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New molecular technics reveal the function of Arabidopsis genome

Photosynthetic conversion of solar to chemical energy and oxidation of water to form oxygen are inormously important life processes. They are catalyzed by photosynthetic reaction centres composed of chlorophyll-containing proteins in plant cells. By sequencing the entire genome of Arabidopsis thaliana in 2000., studying of photosynthetic genes was somewhat simplified. Recent development of new genomic technics allowed scientists to investigate expression of thousands of genes in a single reaction. Molecular technologies of forward (identifying mutations that produce a certain phenotype) and reverse (determining the phenotype that results from mutating a gene) genetics, including microarray technology approach, were used in this work to elucidate the function of TLP40 and TROL proteins. Chloroplast protein TLP40 (thylakoid lumen PPIase of 40 kDa) cychlophilin-like PPIase, located in thylakoid lumen shows peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins. Gene coding CYP38 protein, ortholog of TLP40 from Spinacia oleracea is located on the 3rd chromosome of Arabidopsis thaliana. To characterize the function of this protein in vivo, we constructed antisense Arabidopsis thaliana plants. CYP38 gene was silenced using Gateway technology cloning methods. The Gateway workflow involves cloning of the CYP38 gene into an entry clone and further shifting of DNA fragment to an expression vector system with a one-step, 1-hour site-specific recombination reaction (LR). Part of the T-DNA region with CYP38 gene between LB (left) and RB (right) borders, was inserted to Agrobacterium tumefaciens. Arabidopsis thaliana Col-0 (L. Heynh) plant were infected with Agrobacterium cells by virulence genes on disarmed Ti-plasmid, which helped in transfering gene of interest into plant genome by floral dip transformation method. Nuclear-encoded component of thylakoid membranes, protein TROL (thylakoid rhodanase like-protein of 66 kDa) isolated from cytb6f fraction, is required for sustaining of efficient linear electron flow (from H2O to NADP+) in vascular plants. TROL consists of two distinct modules ; a centrally positioned rhodanese-like domain and a C-terminal hydrophobic FNR binding region. Flavoenzyme ferredoxin: NADP+ oxidoreductase or FNR ensures the final electron transfer from ferredoxin to NADP+. So, TROL is considered necessary for anchoring FNR to the thylakoid membranes. As FNR interacts with redox sensible ferredoxin we can conclude that TROL could be the sensor which responds to redox changes in chloroplast. Affymetrix ATH1 GeneChip&#61650; analysis of TROL deficient plants compared to wild-type plants, showed modulation of specific nuclear-encoded genes. Among 237 differentially expressed genes (selected by q-value cutoff of <0.001) were protochlorophyllide oxidoreductase (POR B) and NADP-malic-enzyme (AtNADP-ME2), chloroplast targeted proteins whose expression was further verified using Real-time PCR method based on TaqMan chemistry.

Arabidopsis thaliana; Agrobacterium tumefaciens; Gateway technology; Affymetrix chip; Real-time PCR

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