16. Oligo microarray analysis of transcription in γ -aminobutyric acid-overaccumulating rice calli
T. AKIHIRO1, T. FUJIMURA2, H. EZURA1 and K. AKAMA3
1) Gene Research Center, University of Tsukuba, Ts ukuba, Ibaraki 305-8572 Japan
2) Graduate School of Life and Environmental Sciences, University of Tsukuba,
3) Department of Biological Science, Shimane Universit y, Matsue, Shimane 690-8504
γ-aminobu tyric acid (GA BA) is a fou r- carbon non-protein amino acid th at is
produced from glutamate by glutamate decarboxylase (GAD) and is ubiquitously present
in organisms ranging from bacteria to plants and vertebrates (Bouché and Fromm 2004).
GAB A is known to function as an inhibitory neurotransmitter in animals, but its role in
plants still remains unclear. Interestingl y, only plant GADs have a C-terminal extension
that acts as a calmodulin binding domain (CaMBD) (Baum et al. 1993). Therefore, it is
thought that transient accumulation of intracellular calcium in response to various stresses
may activate GAD via interaction of the CaMBD with a Ca2+/CaM complex. We recently
identified a cDN A encoding a novel GAD isoform in rice (OsGAD2) (Akama et al. 2001).
Like the GADs from dicotyledonous plants, OsGAD2 has a C-terminal extension, but it
has no ability to bind to Ca2+/CaM. In transgenic rice cells that overexpressed OsGAD2
lacking the coding region for the C-terminal 30 amino acids (OsGAD2
high levels of GABA were accumulated (300 pmol/mg FW and 30,000 pmol/mg FW in
wild-type and OsGAD2ΔC calli, respectively), whereas the remaining free amino acids
analyzed were present at almost the same levels as in wild-type cells, except for a
several-fold increase in Gln and a several-fold decrease in Glu, Asp and Asn. Given tha t
GAB A production is induced by stress (Bouché and Fromm 2004), and that GAB A
functions to guide the pollen tube in Arabidopsis (Palanivelu et al. 2003), it is prob able
that GAB A plays a key role in physiological regulation, for example by acting as a
phytohormone, in plant cells. In order to investigate this possibilit y, we compared the
transcription patterns of wild-type and GABA-accumulating calli using oligo microarray
Plant binary vector pCAMBIA1302 (Cambia) or pCAMBIA1302 carrying
CaMV35S::OsGAD2ΔC were introduced into fresh rice calli (Oryza sativa L. cv. Kitaake) that had been maintained in 2N6 solid media. The cells were then transferred to 100 ml
flasks containing 20 ml of liquid 2N6 media to initiate a suspension culture. The flasks
were placed on a shaker 120 rpm at 28 °C in the dark. To maintain uniformity of cell size
in the rice suspension cultures, cells were passed through mesh (pore size: 1 mm) and
were transferred to fresh medium every 7 days to repeat for one month. Then, two days
after transfer of the cells to the same fresh medium, total RNAs were extracted from
cultured cells from these two different lines. For expression profiling by microarray
analysis, we used 800 ng of total RNA per microarray slide (Rice Oligo Microarray Kit,
22K; Agilent). To eliminate false-positive results, we performed a dye-swap-experimen t
using two arrays. The procedure used for the microarray analysis was essentially as
described by Yazaki et al. (2000). After hybridization and washing, the arrays were
scanned using an Agilent Microarray Scanner. For spots that showed more than 5.55-fold
higher signal intensity (see below) after normalization in samples corresponding to the
GABA-accumulating cells, a BLAST search was performed. cDNAs from a total of 60
spots were selected and Table 1 lists these putative genes with elevated expression
according to function. First, OsGAD2 expression was up-regulated 5.55-fold. Endogenous
and introduced GAD2 genes were both monitored in the present system, and the observed
up-regulation was considered to be due to overexpression of the truncated form of
OsGAD2. The observed up-regulation of ACC oxidase (AK058296), a key enzyme in th e
synthesis of ethylene, is to be expected, given that GAB A induces accumulation of ACC oxidase mRNA (Kathiresan et al. 1998). These results suggest that expression patterns of
the GABA-inducible genes could be faithfully monitored in this experiment. Of the
functional classes of proteins shown in Table 1, the lipid and cell wall-related proteins are
the most numerous, with ten different genes found to be up-regulated. Because previous
reports have indicated that GABA controls the elongation of stems and pollen tubes
(Kathiresan et al. 1998, Palanivelu et al. 2003), it is tempting to conclude that the
GABA-triggered expression of genes related to cell wall structure is involved in this
dynamic growth. The transcription factor, transporter and stress-response-related classes
of proteins each comprised three or four genes that were up-regulated. In particular,
expression of YABBY ortholog (AK070205), which is known to play an important role in
leaf and flower formation in plants (Bowman et al. 2002), was up-regulated about 20-fold
in the GABA-accumulating cells. To date, little is known about the relationship between
GAB A and regulation of gene expression, so in future studies it would be worth
investigating the possibility that GABA functions as a signal molecule that induces
expression of transcription factors such as YABB Y.
Table 1. Genes that were overexpressed in GABA-overaccumulating rice calli
Accession Accession Putative Gene Function Putative Gene Function increase increase Transcription factor (continued)
carboxylate oxidase (ACC oxidase)Avr9/Cf-9 rapidly elicited protein
AK068359 DNA binding / transcription factor 2.00E-09
Transporter
fragment 20 protein 3similar to myosin, heavy
AK060520 transporter/malic acid transport
Lipid and cell wall related UnKnown function
protein PM 19putative receptor-like protein
AK064117 ankyrin-like protein-like protein
Stress responsive related Unclasiffied
AK069636 GDP dissociation inhibitor protein 0.00E+00
Acknowledgment
We thank Dr. Y. Nagamura (Rice Genome Research Center, National Institute of
Agrobiological Sciences, Tsukuba, Japan) for his help with carrying out the oligo
References
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PEER 2 PEER SEMINAR ROYAL COLLEGE OF ART APRIL 2nd 2001 DOCUMENTATION NOTES INTRODUCTION These notes are intended to indicate the breadth of issues discussed during the event. They are not exhaustive, but are culled from notes taken by participants during the group discussions. Additional contributions have been added at the end of this document. Video documentation (in Quicktime fo
A fishy recommendation: omega-3 fatty acidintake in pregnancyDepartment of Obstetrics and Gynecology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, CanadaCorrespondence: Dr SJ Genuis, University of Alberta, 2935-66 Street, Edmonton, Alberta, Canada T6K 4C1. Email [email protected] cite this paper as: Genuis S. A fishy recommendation: omega-3 fatty acid intake in pregnanc