75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

247 Large-Scale Screen and Isolation of Genes Induced by Photooxidation and Photoinhibition
Stresses in Rice
Ji-Sung Han 1 , Sun-Ho Kim 1 , Dong-Hyouk Woo 1 , Ja-Myoung Lee 1 , Byoung Yong Moon 2 , Gynheung An 3 , Choon-Hwan
Lee 1 , Yong-Hwan Moon 1
1
Department of Molecular Biology, Pusan National University, Busan 609-735, Korea, 2 School of Biotechnology
and Biomedical Science, Inje University, Gimhae 621-749, Korea, 3 Division of Molecular and Life Sciences,
Pohang University of Science and Technology, Pohang 790-784, Korea
Light is one of the most important environmental factors that control the growth and development of plants. But
excess light is harmful to plants and causes the photooxidation of pigments and the photoinhibition of photosynthesis. In
this study, to isolate genes induced by photooxidation and photoinhibtion stresses, we have performed two experiments
that involve a new reverse transcription-polymerase chain reaction (RT-PCR) using annealing control primers (ACPs)
and analysis of promoter activity in promoter trap lines.
First of all, we have isolated 37 differentially expressed genes (DEGs) under photooxidation and photoinhibition
stresses using 20 ACPs. Among the 37 DEGs, 22 DEGs showing strong up-regulation (16 DEGs) and down-regulation
(6 DEGs) under both stresses were selected for sequence analysis. Basic Local Alignment Search Tool (BLAST) searches
revealed that the 22 DEGs represented 21 different genes including S-adenosylmethione synthetase, early nodulin,
temperature stress-induced lipocalin, and etc. On the basis of expected functions of the 21 genes, we selected 14 genes for
further study. To confirm the results of ACPs, semi-quantitative RT-PCR was performed, and so far 5 genes showed the
same expression patterns as in the ACPs results. On the other hand, we analyzed promoter trap lines including pGA2717,
a promter trap vector with GFP, to isolate promoter induced by highlight and low oxgen. Out of 3500 lines, we selected
103 lines using bioinformatical knowledge that GFP is inserted within exon or intron of genes and the orientation of
GFP is the same as that of gene. We analyzed GFP activity in the 103 lines under highlight and low oxygen conditions
in large scale. As results of the analysis, 13 and 5 lines showed the increase of GFP activity under highlight and low
oxygen conditions, respectively, and 5 lines showed the increase of GFP activity under both conditions.
Now, we are investigating the biological functions of selected genes especially under photooxidation and
photoinhibition conditions.
248 Function of Coactivator Proteins ADA2 and GCN5 in Cold Acclimation in Arabidopsis
Kanchan Pavangadkar 1,2 , Nathan Lord 2 , Michael Thomashow 1,3,4 , Steven Triezenberg 1,2
1
Genetics Program, Michigan State University, East Lansing, MI 48824, 2 Department of Biochemistry and
Molecular Biology, Michigan State University, East Lansing, MI 48824, 3 DOE-Plant Research Laboratory,
Michigan State University, East Lansing, MI 48824, 4 Department of Crop and Soil Science
Covalent modifications of histones play important roles in the regulation of transcription. Acetylation of lysine residues on
the amino-terminal tails of histones is associated with transcriptionally active genes and is catalyzed by histone acetyltransferases
(HAT). GCN5 (a HAT) and ADA2 are components of coactivator complexes such as SAGA in yeast. The coactivator protein
ADA2 is essential for the HAT activity of GCN5 in yeast. The Arabidopsis genome encodes one homologue of GCN5 and
two homologues of ADA2 (ADA2a and ADA2b). Null mutants of GCN5 and ADA2b have pleiotropic effects on plant growth
and development whereas ada2a mutants show no aberrant phenotype.
Arabidopsis ADA2 and GCN5 physically interact with the transcriptional activator CBF1 which activates the expression
of cold-regulated (COR) genes during cold acclimation. Cold acclimation is the process by which plants increase freezing
tolerance upon exposure to low non-freezing temperatures. CBF1 binds to the cold/dehydration responsive element (CRT/
DRE) present in COR gene promoters. ada2b and gcn5 mutants show a delay in activation and a reduction in expression of
COR genes during cold acclimation. Chromatin immunoprecipitation assays show that the acetylation of histone H3 at the
COR promoters increases upon cold acclimation. ADA2b and GCN5 may also be essential in maintaining the basal expression
levels of COR genes. We hypothesize that these proteins ‘poise’ the promoter of COR genes in the uninduced state and thus
potentiate stronger activation upon induction.
The Arabidopsis genome lacks obvious homologs of several SAGA subunits, and thus plant coactivator complexes are
likely to be distinct from human or yeast complexes studied previously. To identify components of GCN5-containing coactivator
complex(es) in plants, a tandem affinity purification (TAP) cassette encoding a calmodulin binding peptide and IgG binding
domain was fused to the N- or C- termini of AtGCN5. The fusion genes were transformed into an Arabidopsis gcn5 mutant.
Lines in which TAP-GCN5 complements the mutant phenotype were used for affinity purification. Western blot analysis of
purified fractions suggests that the transcriptional coactivators ADA2a and ADA2b copurify with TAP-GCN5. This study
provides a foundation for understanding the biological role and biochemical mechanism of GCN5 in Arabidopsis.