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Abstract
New molecular technics reveal the function of Arabidopsis genome
Ana Tomašić, Snježana Jurić, Hrvoje Fulgosi
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 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.
Key words: Arabidopsis thaliana, Agrobacterium tumefaciens, Gateway technology,
Affymetrix chip, Real-time PCR