Assistant Research Fellow
Ph.D. Plant Molecular and Cellular Biology Program University of Florida
M.S. Horticulture National Taiwan University
B.S. Horticulture National Chung-Hsing University
Research Interests
Sugar sensing and signaling; Sugar signal interaction with phytohormones (ABA and ethylene); ABA biosynthesis and signaling
I. GLUCOSE INSENSITIVE (GIN) gene function
Sugars not only serve as carbon source for the basic metabolism in all organisms, but also act as signal molecules in regulating a large spectrum of gene expression, and eventually in controlling plant growth and development. The components involved in sugar signal pathway have been well-characterized in microorganisms, but it largely remains obscure in higher plants. To better understand the sugar signal components, we successfully isolated several glucose insensitive (gin) mutants from a genetic screen of T-DNA tagged seed pools on 6% glucose agar plates. Upon this glucose concentration, the wild-type seeds germinate with further developmental arrest, whereas the gin mutants may grow steadily with true leaf development (Fig. 1A). Genetic allelism test demonstrates that gin1 and gin4 are an allele of aba2 and ctr1, respectively, indicating the intimate interlink among sugar, ABA, and ethylene signaling. The glucose-insensitive1 (gin1) showing early germination, flowering (Fig. 1B), and wilty phenotypes (Fig. 1C), has been cloned by positional cloning. The GIN1 (or ABA2) locus encodes a unique short-chain dehydrogenase/reductase (SDR1) that catalyzes the multiple-step conversion of xanthoxin to abscisic aldehyde in the last 2nd step of ABA biosynthesis in the cytosol. Further analyses of gin1/aba2 null mutants and transgenic complementation define dual functions of ABA in inhibiting post-germination growth by distinct sugar and stress signal, and in promoting growth in the absence of exogenous sugars and severe stress.
The GIN1/ABA2 has been considered a constitutive expression gene; thus, its expression was predicted to be in all tissues and its regulation through genetic manipulation will not affect the ABA levels in plants. However, transgenic expression analyses of GIN1::GUS fusions revealed the surprising tissue-specific and vascular-restrictive expression of GIN1/ABA2 (Fig. 1D), reflecting spatial and temporal expression of GIN1/ABA2 and a dynamic mobility of ABA precursors and/or ABA to the target sites (guard cells or seeds). In addition, GIN1/ABA2 over-expression transgenic plants with elevated levels of ABA promote seed dormancy and stress tolerance (Fig. 1E).
To further dissect the components involved in ABA signaling pathway, aba2/gin1 suppressor (abs) mutants were isolated and characterized based on their glucose and salt sensitivity (Fig. 1F). This study will shed light on the better understanding of ABA function in response to sugar and salt signaling. The cloning of these aba2/gin1 suppressors is undergoing.
Further studies of sugar signaling crosstalk to ABA and ethylene are carrying out. The microarray will be used to study the global gene expression profile regulated by glucose, GIN1 and ethylene at genomic scale.
II. GLUCOSE OVERSENSITIVE (GLO) gene function
The glucose oversensitive (glo) mutants were isolated by the screens of T-DNA tagged seed pools on 4% glucose, a concentration that wild-type seeds may germinate and grow normally, but the glo mutants showed germination followed by developmental arrest or stunted growth (Fig. 2A). One of glo mutants was designated as glucose-inhibited root elongation (gir). This mutant displayed near normal root growth in soil or agar plates without sugar, whereas it showed short and swollen roots at the presence of sugar (Fig. 2B). In spite of the reduced root elongation in the presence of sugar, the Atgir mutant also displayed severe sterility, more flower shoots, senescence delay (Fig. 2C), and strong resistance to drought and salt stresses, which are useful traits for some vegetative crops. Anatomical analysis of Atgir mutant exhibited the defects of root structure, pollens and flower organs (Fig. 2D). Molecular cloning of GIR in Arabidopsis has been done. The AtGIR gene encodes a C2-domain containing protein presumably with phospholipid- associated and protein signaling or trafficking functions. GIR::GUS transgenic plants reveal the GIR gene primary expression in root and mature pollens (Fig. 2E). GIR::GFP-GIR transgenic plants further support the subcellular localization of GIR protein present in the plasma membrane of roots (Fig. 2F). Genechip analysis of Atgir mutant vs. wild type revealed that the knockout mutant affected the genes involved in cell wall modification, PR gene expression, transcriptional regulation, and other signal transduction pathways. Further studies are focus on the protein-protein interaction and the signal pathways involved in and causing the pleiotropic effects in the mutant.
Figure 1. GIN1/ABA2 function, interaction with phytohormones, and gin1/aba2 suppressor isolation
1A. Genetic screens of glucose insensitive (gin) mutants.
1B. gin1/aba2 mutant with earlier flowering than that of wild type.
1C. gin1/aba2 mutant with wilty phenotype.
1D. GUS staining with mature plant and silique of GIN1::GUS transgenic plant.
1E. Phenotypic comparison of wild type, gin1/aba2, and overexpression line 4-4 in response to salinity. Plants were watered with NaCl solution for 16 days.
1F. EMS-treated gin1/aba2 were screened on 6.5% Glc or 250 mM NaCl for isolation of gin1/aba2 suppressors (abs).
Figure 2. Isolation of glucose oversensitive (glo) mutants and functional study of GLUCOSE-INHIBITED ROOT ELONGATION (GIR) gene
2A. The glo mutants were isolated from T-DNA tagged seeds grown on 4% Glc agar plates.
2B. One of glo mutants is designated as glucose-inhibited root elongation (gir) with developmental arrest on 6% Glc (left panel) and short root on 4% Glc (right panel).
2C. Phenotypic comparison of wild type and gir.
2D. Comparison of flower organs and pollens between wild type (WT) and gir.
2E. GIR::GUS transgenic plant showing GUS staining in roots and mature pollens.
2F. GIR::GFP-GIR transgenic plants showing GFP signal in plasma membrane of root epidermal cells.
Cheng, W.-H., Endo, A., Zhou, L., Penney, J., Chen, H.-C., Arroyo, A., Leon, P., Nambara, E., Asami, T., Seo, M., Koshiba, T., and Sheen, J. (2002). A unique short-chain dehydrogenase/ reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14: 2723-2743.
Moore, B., Zhou, L., Rolland, F., Hall, Q., Cheng, W.-H., Liu, Y.-X., Hwang, I., Jones, T., and Sheen, J. (2003). Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 300: 332-336.
Lin P.-C., S.-G. Hwang, A. Endo, M. Okamoto , T. Koshiba, and W.-H. Cheng. (2007). Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance. Plant Physiol. 143: 745-758.
