Abiotic stress regulates expression of galactinol synthase genes post-transcriptionally through intron retention in rice.

MAIN CONCLUSION: Expression of the Galactinol synthase genes in rice is regulated through post-transcriptional intron retention in response to abiotic stress and may be linked to Raffinose Family Oligosaccharide synthesis in osmotic perturbation. Galactinol synthase (GolS) is the first committed enzyme in raffinose family oligosaccharide (RFO) synthesis pathway and synthesizes galactinol from UDP-galactose and inositol. Expression of GolS genes has long been implicated in abiotic stress, especially drought and salinity. A non-canonical regulation mechanism controlling the splicing and maturation of rice GolS genes was identified in rice photosynthetic tissue. We found that the two isoforms of Oryza sativa GolS (OsGolS) gene, located in chromosomes 3(OsGolS1) and 7(OsGolS2) are interspersed by conserved introns harboring characteristic premature termination codons (PTC). During abiotic stress, the premature and mature transcripts of both isoforms were found to accumulate in a rhythmic manner for very small time-windows interrupted by phases of complete absence. Reporter gene assay using GolS promoters under abiotic stress does not reflect this accumulation profile, suggesting that this regulation occurs post-transcriptionally. We suggest that this may be due to a surveillance mechanism triggering the degradation of the premature transcript preventing its accumulation in the cell. The suggested mechanism fits the paradigm of PTC-induced Nonsense-Mediated Decay (NMD). In support of our hypothesis, when we pharmacologically blocked NMD, the full-length pre-mRNAs were increasingly accumulated in cell. To this end, our work suggests that a combined transcriptional and post transcriptional control exists in rice to regulate GolS expression under stress. Concurrent detection and processing of prematurely terminating transcripts coupled to repressed splicing can be described as a form of Regulated Unproductive Splicing and Translation (RUST) and may be linked to the stress adaptation of the plant, which is an interesting future research possibility.

Functional characterization of two myo-inositol-1-phosphate synthase (MIPS) gene promoters from the halophytic wild rice (Porteresia coarctata).

MAIN CONCLUSION: The promoter deletion mutants from second isoform of INO1 (gene-encoding MIPS) from Porteresia coarctata of 932 bp (pPcINO1.2.932) and 793 bp (pPcINO1.2.793) prove to be very efficient as salt/drought stress-inducible promoters, while pPcINO1.2.932 is found to be responsive to cold stress as well. The promoters of the two identified myo-inositol-1-phosphate synthase (INO1) isoforms from salt-tolerant wild rice, Porteresia coarctata (PcINO1.1 and PcINO1.2) have been compared bioinformatically with their counterparts present in the salt-sensitive rice, Oryza sativa. PcINO1.2 promoter was found to be enriched with many abiotic stress-responsive elements, like abscisic acid-responsive elements, MYC-responsive elements, MYB-binding sites, low-temperature stress-responsive elements, and heat-shock elements similar to the ones found in the conserved motifs of the promoters of salt/drought stress-inducible INO1 promoters across Kingdom Planta. To have detailed analysis on the arrangement of cis-acting regulatory elements present in PcINO1 promoters, 5' deletion mutational studies were performed in dicot model plants. Both transient as well as stable transformation methods were used to check the influence of PcINO1 promoter deletion mutants under salt and physiologically drought conditions using ß-glucuronidase as the reporter gene. The deletion mutant from the promoter of PcINO1.2 of length 932 bp (pPcINO1.2.932) was found to be significantly upregulated under drought stress and also in cold stress, while another deletion mutant, pPcINO1.2.793 (of 793 bp), was significantly upregulated under salt stress. P. coarctata being a halophytic species, the high inducibility of pPcINO1.2.932 upon exposure to low-temperature stress was an unexpected result.

An evolutionary analysis identifies a conserved pentapeptide stretch containing the two essential lysine residues for rice L-myo-inositol 1-phosphate synthase catalytic activity.

A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-myo-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, Oryza sativa (OsMIPS1). Following this study, OsMIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity.

Significance of galactinol and raffinose family oligosaccharide synthesis in plants.

Abiotic stress induces differential expression of genes responsible for the synthesis of raffinose family of oligosaccharides (RFOs) in plants. RFOs are described as the most widespread D-galactose containing oligosaccharides in higher plants. Biosynthesis of RFOs begin with the activity of galactinol synthase (GolS; EC 2.4.1.123), a GT8 family glycosyltransferase that galactosylates myo-inositol to produce galactinol. Raffinose and the subsequent higher molecular weight RFOs (Stachyose, Verbascose, and Ajugose) are synthesized from sucrose by the subsequent addition of activated galactose moieties donated by Galactinol. Interestingly, GolS, the key enzyme of this pathway is functional only in the flowering plants. It is thus assumed that RFO synthesis is a specialized metabolic event in higher plants; although it is not known whether lower plant groups synthesize any galactinol or RFOs. In higher plants, several functional importance of RFOs have been reported, e.g., RFOs protect the embryo from maturation associated desiccation, are predominant transport carbohydrates in some plant families, act as signaling molecule following pathogen attack and wounding and accumulate in vegetative tissues in response to a range of abiotic stresses. However, the loss-of-function mutants reported so far fail to show any perturbation in those biological functions. The role of RFOs in biotic and abiotic stress is therefore still in debate and their specificity and related components remains to be demonstrated. The present review discusses the biology and stress-linked regulation of this less studied extension of inositol metabolic pathway.