Alkaline alpha-galactosidase degrades thylakoid membranes in the chloroplast during leaf senescence in rice.
Here, we studied the functional role of a chloroplast alkaline alpha-galactosidase (OsAkalphaGal) in the breakdown of thylakoid membranes during rice (Oryza sativa) leaf senescence. We assayed the enzyme activity of recombinant OsAkalphaGal with different natural substrates and examined the effect of ectopic OsAkalphaGal expression in rice plants. Recombinant OsAkalphaGal showed at least a two-fold greater substrate-binding affinity and a 10-fold greater turnover rate to galactolipid digalactosyl diacylglycerol than the raffinose family of oligosaccharides (verbascose, stachyose, raffinose) and melibiose. The OsAkalphaGal null mutant (osakalphagal) displayed a delayed leaf senescence phenotype. OsAkalphaGal complementation in osakalphagal recovered OsAkalphaGal expression and showed a senescence phenotype similar to that of wild-type plants. Transgenic plants overexpressing OsAkalphaGal (UbiP-OsAkalphaGal) exhibited retarded plant growth and development, and showed a pale-green phenotype coupled with a reduced chlorophyll content to 42% in newly unfolded leaves. UbiP-OsAkalphaGal leaves also showed a 29-fold increase in alkaline alpha-galactosidase activity compared with wild-type leaves. An ultrastructural study of Ubi-OsAkalphaGal chloroplasts in newly unfolded leaves revealed abnormal grana organization. Our findings strongly suggest that OsAkalphaGal is a thylakoid membrane-degrading enzyme involved in the degradation of digalactosyl diacylglycerol during rice leaf senescence.
Lee
Ruey-Hua
RH
Department of Life Sciences and Institute of Biodiversity, National Cheng-Kung University, No. 1, University Road, Tainan 700, Taiwan.
Hsu
Jen-Hung
JH
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan.
Huang
Hao-Jen
HJ
Department of Life Sciences and Institute of Biodiversity, National Cheng-Kung University, No. 1, University Road, Tainan 700, Taiwan.
Lo
Shu-Fang
SF
Chiayi Agricultural Experiment Station, TARI, Chiayi, Taiwan.
Grace Chen
Shu-Chen
SC
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan.
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D038983:Galactolipids / Q000378:metabolism; D017353:Gene Deletion; D017343:Genes, Plant; D005816:Genetic Complementation Test; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D012275:Oryza / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure; D010641:Phenotype; D018515:Plant Leaves / Q000201:enzymology / Q000254:growth & development / Q000648:ultrastructure; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020524:Thylakoids / Q000201:enzymology* / Q000648:ultrastructure; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018744:DNA, Plant; D038983:Galactolipids; D010940:Plant Proteins; D011994:Recombinant Proteins; C007388:digalactosyldiacylglycerol; D000519:alpha-Galactosidase
Bachmann M, Keller F. 1995. Metabolism of the raffinose family oligosaccharides in leaves of Ajuga reptans L. Inter- and intracellular compartmentation. Plant Physiology 109: 991-998.
Bachmann M, Matile P, Keller F. 1994. Metabolism of the raffinose family oligosaccharides in leaves of Ajuga reptans L. (cold acclimation, translocation, and sink to source transition: discovery of chain elongation enzyme). Plant Physiology 105: 1335-1345.
Barclay KD, Mckersie BD. 1994. Peroxidation reactions in plant membranes: effects of free fatty acids. Lipid 29: 877-882.
Benning C, Ohta H. 2005. Three enzyme systems for galactoglycerolipid biosynthesis are coordinately regulated in plants. Journal of Biological Chemistry 280: 2397-2400.
Buchanan-Wollaston V. 1997. The molecular biology of leaf senescence. Journal of Experimental Botany 48: 181-199.
Carmi N, Zhang G, Petreikov M, Gao Z, Eyal Y, Granot D, Schaffer AA. 2003. Cloning and functional expression of alkaline α-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolase. Plant Journal 33: 97-106.
Dörmann P, Benning C. 2002. Galactolipids rule in seed plants. Trends in Plant Sciences 7: 112-118.
Dörmann P, Hoffmann-Benning S, Balbo I, Benning C. 1995. Isolation and characterization of an Arabidopsis thaliana mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. Plant Cell 7: 1801-1810.
Dörmann P, Balbo I, Benning C. 1999. Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGDG1. Science 284: 2181-2184.
Fyfe PK, Hughes AV, Heathcote P, Jones MR. 2005. Proteins, chlorophylls and lipids: X-ray analysis of a three-way relationship. Trends in Plant Sciences 10: 275-282.
Gao ZF, Schaffer AA. 1999. A novel α-galactosidase from melon fruit with substrate preference for raffinose. Plant Physiology 119: 979-988.
Gray JC. 1996. Biogenesis of chloroplasts in higher plants. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford, UK: Bios Scientific Publishers, 441-458.
Grossmann GA, Terra WT. 2001. α-Galactosidases from the larval midgut of Tenebrio molitor (Coleoptera) and Spodoptera frugiperda (Lepidoptera). Comparative Biochemical Physiology 128: 109-122.
He Y, Gan S. 2002. A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis. Plant Cell 1: 805-815.
Henrissat B, Coutinho PM, Davies GJ. 2001. A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana. Plant Molecular Biology 47: 55-72.
Hörtensteiner S. 2009. Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. Trends in Plant Science 14: 155-162.
Ikeda N, Bautisuda NS, Yamada T, Kamijima O, Ishii T. 2001. Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice. Plant Molecular Biology Report 19: 27-32.
Jarvis P, Dörmann P, Peto CA, Lutes J, Benning C, Chory J. 2000. Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant. Proceedings of the National Academy of Science, USA 97: 8175-8179.
Jones MR. 2007. Lipids in photosynthetic reaction centres: structural roles and functional holes. Progress in Lipid Research 46: 56-87.
Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauß N. 2001. Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution. Nature 411: 909-917.
Joyard J, Marechal E, Block MA, Douce R. 1996. Plant galactolipids and sulfolipid: structure, distribution and biosynthesis. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford, UK: Bios Scientific Publishers, 441-458.
Keller F, Matile P. 1985. The role of the vacuole in storage and mobilization of stachyose in tubers of Stachys sieboldii. Journal of Plant Physiology 119: 369-380.
Kobayashi K, Kondo M, Fukuda H, Nishimura M, Ohta H. 2007. Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis. Proceedings of the National Academy of Science, USA 104: 17216-17221.
Lee RH, Chen SCG. 2002. Programmed cell death during rice leaf senescence is nonapoptotic. New Phytologist 155: 25-32.
Lee RH, Wang CH, Huang LT, Chen SCG. 2001. Leaf senescence in rice plants: cloning and characterization of senescence up-regulated genes. Journal of Experimental Botany 52: 1117-1121.
Lee RH, Lin MC, Chen SCG. 2004. A novel alkaline α-galactosidase gene is involved in rice leaf senescence. Plant Molecular Biology 55: 281-295.
Lichtenthaler HK. 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Method in Enzymology 148: 351-382.
Lim PO, Kim HJ, Nam HG. 2007. Leaf senescence. Annual Review of Plant Biology 58: 115-136.
Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang W. 2004. Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428: 287-292.
Martínez DE, Costa ML, Guiamet JJ. 2008. Senescence-associated degradation of chloroplast proteins inside and outside the organelle. Plant Biology 10: 15-22.
Matile P. 1992. Chloroplast senescence. In: Baker NR, Thomas H, eds. Crop photosynthesis: spatial and temporal determinants. New York, NY, USA: Elsevier Science Publishers, 413-440.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.
Noodén LD, Guiamet JJ, John I. 1997. Senescence mechanisms. Physiologia Plantarum 101: 746-753.
Nußberger S, Dörr K, Wang DN, Kühlbrandt W. 1993. Lipid-protein interactions in crystals of plant light-harvesting complex. Journal of Molecular Biology 234: 347-356.
Peter GF, Thornber JP. 1991. Biochemical composition and organization of higher plant photosystem II light harvesting pigment-proteins. Journal of Biological Chemistry 266: 16745-16754.
Peterbauer T, Richter A. 2001. Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. Seed Science Research 11: 185-197.
Sallaud C, Meynard D, Van Boxtel J, Gay C, Bès M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M et al. 2003. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theoretical and Applied Genetics 106: 1396-1408.
Stelmach BA, Müller A, Henning P, Gabhardt S, Schubert-Zsilavecz M, Weiler EW. 2001. A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride, from Arabidopsis thaliana. Journal of Biological Chemistry 276: 12832-12838.
Thompson JE, Froese CD, Madey E, Smith MD, Hong Y. 1998. Lipid metabolism during plant senescence. Progress in Lipid Research 37: 119-141.
Webb MS, Green BR. 1991. Biochemical and biophysical properties of thylakoid acyl lipids. Biochimica et Biophysica Acta 1060: 133-158.
Woolhouse HW. 1984. The biochemistry and regulation of senescence in chloroplasts. Canadian Journal of Botany 62: 2934-2942.
Xu CC, Jilian F, Froehlich JE, Awai K, Benning C. 2005. Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell 17: 3094-3110.
Zhao TY, Corum JW, Mullen J, Meeley RB, Helentjaris T, Martin D. 2006. An alkaline alpha-galactosidase transcript is present in maize seeds and cultured embryo cells, and accumulates during stress. Seed Science Research 16: 107-121.