Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato

Asr1, Asr2 andAsr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal thatAsr1, Asr2 andAsr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F₂ population derived from an interspecific hybrid crossL. esculentum × L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of theAsr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.     

Epigenetic marks in an adaptive water stress-responsive gene in tomato roots under normal and drought conditions

Tolerance to water deficits was evolutionarily relevant to the conquest of land by primitive plants. In this context, epigenetic events may have played important roles in the establishment of drought stress responses. We decided to inspect epigenetic marks in the plant organ that is crucial in the sensing of drought stress: the root. Using tomato as a crop model plant, we detected the methylated epialleles of Asr2, a protein-coding gene widespread in the plant kingdom and thought to alleviate restricted water availability. We found 3 contexts (CG, CNG, and CNN) of methylated cytosines in the regulatory region of Solanum lycopersicum Asr2 but only one context (CG) in the gene body. To test the hypothesis of a link between epigenetics marks and the adaptation of plants to drought, we explored the cytosine methylation status of Asr2 in the root resulting from water-deficit stress conditions. We found that a brief exposure to simulated drought conditions caused the removal of methyl marks in the regulatory region at 77 of the 142 CNN sites. In addition, the study of histone modifications around this model gene in the roots revealed that the distal regulatory region was rich in H3K27me3 but that its abundance did not change as a consequence of stress. Additionally, under normal conditions, both the regulatory and coding regions contained the typically repressive H3K9me2 mark, which was lost after 30 min of water deprivation. As analogously conjectured for the paralogous gene Asr1, rapidly acquired new Asr2 epialleles in somatic cells due to desiccation might be stable enough and heritable through the germ line across generations, thereby efficiently contributing to constitutive, adaptive gene expression during the evolution of desiccation-tolerant populations or species.     

Low Sugar Is Not Always Good: Impact of Specific O-Glycan Defects on Tip Growth in Arabidopsis

Mutants of the O-glycosylation pathway of extensins as well as molecular dynamics simulations uncover the effects of the O-glycosylation machinery on root hair tip growth.The hydroxyproline-rich O-glycoproteins (HRGPs) comprise several groups of O-glycoproteins, including extensins (EXTs), ultimately secreted into plant cell walls. The latter are shaped by several posttranslational modifications, mainly hydroxylation of Pro residues into Hyp and further O-glycosylation on Hyp and Ser (Supplemental Fig. S1A). EXTs contain several Ser-(Hyp)₄ repeats, usually O-glycosylated with chains of up to four or five linear arabinosyl units on each Hyp (Velasquez et al., 2011; Ogawa-Ohnishi et al., 2013) and monogalactosylated on Ser residues (Saito et al., 2014). O-Glycosylated Ser-(Hyp)₄ repeats are not only present in EXTs but can potentially decorate several other EXT-like chimeras and hybrid EXT glycoproteins that contain other domains, such as arabinogalactan protein-EXTs, Pro-rich protein-EXTs, Leu-rich repeat-EXTs, Pro-rich kinases, and formins with an extracellular EXT domain, etc. In addition, Hyp-O-arabinosylation also occurs in single Hyp units in the small secreted glycopeptide hormones (e.g. CLAVATA3 [CLV3]) with up to three arabinosyl units (Ohyama et al., 2009; Matsubayashi, 2010; Shinohara and Matsubayashi, 2013). In this context, three groups of arabinosyltransferases (AraTs), hydroxyproline O-arabinosyltransferase1 (HPAT1) to HPAT3 (classified as GT8 in the Carbohydrate Active Enzymes database), Reduced Residual Arabinose1 (RRA1) to RRA3, and Xyloglucanase113 (XEG113; GT77 family), have recently been implicated in the sequential addition of the innermost three l-arabinosyl residues (Egelund et al., 2007; Ogawa-Ohnishi et al., 2013; Supplemental Table S1). In addition, one novel peptidyl-Ser galactosyltransferase named SERGT1 has been reported to add a single α-galactopyranose residue to each Ser residue in Ser-(Hyp)₄ motifs of EXTs, thus belonging to the GT96 family within the Carbohydrate Active Enzymes database (Supplemental Table S1). Finally, glycosylated EXTs are possibly cross-linked by putative type III peroxidases at the Tyr residues, forming EXT linkages (Cannon et al., 2008) able to build a three-dimensional network likely to interact with other cell wall components like pectins (Cannon et al., 2008). EXT assembly into a putative glycoprotein network seems to be crucial for cell expansion of root hair, and several EXT and EXT-related mutants (e.g. ext6-7, ext10-12, and Leu-rich repeat extensin1, etc.) were previously isolated with abnormal root hair cell expansion phenotypes (Ringli, 2010; Velasquez et al., 2011).Here, by using mutants of several known enzymes of the O-glycosylation pathway of HRGPs, we addressed to what extent each specific defect on the O-glycosylation machinery impacts root hair tip growth. In addition, we refer only to Hyp-O-arabinosylation and Ser-O-galactosylation modifications of EXT and EXT-related proteins, while we have excluded Hyp O-(arabino)galactosylation, commonly present in other types of HRGP like arabinogalactan proteins, from our analysis. Finally, by molecular dynamic simulations, we propose a possible model to explore how these two specific types of O-glycan defects would affect EXT self-assembly and, ultimately, their impact on the polarized cell expansion. We use a classical EXT repetitive sequence to begin to explore how O-glycosylation might affect glycoprotein conformation and possible self-interactions in the context of polarized growth, but we are aware of the complexity and diversity of EXT and EXT-related proteins that offers several other possible scenarios.     

Dimerization and DNA-binding of ASR1, a small hydrophilic protein abundant in plant tissues suffering from water loss

The Asr gene family is present in Spermatophyta. Its members are generally activated under water stress. We present evidence that tomato ASR1, one of the proteins of the family, accumulates in seed during late stages of embryogenesis, a physiological process characterized by water loss. In vitro, electrophoretic assays show a homo-dimeric structure for ASR1 and highlight strong non-covalent interactions between monomers prone to self-assemble. Direct visualization of single molecules by atomic force microscopy (AFM) confirms that ASR1 forms homodimers and that uncovers both monomers and dimers bind double stranded DNA.     

ASR1, a stress-induced tomato protein, protects yeast from osmotic stress

Asr1 , a tomato gene induced by abiotic stress, belongs to a family, composed by at least three members, involved in adaptation to dry climates. To understand the mechanism by which proteins of this family seem to protect cells from water loss in plants, we expressed Asr1 in the heterologous expression system Saccharomyces cerevisiae under the control of a galactose-inducible promoter. In a mutant yeast strain deficient in one component of the stress-responsive high-osmolarity glycerol (HOG) pathway, namely the MAP kinase Hog 1, the synthesis of ASR1 protein restores growth under osmotic stress conditions such as 0.5  M NaCl and 1.2  M sorbitol. In contrast, the rescuing of this phenotype was less evident using a wild-type strain or the upstream MAP kinase kinase (Pbs2)-deficient strain. In both knock-out strains impaired in glycerol synthesis because of a dysfunctional HOG pathway, but not in wild-type, ASR1 led to the accumulation of endogenous glycerol in an osmotic stress-independent and unrestrained manner. These data suggest that ASR1 complements yeast HOG-deficient phenotypes by inducing downstream components of the HOG pathway. The results are discussed in terms of the function of ASR proteins in planta at the molecular and cellular level.