Physiological and molecular mechanisms of tolerance to salinity, alkalinity and alkaline salinity in Brassica juncea

© 2012 Dr. Muhammad JavidSoil characteristics such as alkalinity and salinity are common constraints to crop productivity in arid and semi-arid regions of Australia. B. juncea is recognized as a promising oilseed crop because of its moderate drought and salinity tolerance, resistance to pod shattering and blackleg disease. However, the combined effect of alkalinity and salinity has not been assessed in this important Brassica species. Hence, the aim of this study was to elucidate physiological processes linked to molecular functions involved in the tolerance mechanism(s) to bicarbonate (HCO3-) and NaCl stresses. Initially, a broad range of B. juncea germplasm was hydroponically screened for the combined stresses of 5 mM HCO3- (pH 8.5) and 50 mM NaCl under a controlled growth environment. On the basis of plant survival and biomass production under stress relative to the control, cultivars NDR 8501 and Vaibhav were identified as moderately tolerant and Xinyou 5 highly sensitive. A validation experiment with the three selected cultivars and a slightly acidic (pH 6.0) control showed that combined salinity and alkalinity (alkaline salinity) stress reduced plant growth by 78 to 90% (p≤0.001) in all three accessions. The effects of alkaline salinity were more severe than salinity (10 to 36%) or alkalinity (65 to 76%) alone. The alkalinity level used in the screening and validation experiment (5 mM HCO3-) caused severe impacts on plant growth. A detailed physiological study was conducted to further investigate plant responses of the three selected accessions to a milder alkaline stress (2.5 mM HCO3- / pH 8.25). Even at this moderate level, plant growth was more severely reduced (25 to 33%) than by salinity (9 to 27%). Alkaline salinity reduced growth by 33 to 39% (p≤0.001) and was more damaging to growth than salinity alone. Although the salt-tolerant accessions NDR 8501 and Vaibhav performed significantly better compared to the sensitive accession Xinyou 5 under salinity alone, results demonstrated that alkaline salinity severely compromised the response of the identified salt tolerant cultivars. All three accessions showed partial sodium (Na+) exclusion under both salinity and alkaline salinity. However Na+ concentrations under salinity expressed on a tissue water basis, increased to nearly 50 mM in both NDR 8501 and Vaibhav and 86 mM in Xinyou 5, relative to the control. Similarly, leaf Na+ concentration under alkaline salinity increased to 80, 95 or 131 mM in NDR 8501, Vaibhav and Xinyou 5, respectively. NDR 8501 excluded Na+ more efficiently than the other two accessions under alkaline salinity, but only grew marginally better than them. A need for further screening of B. juncea at moderate alkalinity appears essential to identify better adapted B. juncea accessions to low rainfall areas with alkaline soils or subsoils, with or without additional saline stress. To determine molecular mechanism(s) potentially controlling an adaptive tolerance response to alkalinity, salinity and the combined treatment, the moderately tolerant cv. NDR 8501 was further investigated using microarray analyses. The transcripts of treated leaf tissues vs. those of the untreated control sample were analysed after four weeks of stress. Whilst many differential genes were shared among the stress responses, many were unique to a particular treatment and a far greater number of genes were uniquely differentially transcribed (1107) when alkalinity was part of the stress complex, as opposed to solely salinity (415). Salinity tolerance in NDR 8501 was proposed to be contributed by a member of the Na+/Ca2+ exchanger protein family and a potassium ion transmembrane transporter for intracellular regulation of Ca2+ and K+. Na+ exclusion and ion homeostasis was potentially achieved by induced vacuolar ATPase and small GTPase genes. These vacuolar ATPase and small GTPase genes transcribed in response to salinity however were not transcribed under alkalinity or the combined treatment. Similarly, vacuolar ATPase; AVA-P2 and VHA-B1 were only transcribed and up-regulated when alkalinity was present in the stress complex with salinity. Microarray results were validated for specificity and reproducibility by quantitative real time polymerase chain reactions. These molecular responses clearly demonstrated that different genes and genetic pathways are involved in responses to alkalinity, salinity or the combined stress, and that the effects of alkalinity dominate when the two stresses are combined. They also provide supporting evidence for the physiological responses that occur in the plant, with massive reprogramming of the transcriptome leading to partial ion exclusion, shuttling and compartmentation. Following further functional validation, genes identified in the key pathways may be targeted for future selective breeding purposes or for molecular manipulation to enhance tolerance to these subsoil constraints in B. juncea and possible relative species