Defining Transcriptional Networks Associated with Plant Salinity Tolerance

Abstract

Salinity is a major issue for the sustainability of agriculture worldwide. Salinity causes an initial hyperosmotic stress and subsequently, secondary nutritional imbalance and oxidative stress through ion toxicity. Many studies focus on identifying genes and the molecular mechanisms involved in salinity tolerance. The identification of such genes may then be used in the development of more salt tolerant crops required for a sustainable global food production. Calcineurin B-like protein interacting protein kinases (CIPKs) are key regulators of pretranscriptional and post-translational responses to abiotic stress. Arabidopsis thaliana CIPK16 (AtCIPK16) was identified from a forward genetic screen as a candidate gene that mediates lower shoot salt accumulation and improves salinity tolerance in Arabidopsis and transgenic barley. However, relatively little is known about the pathways in which CIPK16s operate to affect salinity tolerance and even about the presence of orthologues in cereals. A transcriptomic study was conducted using Arabidopsis thaliana plants subjected to salt stress. The experiment included overexpressing AtCIPK16 and null transgenic plants that were salt stressed or controls. Our analysis characterizes the transcriptional landscape of AtCIPK16 overexpression dependent salt responsiveness in Arabidopsis. These transgene-dependent salt responsive genes suggest an involvement of transcription factors and phytohormones, such as ethylene, jasmonic acid and auxin in downstream signaling pathways. Whether these transcription factors and possible hormone changes have an impact on the plants’ physiological aspect needs to be experimentally determined. Although enhanced salt tolerance has been demonstrated in transgenic barley plants overexpressing AtCIPK16, the presence of a CIPK16 orthologue in barley has not been established. The second part of the project therefore was involved with a molecular phylogenetic analysis of CIPK16 homologues in terrestrial plant species. We mined genome sequence databases, including monocot and dicot species, for CIPK16 homologues. The subsequent phylogenetic analysis revealed a clade containing AtCIPK16 along with two segmentally duplicated CIPKs: AtCIPK5 and AtCIPK25. We found no evidence for an AtCIPK16 orthologue in any monocots but instead found homologues which formed a group basal to the entire CIPK16, 5 and 25 clade. Our analyses also revealed that CIPK16s contain a unique inDel (MMPEGLGGRRG) and a putative nuclear localization signal (PPTKKKKKD). Whether these synapomorphic characters have a biological function will require further experimental validation.We investigated the transcriptome of a subset of six barley cultivars with varying Na+ accumulation in the leaf blade and sheath using the RNA-Seq data generated for the leaf blade, leaf sheath and root tissues from plants grown in saline conditions. Based on prior knowledge we specifically investigated genes involved in sodium transport and salt response and examined their expression and genetic variation (SNPs and indels) across the 6 accessions. Our results showed that allelic variations in HvHKT1;5 may be one of the crucial factors in determining the level of Na+ in the shoots of barley. We hypothesise that for high shoot Na+ accumulating cultivars such as Alexis, Commander and Maritime genes such as HvNHXes (e.g. HvNHX4) may play a role in dealing with high levels of Na+, through sequestrating Na+ into the vacuole or K+ homeostasis.