Journal of Experimental Botany, Vol. 51, No. 349, pp. 1479-1480,
August 2000
© 2000 Oxford University Press
Gene Note |
Abg1: a novel gene up-regulated by abscisic acid in guard cells of Vicia faba L.
1 Department of Biological Science, Florida State University, Tallahassee, FL 32306–4370, USA
2 Institute of Paper Science and Technology, 500 10th Street NW, Atlanta, GA 30318–5794, USA
3 Department of Nutrition, Food and Exercise Science and Program in Neuroscience, Florida State University, Tallahassee, FL 32306–4340, USA
Received 8 June 2000; Accepted 9 June 2000
Abstract
A novel gene (abg1) was isolated by differential display RT-PCR from guard cells of Vicia faba L. Abg1 transcript accumulated in guard cells that were incubated with 5 µM S(+)-ABA for 1 h. The full-length abg1 cDNA was 753 bp, which included a 513 bp coding region. The deduced 17.8 kDa protein shared sequence similarity with several desiccation-related proteins reported in plants.
Key words: Abscisic acid, guard cell, gene expression, Vicia faba.
Guard cells modulate stomatal pore size by co-ordinating responses to environmental and physiological factors. These cells are at the distal end of the transpiration stream; thus, apoplastic solutes accumulate there by peristomatal evaporation (Ewert et al., 2000
). For example, in Vicia faba L., sucrose, 5 mM in the leaf apoplast, accumulates in the guard cell apoplast over the photoperiod to a maximum concentration of 300 mM and lowers stomatal conductance (Lu et al., 1995, 1997).
Water stress causes guard cell ABA accumulation and consequent stomatal closure. During root water stress, guard cell apoplastic ABA concentration reaches 2.3 µM, a 20-fold increase over leaf apoplastic ABA (SQ Zhang and WH Outlaw Jr, unpublished results); during severe leaf water stress, guard cell symplastic ABA can reach 10 µM (Harris et al., 1988). The short-term responses of guard cells to ABA are well known, and the underlying mechanisms are being elucidated (Leung and Giraudat, 1998).
In both embryonic and vegetative tissues, ABA induces long-term responses by regulation of gene expression, which may enhance stress tolerance (Chandler and Robertson, 1994; Ingram and Bartels, 1996). ABA and water stress also regulate the expression of guard cell transcripts (Shen et al., 1995; Taylor et al., 1995; Kopka et al., 1997). The objective of this study was to identify and characterize genes induced by ABA in guard cells.
For this purpose, differential display RT-PCR (Liang and Pardee, 1992) was employed. Poly-A+ RNA was extracted (oligo-dT25 coated magnetic beads, Dynal) from guard cells, which were obtained by sonicating epidermal peels. Differential display followed Xu et al. (Xu et al., 1997); the RNA was reverse transcribed with an anchored oligo-dT11 primer (Genhunter). The products were amplified in the presence of 35S-
-dATP (NEN), with an oligo-dT11 primer and one of eight 13-mers (Genhunter). Several differentially expressed transcripts were identified by autoradiography after separation of the cDNA products in a denaturing polyacrylamide gel. Figure 1 shows a 261 bp band, which was excised, reamplified, cloned into pCR 2.1 vector (Invitrogen). It detected a ~700 base transcript (abg1), which was up-regulated within 1 h by 5 µM S(+)-ABA, used as the racemic mixture (Fig. 2). In addition, whole leaf showed up-regulation of the transcript by ABA (not shown).
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5' RACE PCR (Life Technologies) was used to isolate the full-length cDNA of abg1. A gene-specific primer, derived from the sequence of the differential-display fragment, and a 5' anchor primer (5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3', Life Technologies) amplified a 561 bp cDNA, which was cloned and sequenced. This sequence represented the 5' end of abg1. The overall length of abg1 matched the corresponding transcript (Fig. 1
), and included a 513 bp open reading frame. Neither the 77 bp 5' UTR nor the 163 bp 3' UTR contained any known elements.
The hydrophilic core of putative ABG1 protein (17.8 kDa) showed some sequence similarity to a diverse set of plant desiccation-related proteins (Fig. 3). The expression kinetics of abg1 mRNA and further details of ABG1 are being studied.
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Acknowledgments
The authors thank Steve Thompson for advice, the Arabidopsis Biological Resource Center for sharing the actin cDNA clone, and the US Department of Energy for a grant to WHO.
Notes
4 To whom correspondence should be addressed. Fax: +1 850 644 0481. E-mail: outlaw{at}bio.fsu.edu 
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