Functional Characterization of the Stipa purpurea P5CS Gene under Drought Stress Conditions

Free proline has multiple functions in plant cells, such as regulating osmotic potential and protecting both proteins and cell membranes. The expression of Δ1-Pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway, increases under drought, salt and cold stress conditions, causing plant cells to accumulate large amounts of proline. In this study, we cloned and identified the P5CS gene from Stipa purpurea, which has a full-length of 2196 bp and encodes 731 amino acids. A subcellular localization analysis indicated that SpP5CS localized to the cytoplasm. The ectopic overexpression of SpP5CS in Arabidopsis thaliana resulted in higher proline contents, longer roots, higher survival rates and less membrane damage under drought stress conditions compared with wild-type controls. SpP5CS-overexpressing A. thaliana was more resistant to drought stress than the wild type, whereas the deletion mutant sp5cs was less resistant to drought stress. Thus, SpP5CS may be a potential candidate target gene for increasing plant resistance to drought stress.

Uncovering the role of a positive selection site of wax ester synthase/diacylglycerol acyltransferase in two closely related Stipa species in wax ester synthesis under drought stress

Natural selection drives local adaptations of species to biotic or abiotic environmental stresses. As a result, adaptive phenotypic divergence can evolve among related species living in different habitats. However, the genetic foundation of this divergence process remains largely unknown. Two closely related alpine grass species, Stipa capillacea and Stipa purpurea, are distributed in different rainfall regions of northern Tibet. Here, we analyzed the drought tolerance of these two closely related Stipa species, and found that S. purpurea was more resistance to drought stress than S. capillacea. To further understand the genetic diversity behind their adaptation to drought environments, a comprehensive gene repertoire was generated using PacBio isoform and Illumina RNA sequencing technologies. Bioinformatics analyses revealed that differential transcripts were mainly enriched in the wax synthetic pathway, and a threonine residue at position 239 of WSD1 was identified as having undergone positive selection in S. purpurea. Using heterologous expression in the Saccharomyces cerevisiae mutant H1246, site-directed mutagenesis studies demonstrated that a positive selection site results in changes to the wax esters profile. This difference may play an important role in S. purpurea in response to drought conditions, indicating that S. purpurea has evolved specific strategies involving its wax biosynthetic pathway as part of its long-term adaptation to the Qinghai–Tibet Plateau.

Seed Germination in Alpine Meadow Steppe Plants from Central Tibet in Response to Experimental Warming

Clarifying the effects of climate warming on seed germination is critical for predicting plant community assembly and species renewal, especially in alpine grassland ecosystems where warming is occurring faster than in other biomes globally. We collected matured seeds of 19 common species from a typical alpine meadow steppe community in Central Tibet. Seeds were germinated in three incubators with manipulated day-night temperatures to impose three treatments: (1) theoretically optimal values of 25/15 °C, (2) temperatures observed in the field (control), and (3) a warming of 3 °C above the observed temperatures. We calculated seed germination percentage (SGP) and mean germination time (MGT) per species at different treatments. Our results showed that SGPs of Stipa capillacea, Kobresia macrantha, Potentilla saundersiana, Saussurea tibetica, Pedicularis kansuensis, and Androsace graminifolia were higher under the warming treatment than under control. Among them, the MGTs of S. capillacea, K. macrantha, and And. graminifolia were significantly shortened, while the MGT of Pe. kansuensis was significantly lengthened by warming of 3 °C. Significant decreases in MGT induced by warming were only observed for Festuca coelestis and Anaphalis xylorhiza. Additionally, the treatment with theoretically optimal temperatures restrained germination of Stipa purpurea, S. capillacea, F. coelestis, and Sa. tibetica seeds but promoted germination of K. macrantha, Astragalus strictus, P. saundersiana, Potentilla bifurca, Pe. kansuensis, Swertia tetraptera, Pleurospermum hedinii, and And. Graminifolia seeds, when compared with the control and warming treatments. Therefore, the response of seed germination to warming differs among alpine species, implying that future warming could result in significant changes in community assembly of alpine grasslands on the Tibetan Plateau.

Cold tolerance of ScCBL6 is associated with photosynthesis and tonoplast transporters in Arabidopsis

Plant adapted in the fragile zone offer enormous opportunity to understand the stress biology under ecological scenario . Stipa capillacea is widely distributed in the frigid and arid region of Tibet Plateau, but its signal system has never been investigated yet. In this study, we isolated a signal transduction gene, ScCBL6 , in Stipa capillacea , to characterize its cold tolerance capacity by ectopic expression in Arabidopsis . The results suggested that full length ScCBL6 encodes 227 amino acids, and phylogenetically clustered with CBL6 protein in Stipa purpurea and Oryza sativa . In comparison with wild type (WT) plants, ScCBL6 overexpressing plants ( ScCBL6- OXP) are tolerant to cold stress but not drought stress, which attested by the higher photosynthetic capacity (Fv/Fm) and survival rate of ScCBL6- OXP under cold stress. We further compared their cold-responsive transcriptome profiles through RNA-Seq. Totally, 3931 genes were differentially expressed by introduction of ScCBL6. They are participated in multiple processes like immune system, lipid catabolic, secondary metabolic and mainly enriched in plant hormone signal transduction and biomacro-molecule metabolism as regard to KEGG pathway. Differential expressed genes (DEGs) were predicted to locate in chloroplast, mitochondrion, vacuole, and so on, suggesting multitudinous function of ScCBL6. Based on the integrated analysis of ScCBL6-OXP, we inferred that ScCBL6 improve plant cold stress tolerance via regulate photosynthesis redox and vacuole metabolites transport in Arabidopsis .