Proteomic Analysis Of Thellungiella Salsuginea

Salt cress (Thellungiella salsuginea) is a kind of halophyte which belongs to the crucifer family (Brassicaceae), and it is very tolerant to cold, drought, salt and low nitrogen. Meanwhile, it is vey similar to the model species Arabidopsis such as small size, short life cycle, self-fertilization, copious seed production, small genome, and easy to transform and mutagenize. In cDNA level it has 90-95% identity to Arabidopsis, and has emerged as a new model system for abiotic stress study.Research of Thellungiella has made enormous progress over the last few years, and the studies were mainly focused on ion transport, expression profiling, gene identification, and some physiological analysis. These studies have got the preliminary knowledge of stress tolerance in Thellungiella. Compared with Arabidopsis, many stress-responsive genes in Thellungiella are constitutively expressed at higher level, ion transport mechanism is more selective to K+ than Na+, and the species and metabolism of protection materials are also different. So the higher stress tolerance of Thellungiella must be regulated by complex process.Proteins are the functional machine of cell, and the proteomic analysis can help us to get a further intuitionist and exact knowledge of stress tolerance variation. However, to the best of our knowledge, it has not been reparted that comparing Arabidopsis and Thellungiella at proteomic level, as well as the proteomic comparision between Thellungiella ecotypes.In this study, Arabidopsis Columbia ecotype, Thellungiella Shandong ecotype, and Thellungiella Xinjiang ecotype were selected as research materials, and all the materials were cultured in hydroponic system. In the first part of this study, the Arabidopsis and Thellungiella leaf proteome were compared, and many higher abundant protein spots in 2-D gel were selected and then identified by mass spectrum (MS). This part aimed to find the basal element for higher stress tolerance of Thellungiella. In the second part, the proteome of leaves and roots were compared between Shandong ecotype and Xinjiang ecotype. The varied expression protein spots were identified, and the differences were analyzed and compared at the optimal growth status, aiming to discover the evolution differentiation at protein level.The main results of this study were summarized below:1. Proteome analysis of Thellungiella and Arabidopsis leaves. After grown in hydroponic culture system for several weeks (4 weeks of Arabidopsis, 6 weeks of Thellungiella), leaves were harvested, and then undergone TCA/Acetone procession for total protein extraction. Total protein were separated by 2-D electrophoresis, 24 cm pH 4-7 IPG dry strips were used for IEF and 12.5% SDS-PAG were used for the second dimension. 2-D protein images showed a higher level difference between Arabidopsis and Thellungiella leaves proteome.Protein spots were arranged by their volume values. Spots with bigger vol. value were indentified by MS, some smaller value spots were also selected according to their similar relative position in the gel with other specie. 87 spots in Arabidopsis gel were selected to analyze by MS, and 70 spots belonging to 57 genes were successfully indentified. 84 spots in Thellungiella were analyzed by MS, and 67 spots belonging to 58 genes were successfully identified. Then these proteins were classified by their function.By comparing the function catalog and spots pattern, we found some proteins had similar expression pattern. All these proteins belong to key housekeep proteins, such as photosynthesis related proteins: PSBO2, PSBP1, Phosphoribulokinase, ROC4, thylakoid membrane binding protein, SBPase, CSP41A, HCF136, and ATGLDP1; glycolysis related proteins: FBA1, FBA3, and TPI; all life activity related proteins: NDPK1, SAMS1, and ADK1; and a new reported At1g13930 protein.Some proteins of photosynthesis and photorespiration had diversity between Arabidopsis and Thellungiella. Photosynthesis related several proteins had different expression pattern between these two species, such as RbcL, RbcS, and RCA. Photorespiration related enzymes, GGT1 and glycine cleavage system H protein 2, which were expressed higher in Thellungiella than in Arabidopsis. Based on these facts, at least, we could find a viewpoint to understand the mechanism of lower growth rate in Thellungiella compared with Arabidopsis. Thellungiella may have a higher level photorespiration, which limited the growth rate and reduce photosystem damage, and to provide substrate for protective molecule synthesis.Many stress related or stress induced proteins in Arabidopsis had higher expression level in Thellungiella in normal condition. Oxidative reagents or other stresses induced antioxidative enzymes in Arabidopsis, such as Apx1, CZSOD2, and several Prxs, had higher expression level in Thellungiella. System defense related proteins, such as Jacalin lectin family protein, MLP328 protein, and curculin-like lectin family protein, also were expressed at higher level in Thellungiella. Pathogenesis-related protein PR-5 had a much higher expression level in Thellungiella. Beta -1, 3-glucosidase 1, disulfide isomerase 1, and hsp70 were highly expressed in Thellungiella. Myrosinase was highly expressed in both Arabidopsis and Thellungiella, but their pI and sequence similarity were different.All these differences could be recapitulated as that Thellungiella took an"against a rainy day"surviving strategy. Based on limited growth rate in normal condition, Thellungiella accumulated many anti-stress materials to prevent stress scare. This strategy may be acquired during evolution according to their habitat2. Comparsion of basic physiological characteristics and proteomic analysis of leaves and roots between Shandong ecotype and Xinjiang ecotype.After grown in hydroponic culture system for 6 weeks, some differences were shown between Shandong (SD) ecotype and Xinjiang (XJ) ecotype. SD ecotype leaves were green and shorter; roots were tap root system with preponderant tap roots and lateral roots sparsely brached from tap root. In contrast, XJ ecotype leaves were jade-green and longer; roots were tap root system with less preponderant tap roots and very developed lateral roots. XJ leaves outspreaded more upright than SD leaves.Leaves and roots were harvested respectively, and then longest leave length, longest root length, fresh weight, dry weight, and ash weight were measured. Data showed that fresh weight, ash weight, water content, and ash content of XJ leaves were higher than that of SD leaves. Dry weight of XJ leaves was a bit higher than SD leaves, but not significantly. Fresh weight and dry weight of XJ roots were similar with SD roots; water content of XJ roots was higher than that of SD roots; ash weight and ash content of XJ roots were lower than that of SD roots. In conclusion, whole plant water content of XJ ecotype was higher than that of SD ecotype; XJ leaves biomass was bigger than SD leaves; roots biomass of XJ was similar with SD roots. Ion content of leaves was higher than that of roots in XJ ecotype, and ion content of leaves was similar with that of roots in SD ecotype.2-D protein images showed a higher level similarity between SD and XJ proteome. There were 3.4% of total leaves protein spots and 2.2% of total roots protein spots different between SD and XJ ecotype. These diversity protein spots were picked from the gel and then indentified by MS, using Arabidopsis protein MS database as reference. The successful MS identification ratios were 40.9% of leaves, and 85.3% of roots. The successful ration difference maybe due to that, upground tissues (leaves) must confront higher selective pression than underground organs (roots), thus leaf proteins maybe less conserved compared with Arabidopsis than root proteins. Comparing the 2-D maps of leaves and roots between SD and XJ ecotypes, several expression difference spots were found. According to these proteins functions, we got a glance of the mechanism controlling the phenotype diversity in protein level.Compared with SD ecotype, XJ leaves stretched more vertically, rap roots showed less predominant, and lateral roots were dense. These different phenotypes maybe caused by difference in phytohormone content or in phytohormone response elements. ACO, the key enzyme of the last step of ethylene synthesis, expressed higher in XJ leaves, and leaves may release more ethylene. Higher level ethylene may stimulate a more vertical orientation of the petioles (hyponasty), enhance elongation, and induce senescence. Auxin may induce root system formation, and plants had shorter rap root and thicker lateral roots with higher auxin level. In XJ roots, two auxin induced proteins, MMSDH and FQR1, had higher expression level, and FQR1 also was a fast and primary auxin response protein. According to these two auxin response proteins'expression pattern, the auxin level in XJ roots may be higher, and higher level may affect root system architecture.Some proteins closely related to growth, such as KARI and Cpn20, were highly expressed in XJ leaves; and some defense related proteins, PCAP1, PR-5, endochitinase, and beta-1, 3-glucanase 1, had higher expression level in SD leaves. More energy and material were used for defense proteins synthesis, which may reduce the growth rate. XJ leaves may have more ethylene, and ethylene could induce growth. The most significant difference was that, although they had similar biomass, XJ roots may produce more energy than SD roots. Some respiration related important proteins, such as PGMI, PK1, Complex I-75kD, and pdh-e1 beta, were highly expressed in XJ roots, which may provide more energy for root metabolism and ion uptake and transportation for upground tissue fast growth.In conclusion, differences in phytohormone content or phytohormone response elements may be the reason of different phenotype, and this needed to be further studied. The differences in roots energy production capability and leaves energy/materials distribution may be the explanation why XJ leaves had larger biomass than SD leaves.The main innovation points of this study were generalized as follows:1. Total protein of Arabidopsis and Thellungiella leaves were separated by 2-D electrophoresis, and some abundant protein spots were selected and identified by MS. According the function catalog and comparision, the mechanism controlling the growth rate and stress tolerance differences was uncovered. Net photosynthesis and energy/materials distribution differences limited Thellungiella growth rate; more energy and materials were used for defense and stress tolerance related protein synthesis, so Thellungiella could have higher stress tolerance in normal condition.2. Phenotype and some physiological characters of Shandong ecotype and Xinjiang ecotype, under normal condition, were analyzed and compared. Some different aspects of two ecotypes were quantitatively analyzed, such as biomass, water content, and root-crown ratio.3. Total protein of leaves and roots were separated, and comparision between Shandong ecotype and Xinjiang ecotype were conducted. Some differences were discovered for the two ecotypes in protein level, and some proteins were identified, which were related to phenotype and physiological difference. The phenotype differences may be determined by phytohormone level difference, and leaves biomass differences were caused by energy/materials distribution and energy production capability differences.