant also were more prone to wilting when grown with limited soil moisture compared with wild-type plants. The stomata of osm1 plants were insensitive to both ABA-induced closing and inhibition of opening compared with wild-type plants. The T-DNA insertion appeared in the first exon of an open reading frame on chromosome 1 ( F3M18.7 , which is the same as AtSYP61 ). This insertion mutation cosegregated closely with the osm1 phenotype and was the only functional T-DNA in the mutant genome. Expression of the OSM1 gene was disrupted in mutant plants, and abnormal transcripts accumulated. Gene complementation with the native gene from the wild-type genome completely restored the mutant phenotype to the wild type. Analysis of the deduced amino acid sequence of the affected gene revealed that OSM1 is related most closely to mammalian syntaxins 6 and 10, which are members of the SNARE superfamily of proteins required for vesicular/target membrane fusions. Expression of the OSM1 promoter:: ␤ -glucuronidase gene in transformants indicated that OSM1 is expressed in all tissues except hypocotyls and young leaves and is hyperexpressed in epidermal guard cells. Together, our results demonstrate important roles of OSM1/SYP61 in osmotic stress tolerance and in the ABA regulation of stomatal responses.

TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis

“…Thus, vesicle fusion appears to directly or indirectly affect salt tolerance at the PVC and vacuoles. By contrast, the tno1 mutant and a mutation in SYP61 (Zhu et al, 2002) showed a salt-sensitive phenotype. TNO1 and SYP61 colocalize to the TGN and are likely to exist in the same protein complex.…”

“…4, A and B). Lack of TNO1 protein expression was confirmed by immunoblotting of total protein extracts from wild-type and tno1 mutant plants using affinity-purified TNO1 antibodies ( SYP61/OSM1, a t-SNARE in the SYP41 complex, has been reported to be involved in general salt/osmotic stress responses (Zhu et al, 2002). As TNO1 also interacts with SYP41, TNO1 was hypothesized to be involved in similar stress responses.…”

“…In the tno1 mutant, less SYP61 colocalized with VHAa1-GFP-labeled TGN compared with wild-type plants, leading to possible reduced SYP61 function in salt tolerance. It was hypothesized that a cation transporter in the osm1/syp61 mutant may not function correctly, affecting guard cell turgor and salt sensitivity (Zhu et al, 2002). Extensive investigations of cation transporters in yeast, plants, and mammalian cells have shown that cation/H + antiporters in the Golgi, TGN, endosome, and PVC regulate the pH and cation concentration in these organelles, affecting vesicle trafficking, and knockdown of VHAa1 in Arabidopsis also leads to increased salt sensitivity (Brett et al, 2002(Brett et al, , 2005Nakamura et al, 2005;Pardo et al, 2006;Fuji et al, 2007;Krebs et al, 2010).…”

“…One-week-old wild-type and tno1 mutant seedlings were treated with 300 mM NaCl, 40 mM LiCl, 300 mM KCl, or 600 mM mannitol for 5 h. Total RNA was extracted from the seedlings, and expression of SOS1, DREB2A, RD29, TNO1, and SYP61 was analyzed by RT-PCR using gene-specific primers (Table I; Yamaguchi-Shinozaki and Shinozaki, 1993a;Liu et al, 1998;Shi et al, 2000;Zhu et al, 2002).…”

“…However, they form separate complexes with AtVPS45 in different subdomains of the TGN and are not functionally redundant, as individual syp41 and syp42 knockout mutants are gametophytic lethal (Sanderfoot et al, 2001b). A mutation in SYP61, another t-SNARE in the SYP41 complex (Zhu et al, 2002), caused defects in osmotic stress tolerance and abscisic acid regulation of stomatal responses. The v-SNARE VTI12 is involved in trafficking of storage proteins (Sanmartín et al, 2007) and a vti12 mutant showed an early-senescence phenotype under starvation conditions, suggesting that VTI12 is also involved in the autophagy pathway for the degradation of cellular contents (Surpin et al, 2003).…”