Natural Variation Of Arabidopsis Thaliana Accessions In Response To Salinity Stress

Salinity stress is one of the most serious environmental constraints to plant growth and crop productivity. It causes ionic and osmotic stresses, and the inhibition of leaf expansion, restricts photosynthesis and limits the accumulation of biomass. In response to salinity stress, plants employed many adaptive stratigies such as the active exclusion of sodium (Na4) ions and/or their sequestration into the vacuole, the production of compatible solutes and the ROS scavenge of reactive oxygen species (ROS). Understanding the physiological and molecular mechanisms of salt tolerance not only has important scientific significance, but also provides important theoretical guidance for plant salt resistant breedings in the future.Arabidopsis thaliana as a model plant for scientific research, the Arabidopsis accessions are widely distributed in various parts of the world and diversified. Therefore, different Arabidopsis accessions were widely used to study the mechanism of plant adaption to various surrounding environmental stresses. It was previously reported that salt tolerance is a complex trait, which is controlled by multiple genes and involves various biochemical and physiological mechanisms. Hovever, so far, it is not well known about different Arabidopsis accessions of natural variation in response to salt stress and its regulation mechanisms, and this still needs further study.Using 82 diverse accessions in our lab as experiment materials and Col-0 as the reference accession, seven tolerant accessions were selected under salt stress condition. To explore the role of natural variation in salt tolerance and its regulation mechanisms, the physiological and biochemical characteristics of these selected accesions were studied in response to salt stress. The main results and conclusions were as follows:1. Identification of salinity tolerant accessions and the physiological basis of their toleranceEighty-two Arabidopsis accessions were collected from Europe, Asia, East Africa and North America, etc. These collected accessions were challenged by growing the plants on a medium containing 150 or 200 mM NaCl, with Col-0 as the reference accession, seven entries from Europe comprised of Bs-1 (Switzerland), Mog-11 (France), Looe-2 (UK), Got-1 (Germany), Wi1-1 (Russia), Nd-1 (Germany), and Sav-0 (Czech Rep), were selected as the salt-tolerant ones in this study. To further evaluate their response to salt stress, agar medium supplemented with four different NaCl concentrations (0,150,180 and 200 mM) were used. As a result, the selected accessions exhibited obvious tolerant phenotypes under the above stress conditions. In addition, the improved tolerance of these selections was also confirmed by using soil or hydroponics assays. The tolerance of the selected seven accessions was manifested by a better ability to maintain root growth, a weaker tendency for the cotyledons to become bleached and higher chlorophyll content than that of Col-0. Moreover, the selections had less malondialdehyde (MDA) accumulation under salt stress, indicating the decreased cell membrane damage compared to Col-0. Therefore, these results indicated that the selected seven accessions have higher salt tolerance than Col-0.2. The Na+and K+ homeostasis control in the selected salt tolerant accessionsPreviously, it was reported that the maintenance of high K+levels and a low Na+/K+ratio in the cytoplasm were essential for salt tolerance. The results showed that there was no discernible difference in the Na+contents of the whole plants harvested from the selected accessions compared to Col-0, whether or not the plants had been exposed to salinity. This result indicated that Na+level was not a key factor for the elevated salinity tolerance. Although the K+contents of all seven accessions were reduced under salinity stress, they were still able to maintain a higher level than that was achieved by Col-0, maintaining a lower Na+/K+ratio than that of the standard accession Col-0. This result suggested that the maintenance of higher K+contents in the tolerant selections contributed significantly to higher salinity tolerance.Then the net K+flux among all the selected accessions were measured by using non-invasive micro-test technology. It was found that all the tested accessions have K+ efflux whether treated with salt stress or not. However, the salt-tolerant selections showed less K+effluxes than Col-0 under salt stress, suggesting that the higher K+ content in the selections corresponded to the decreased K+effluxes in them. The salt tolerant accessions might have higher ability to retain K+or prevent K+ loss under salt stress. We further analyzed the transcriptional levels of some important K+ transporter coding genes such as AtHAK5, AtCHX17 and AtKUPl by qRT-PCR. The comparative analysis of AtHAK.5, AtCHX17 and AtKUPl expression levels indicated that there is a positive relationship between the expression levels of these genes and high K+ content in the selected accessions. The AtHAK5 transcript abundance was significantly higher than in Col-0 among the selected salt tolerant accessions after salt treatment, showing a more than 10-fold increase in Mog-11 and Got-1 compared to that of Col-0. AtCHX17 and AtKUP1 were both inducible obviously by salinity, especially in the tolerant accessions including Mog-11, Looe-2 and Wil-1.The SOS signaling pathway related factors, NHX and HKT protein families are important factors for salt tolerance in plants. AtSOSs was reported to export Na+out of the cell and AtNHXl to sequester Na+ within the vacuole, while AtHKT1;1 is essential for root-to-shoot Na+partitioning, thus involve in plant salt tolerance. To examine whether these genes have different roles for confronting with salinity stress in the selected accessions, of which AtSOS genes were re-sequenced, and the sequence comparison among the selected accessions identified that all but one carried the alternative allele to Col-0 at position 1116 (T1116A), which lies within the key C terminal autoinhibitory domain. However, this allele cannot be critical for gene function, as it is also carried by the sensitive accession. Similarly, there were also no critical variations in the AtSOS2, AtSOS3, AtNHX1 and AtHKT1;1 of the selected accessions. Moreover, the transcription levels of AtSOS genes analyzed by qRT-PCR in the tolerant selections showed no significant changes when challenged by NaCl, thus, it was concluded that there was no obvious difference in the efficiency of regulating Na+ efflux at cellular level among the tested accessions, which correspond to the similar Na+ levels of the tested accessions. In addition, the transcription level of AtNHX1 decreased in Mog-11, Looe-2 and Sav-0 when exposed to 100 mM NaCl for 3 h, and the AtHK1;1 gene was expressed obviously lower in most of the tolerant ones than in Col-0 when exposed to salt stress. In all, our results supported that the SOS signaling pathway, as well as AtNHX1 and AtHK1;1, could be as a fundamental and conserved mechanism to the elevated salinity tolerance displayed by the selected accessions, but not a key special factor to distinct them from Col-0.3. The different accumulation of compatible solutes in the salt tolerant accessionsThe accumulation of compatible solutes biosynthesized in plant could protect cell from damage, especially during salinity stress. In this study, an elevated level of such solutes was found in all tolerant accessions under salinity stress, in particular that of proline. Furthermore, consistent with this physiological difference, the lower transcription kinetics of two proline degradation-related genes AtProDHl and AtP5CDH were noted in the tolerant accessions, yet the proline biosynthsis-related genes AtPSCR, AtP5CSl and AtP5CS2 were not changed positively compared to that of Col-0.4. The different red ox response after salt treatment in the salt tolerant accessionsROS is one of important signaling molecules mediated the response of salinity stress. In order to clarify the mechanism of ROS signaling pathway involving in salinity stress in the selected accessions, we examined the ROS levels and analysized the expression profile of some relevant genes in the tolerant selections and Col-0. The results showed that the tolerant accessions indeed exhibited a lower level of ROS than Col-0 detected by NBT and DAB staining. Moreover, using the superoxide dismutase activity assay, a higher SOD activity was found in the tolerant accessions compared to Col-0. Furthermore, a higher transcript level of AtCSDl encoding one type of SOD, was found in all tolerant accessions (except Mog-11). Similarly, there were also higher transcript abundance of AtCAT2 and AtAPX2 in them, especially, the high expression levels of AtAPX2 emerged remarkably in Mog-11, Wil-1 and Sav-0. Moreover, two upstream transcription factors AtZAT10 and AtZA12 in the ’ROS gene network’, were also up-regulated significantly in the tolerant accessions. Therefore, we concluded that the tolerant accessions all have the stronger ability of ROS scavengers, which is one of mechanisms of higher salinity tolerance.