| Cold stress is always the crucial limitation for crops distribution and product yield. Arabidopsis Athalia, as the model plant, is extensively studied in many aspects of physiology, biochemistry, gene regulation, and protein expression. Over the last decade, Studies on cold stress of Arabidopsis led to considerable progress in either transcriptomic or proteomic levels. In the respect of proteome, the sub-cellular protein profiling pattern induced by cold stress, especially those of membrane nucleus and chloroplast, have elucidated important biological regulation mechanisms. Due to the technical limits and developmental imbalance of proteome, two problems needed to be solved urgently: firstly, proteins extracted from the cell debris using routine plant extraction buffer might not suitable for typical 2-DE/chromatography, whereas, the debris maybe contain crucial cold responsive proteins. Secondly, as the important homoenzymes in energy flux, Rubisco interacting proteins have not yet explored.Extraction of plant proteins using a typical extraction buffer leaves behind insoluble homogenate debris that cannot be investigated by conventional 2-DE technologies. In this thesis, we present a scalable, off-line procedure for extraction of Arabidopsis thaliana homogenates that can be used in combination with both in-gel digestion and mass spectrometry. Based on sequential [NaCl] gradients and strong detergent fractionation, this new strategy allowed detection of 11 novel proteins from Arabidopsis thaliana that were altered in response to cold stress. More than that, Using the statistical analysis, the 23% of total proteins presents more than 26% changes under cold stress, suggesting the complexity of signal transduction pathways. To characterize the molecular response of Arabidopsis thaliana to cold stress and its capacity to recovery, four-week-old A. thaliana were subjected to 4 treatments: (1) continuous incubation at 20°C (control plants), (2) 4°C chill for 4 h, (3) 4°C chill for 24 h, and (4) incubation for 24 h followed by a 24-h chill (recovery). Changes in the debris protein patterns in response to cold and recovery treatments were analyzed by a proteomic approach. Eleven proteins with more than a 2-fold expression change were identified using PMF/MS, which were cross-checked against mRNA expression changes from the GEO profile. Examining the promoter sequences that correspond to the genes of these 11 cold-responsive proteins showed that 9 of them had DREB, ABRE, MyBR, and MyCR promoter elements in the context of cold or osmic-stress responses, highlighting the in-depth understanding of the mechanism involved in ABA-dependent and ABA-independent pathways. The increased cold responsive proteins were putative protein kinase (AT5g47750), cell signal transduction; nucleoid DNA-binding–like protein (At5g07030), homologue to cold-shock DNA-binding family proteins; and glycosyl hydrolase family 3 protein (At5g20950), involved in cell wall metabolism. The stimulated 3 proteins in recovery were putative snRNPs (At4g20440), mRNA splicing; transcription elongation factor-related (gi|15239051), similar to chromatin-specific transcription factor; and pectin methylesterase-like protein (At3g14310), controlling cell wall metabolism during fruit ripening. The six proteins should be credited the priority for further studies.Protein separation plays an increasing role in life science and biomedical research, the most outstanding of which is immunoproteomics. Developments in the area are unveiling and fostering a new discipline: putting protein separation or enrichment into a context of proteomics and systems biology. However, overwhelming Ab and Ag conceal Ag interacting proteins as the research targets, which is the rate-limiting step in the process of comparative proteomic analyses. We present a convenient and accurate method for Arabidopsis immunoprecipitates to tackle this problem.Rubisco (Ribulose 1, 5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39), despite its crucial biological role, shows a substantial varience depending on environmental fluctuations. Although disassembly, or even degradation of Rubisco is seen after cold treatment, little is known about interacting proteins that may be involved in this process. In this thesis, both the repression of net photosynthesis rate and the disassembly of Rubisco large subunits (Ru-L) were investigated in Arabidopsis thaliana (Col-0) induced by treatment at 4℃for 4 h or 24 h, as well as after recovery from cold stress for 24 h at 20℃. A total of 5 proteins were profiled and identified as interacting candidates. A AAA-type ATPase family protein (AT4G24860) and a glycosyltransferase (AT5G01250), essential for Rubisco activity, were strongly correlated with cold stress, Rubisco breakdown. To guarantee a constant repair of photosynthesis complexes, and thereby to avoid damage, it is crucial to maintain active protein synthesis in chloroplasts, Hypothetical protein (gi|7523687), homologue to protein related to translation machinery, was more active at chill 4 h and 24 h recovery. Therefore, we suggest that the specific association of Rubisco with thylakoid-bound ribosome nascent chain complexes exposes a chaperone-like function of Rubisco, which is crucial in maintaining the translation activity at transient low temperatures. Meanwhile, SEC14 cytosolic factor family protein/ phosphoglyceride transfer family protein AT1G55690, involving in lipid synthesis, cooperate into this process. Using the three-dimensional structure of macromolecular from PDB and online Z-dock, Photosystem 1 seemed to be suppressed through PSAD-1 (photosystem I subunit D-1) interacting with Ru-L resulting in photoinhibition. These results suggest that the disassembly of Rubisco maybe the main cause of photosynthesis rate reduction under chilling conditions, rather than low temperature effects on photosystems or protein biogenesis per se.Plant physiology under cold stress have long been investigated, focusing on the plant phenotypes and metabolism; since 1990's, the transcriptome have achieved great progress in the level of mRNA expression, by exploring Arabidopsis 25,000-30,000 gene transcription under cold stress using microarray, revealing the dominant ABA dependant and independent pathways. These several years, Proteome is now attracting more eyes, because of elucidating the expression changes of functional executive molecules-proteins. Due to technological limitation and imbalance of works, there are some problems waiting for further research, out of which, debris after typical plant protein extraction and Rubisco interacting proteins are important issues. In our work, we found 11 novel cold responsive proteins by studying the debris of typical plant extraction and Rubisco interacting proteins. Moreover, our results were undergone to incorporating to transcriptome results, which is a valuable pioneering work. At the same time, the expression changes of Rubisco interacting proteins revealed the important mechanism of photosynthesis under cold stress, verifying the hypothesis the relationship between Ru-L deassembly and net photosynthesis rate repression. These works sound the basis of gene exploration for crop cold tolerance.
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