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Studies On Proteome Of Chloris Virgata (Swartz.) Leaves Under Carbonate Stress

Posted on:2007-10-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:Q X LuoFull Text:PDF
GTID:1100360215993768Subject:Garden Plants and Ornamental Horticulture
Abstract/Summary:PDF Full Text Request
Soil salinity is one of the main abiotic stresses that restrict the development of agriculture worldwide. How to improve crop salt tolerance, make good use of salinity-alkalinity soil has become the significant thesis for solution. Up to date, studies about plant salt tolerance were focused on plant ecology, plant physiological and biochemical characters, plant salt tolerance related genes and their use on improvement of glycophyte salt tolerance by biotechniques. Research and utilization of halophytes were relatively rare. Compared to NaCl salt stress, studies about carbonate stress, in which the main salts of soil are Na2CO3 and NaHCO3 that are more severe to plants, are very weak, especially in molecular biology. There is no report on proteomic analysis about plant carbonate stress yet.In present study, we investigated the growth characters of an annual C4, herbaceous graminoid Chloris.virgata (Swartz.) under carbonate stress, compared with a rice cultivar Nipponbare (Oryza sativa L.), which was classified as moderate salt tolerance among eight rice cultivars tested for their relative sensitivity. Further, we analyzed the changes of 2-DE gels pattern of C. virgata leaves under sodium carbonate stress in matrix cultural system and the proteome of C. virgata leaves under sodium bicarbonate stress in hydroponic cultural system. Also we analyzed 7 novel salt stress protein genes and GAPDH, a known salt stress responsive gene, at the transcript level. The main results were as follows:The germination rates of both C. virgata and rice were declined with the concentration increasing of Na2CO3, NaHCO3 and NaCl solution. In the same [Na+] levels, the germination rates of both C. virgata and rice followed the order of NaCI>NaHCO3>Na2CO3, so the toxic effect on the germination rates of C. virgata and rice followed the order of Na2CO3>NaHCO3>NaCl. C. virgata seeds were more tolerant to salt stress than rice.Both root and shoot length of C. virgata were declined with the the concentration increasing of Na2CO3, NaHCO3 and NaCl solution. At the same [Na+] levels, both root and shoot length C. virgata followed the order of NaCI>NaHCO3>Na2CO3, so the toxic effect on the root and shoot length of C. virgata followed the order of Na2CO3>NaHCO3>NaCI. Compared to shoot length, three sorts of salts had more toxic effect on root length.With the increasing of NaHCO3 concentration, the dry mass weights of root and shoot of rice seedlings were declined significantly, while the changes of C. virgata seedlings were not obviously. After treatment of 80mM NaHCO3, both root and shoot dry mass weights were lower than controls, and this trend was exaggeratd with the prolonging of stress time. This implied that as a halophyte, C. virgata was more tolerant to NaHCO3 treatment than rice.After SDS-PAGE analysis of soluable protein of C. virgata leaves stressed by different concentrations of Na2CO3 in matrix cultural system, it was clear that the protein bands of C.virgata leaves under 50mmol/L Na2CO3 treatment for 24h was the most plentiful. There were about four bands that were induced or upragulated by 50mM Na2CO3 treatment. Thus the total protein component of C.virgata leaves must be changed by this treatment as soluable protein was one part of total protein. Then we performed 2-DE analysis of total protein of C.virgata leaves under treatment of 50mmol/L Na2CO3 for 24h. By the aid of Image Master Elite 3.01 analysis software, there were 42 protein spots in the gels that were changed significantly compared to control and distributed in the region of pl 3.63-9.60 and Mw 17.12- 69.19kDa. The function of these proteins (or polypeptides) needed to be identified by MS analysis and/or further functional research.Treatment of 80mmol/L NaHCO3 for 3 days was a threshold condition for tolerant responsive of C.virgata cultured by hydroponic system and might taken as an appropriate treatment for experiment of C.virgata research of physiology, biochemistry and molecular biology. Proteomic analysis of C.virgata leaves under 80mmol/L NaHCO3 treatment for 24, 48 and 72h, the results showed that there were about 500 reproducible imaged by eye within all samples. 56 proteins showed significant and producible changes in at least one stage of the carbonate stress cycle compared with controls harvested at the same time points. 30 protein spots with relatively high abundance were analyzed by MALDI-TOF MS and/or TOF-TOF MS. 23 of them were identified by MS analysis successfully.These proteins were classified to six categories according to their potential biological function in the cell, i.e., photosynthesis, metabolism, protease, transcriptional and translational factors, unknown proteins and other proteins. As a C4 metabolism specie, 9 out of 23 proteins (39%) identified as carbonate stress responsive proteins are submitted to photosynthesis, indicating that regulation of photosynthesis may play an important role on the C. virgata salinity-alkalinity tolerance mechanisms. 5 out of 23 spots were identified as proteins functionally related to carbohydrate and nitrogen metabolism, suggested that the maintainning of the matabolism stabilization might be important to carbonate tolerance of C. virgata. Also there had one putative FtsH-like protein Pftf precursor and act as a Zn2+ -dependent metalloprotease, three transcriptional factors, three unknown proteins and two other proteins that were related to carbonate tolerance of C. virgata.Among the 23 proteins identified by MS, RubisCO large subunit, GAPDH and UDPase were related to plant salt tolerance reported previously; photosystemⅡoxygen evolving complex (OEC) protein 1 precursors, aspartate amino transferase, putative FtsH-like protein Pftf precursor, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase, pentatricopeptide (PPR) repeat-containing protein-like, transcription initiation factor IIF and alpha subunit family protein responsive to cold, high temperature, ozone stress, redox stress high light and imgration of macrograms and there was no reported about the relationship between them and salt tolerance yet; PEPCK, methyl-CpG binding protein-like, predicted OJ1767D02.15-2 gene product and 3 unknown proteins were novel proteins response to salt stress and maybe, to other stress too. The identification of these proteins enriched the gene resource of plant salt tolerance.As some proteins identified in present study were new salt responsive ones that hadn't been reported before, to confirm the existence of these proteins and understand the expression profile of their genes, we analyzed 7 of them and GAPDH, a known salt stress responsive gene, at the transcript level. The C. virgata seedings were treated with 100mmol/L NaCI or 80mmol/L NaHCO3 for Oh, 6h, 12h, 24h and 48h, respectively. The mRNA expression of 5- methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase, 2 PEPCK genes, ASP, GAPDH, PSII OEC protein 1 precursor and transcription initiation factor IIF, alpha subunit family protein genes were increased under salt stress, which correlated with that of protein level. Putative FtsH-like protein Pftf precursor gene was down-regulated by both salts stress, which also correlated with that of protein level.There are many intricate mechanism involved in transcript expression of plant genes under salt stress. The expression character of carbonate stress responsive genes under salt stress and the salt stress mechanism of C.virgata need to be investigated further.To our best knowledge, this is the first study on proteomics of a halophyte response to carbonate stress. The proteins identified in this work represent only a small part of the C. virgata proteome response to carbonate stress, the others remain to be identified. Meanwhile, further functional analysis of these proteins may be significance in understanding of carbonate tolerance mechanisms of plants. This research will give new insights into salinity-alkalinity stress response in plants and provide a new method in ultilization and exploitation of halophyes.
Keywords/Search Tags:Proteomics, Chloris.virgata (Swartz.), Salt Tolerance, Carbonate Stress
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