Stachyose triggers apoptotic like cell death in drought sensitive but not resilient plants

Programmed cell death (PCD) is one of the most intensively researched fields in modern mammalian biology with roles in cancer, aging, diabetes and numerous neurodegenerative diseases. It is becoming increasingly clear that PCD also plays significant roles in plant defence and responses to the environment. Given their unique ability to tolerate desiccation (cells remain viable even after they’ve lost 95% of their water), resurrection plants make ideal models to study the regulation of plant PCD pathways. Previously, we showed that the Australian resurrection plant, Tripogon loliiformis, suppresses plant PCD, via trehalose-mediated activation of autophagy pathways, during drying. In the present study, we created a full-length T. loliiformis cDNA library, performed a large-scale Agrobacterium screen for improved salinity tolerance and identified Stachyose synthase (TlStach) as a potential candidate for improving stress tolerance. Tripogon loliiformis shoots accumulate stachyose synthase transcripts and stachyose during drying. Attempts to generate transgenic plants expressing TlStach failed and were consistent with previous reports in mammals that demonstrated stachyose-mediated induction of apoptosis. Using a combination of transcriptomics, metabolomics and cell death assays (TUNNEL and DNA laddering), we investigated whether stachyose induces apoptotic-like cell death in T. loliiformis. We show that stachyose triggers the formation of the hallmarks of plant apoptotic-like cell death in the desiccation sensitive Nicotiana benthamiana but not the resilient T. loliiformis. These findings suggest that T. loliiformis suppresses stachyose-mediated apoptotic-like cell death and provides insights on the role of sugar metabolism and plant PCD pathways. A better understanding of how resilient plants regulate sugar metabolism and PCD pathways may facilitate future targeting of plant metabolic pathways for increased stress tolerance.

Response of Alfalfa (Medicago sativa L.) to Abrupt Chilling as Reflected by Changes in Freezing Tolerance and Soluble Sugars

Abrupt-chilling events threaten the survival of alfalfa plants, the ability to cope with such condition should be considered during cultivar selection in the production. To assess biochemical and molecular responses of alfalfa to abrupt chilling, the cultivars “WL440HQ” (WL) and “ZhaoDong” (ZD) were subjected to a five-phase experimental regime that included two abrupt-chilling events. The freezing tolerance of the crown was determined as the semi-lethal temperature (LT50) calculated from electrolyte leakage. Soluble sugar concentrations were quantified by ion chromatography. The mRNA transcript levels of four genes encoding enzymes (β-amylase, sucrose phosphate synthase, galactinol synthase, and stachyose synthase) involved in sugar metabolism and two cold-regulated genes (Cas15A and K3-dehydrin) were quantified using quantitative real-time PCR analysis. During the abrupt-chilling events, the LT50 decreased significantly in ZD but not in WL. The rapid response of ZD to abrupt chilling may have been due to the large increases in raffinose and stachyose concentrations, which were consistent with increased transcript levels of the galactinol synthase and stachyose synthase genes. Transcript levels of the cold-regulated genes Cas15A and K3-dehydrin were correlated with increased freezing tolerance under abrupt chilling. The results provide a reference for selection of appropriate cultivars to reduce the risk of crop damage in production areas where early autumn or late spring frosts are likely.

Biosynthesis of raffinose family oligosaccharides and galactosyl pinitols in developing and maturing seeds of winter vetch (Vicia vlllosa Roth.)

Changes in the accumulation of two types of α-D-galactosides: raffinose family oligosaccharides and galactosyl pinitols were compared with changes in the activities of galactosyltransferases during winter vetch (<em>Vicia villosa</em> Roth.) seed development and maturation. Occurrence of galactinol and raffinose in young seeds and changes in activities of galactinol synthase and raffinose synthase during seed development indicated that formation of raffinose oligosaccharides (RFOs) preceded synthesis of galactopinitols. Although transfer of galactose residues into raffinose oligosaccharides increased as seeds were maturing, at late stages of seed maturation the accumulation of galactopinitols was preferred to that of RFOs. In the present study, activities of enzymes transferring galactose moieties from galactinol to D-pinitol forming galactopinitol A, and further transfer of galactose moieties from galactinol to mono- and di-galactopinitol A were detected throughout seed development and maturation. This is a new observation, indicating biological potential of winter vetch seeds to synthesize mono-, di- and tri-galactosides of D-pinitol in a pathway similar to RFOs. The pattern of changes in activities of stachyose synthase and enzymes synthesizing galactopinitols (named galactopinitol A synthase and ciceritol synthase) suggests that formation of stachyose, mono- and di-galactopinitol A (ciceritol) is catalyzed by one enzyme. High correlation between activities of verbascose synthase and enzyme catalyzing synthesis of tri-galactopinitol A from galactinol and ciceritol (named tri-galactopinitol A synthase) also suggests that biosynthesis of both types of tri-galactosides was catalyzed by one enzyme, but distinct from stachyose synthase. Changes in concentrations of galactosyl acceptors (sucrose and D-pinitol) can be a factor which regulates splitting of galactose moieties between both types of galactosides in winter vetch seeds.