Research Interests Chloroplast development and function; Amino acid sensing and signaling in plants
We are using molecular genetic approaches to identify genes that are involved in chloroplast development and function. To this end, we have isolated a collection of Arabidopsis T-DNA insertion mutants displaying albino, pale green, pale yellow or variegated phenotypes (Fig. 1).
Figure 1. A collection of Arabidopsis pigmentation mutants.
One of the pale yellow mutants, vanilla cream1 (vac1), has a null mutation in an unknown function gene that encodes a pentatricopeptide repeat (PPR) protein (Fig. 2A & B). The PPR protein family is characterized by tandem repeats of a degenerate motif of 35 amino acids. At least 474 PPR proteins have been identified in the Arabidopsis genome and about two-thirds of these proteins are predicted to be chloroplast or mitochondrial proteins. In contrast, PPR proteins have not been identified in prokaryotes and only very few PPR proteins are found in other eukaryotic organisms such as yeast, C. elegans, Drosophila and humans. Some of the PPR proteins have been shown to be involved in RNA metabolism in chloroplasts and mitochondria. We used protoplast transient assay to demonstrate that the VAC1-GFP fusion protein is localized to the chloroplast (Fig. 2C). Our preliminary studies on Arabidopsis vac1 mutants suggest that the Arabidopsis VAC1 PPR protein may be involved in chloroplast RNA editing and processing.
Figure 2. (A) Progeny of a heterozygous vac1 mutant segregate green and albino plants in a 3:1 ratio on a non-selective medium. Plants shown are 14 days old. (B) Schematic diagram of VAC1 PPR (pentatricopeptide repeat) protein. VAC1 belongs to the DYW subgroup of the PPR family. TP, transit peptide. (C) VAC1-GFP fusion protein is localized to the chloroplast in Arabidopsis protoplast transient assay. Scale bar is 10 µm.
In addition, we have isolated three Arabidopsis slow growth (slo) mutants (Fig. 3). One of the slo mutants, slo1, is caused by a T-DNA insertion in an unknown function gene encoding another PPR protein (Fig. 4A & B). Interestingly, the SLO1-GFP fusion protein is also localized to the chloroplast (Fig. 4C). We are in the process of characterizing the functions of Arabidopsis SLO1 PPR protein. Genetic analyses revealed that the slo2 and slo3 mutants did not co-segregate with the T-DNA insertions. We are currently using map-based cloning to identify the molecular lesions of slo2 and slo3.
Figure 3. Arabidopsis slow growth (slo) mutants.
II. Amino acid sensing and signaling in plants
Compared to yeast and E. coli, little is known about proteins or regulatory domains involved in amino acid sensing and signaling in plants. The ACT domain, an amino acid-binding domain named after bacterial aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase), is a regulatory domain mostly found in enzymes directly or indirectly involved in amino acid and purine metabolism. In a survey of the Arabidopsis genome, we have found several ACT domain-containing protein families (Hsieh and Goodman, 2002). We hypothesize that these novel ACT domain-containing proteins may serve as amino acid sensors in plants. We are taking reverse genetic approaches to study the functions of these ACT domain-containing proteins.
Figure 4. (A) Two weeks old Arabidopsis wild type (WT) and slow growth1 (slo1) mutant. (B) Schematic diagram of SLO1 PPR protein. (C) SLO1-GFP fusion protein is localized to the chloroplast.
Hsieh, M.H., Chang, C.Y., Hsu, S.J. and Chen, J.J. (2008) Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis. Plant Mol Biol (in press)
Hsieh, M.H. and Goodman, H.M. (2006) Functional evidence for the involvement of Arabidopsis IspF homolog in the nonmevalonate pathway of plastid isoprenoid biosynthesis. Planta 223: 779-784.
Hsieh, M.H. and Goodman, H.M. (2005) A novel gene family in Arabidopsis encoding putative heptahelical transmembrane proteins homologous to human adiponectin receptors and progestin receptors. J. Exp. Bot. 56, 3137-3147.
Hsieh, M.H. and Goodman, H.M. (2005) The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiol. 138, 641-653.
Hsieh, M.H. and Goodman, H.M. (2002) Molecular characterization of a novel gene family encoding ACT domain repeat proteins in Arabidopsis. Plant Physiol. 130, 1797-1806.
Lancien, M., Martin, M., Hsieh, M.H., Leustek, T., Goodman, H.M. and Coruzzi, G.M. (2002) Arabidopsis glt1-T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway. Plant J. 29, 347-358.
Oliveira, I.C., Brenner, E., Chiu, J., Hsieh, M.H., Kouranov, A., Lam, H.M., Shin, M.J. and Coruzzi, G. (2001) Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway. Braz. J. Med. Biol. Res. 34, 567-575.
Lam, H.M., Chiu, J.,Hsieh, M.H., Meisel, L., Oliveira, I.C., Shin, M. and Coruzzi, G. (1998) Glutamate receptor genes in plants. Nature 396, 125-126.
Hsieh, M.H., Lam, H.M., van deLoo, F. and Coruzzi, G. (1998) A PII-like protein in Arabidopsis: Putative role in nitrogen sensing. Proc. Natl. Acad. Sci. USA 95, 13965-13970.
Lam, H.M.*,Hsieh, M.H.* and Coruzzi, G. (1998) Reciprocal regulation of distinct asparagines synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J. 16, 345-353. (*first authors)
Lam, H.M., Coschigano, K., Schultz, C., Oliveira, R., Tjaden, G., Oliveira, I., Ngai, N.,Hsieh, M.H. and Coruzzi, G. (1995) Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis. Plant Cell 7, 887-898.
Lin, C.Y., Jinn, T.L.,Hsieh, M.H. and Chen, Y.M. (1994) Class I low molecular weight heat shock proteins in plants: immunological study and thermoprotection against heat denaturation of soluble proteins. Biochemical and Cellular Mechanisms of Stress Tolerance in Plants. NATO ASI Series, Vol. H86: 233-247.
Jinn, T.L., Wu, S.H., Yeh, C.H.,Hsieh, M.H., Yeh, Y.C., Chen, Y.M. and Lin, C.Y. (1993) Immunological kinship of class I low molecular weight heat shock proteins and thermostabilization of soluble proteins in vitro among plants. Plant Cell Physiol. 34, 1055-1062.
Hsieh, M.H., Chen, J.T., Jinn, T.L., Chen, Y.M. and Lin, C.Y. (1992) A class of soybean low molecular weight heat shock protein: immunological study and quantitation. Plant Physiol. 99, 1279-1284.
