Sporopollenin Biosynthetic Enzymes Interact and Constitute a Metabolon Localized to the Endoplasmic Reticulum of Tapetum Cells[W]

Enzymes involved in the biosynthesis of sporopollenin, the main constituent of pollen exine, likely form a metabolon in the endoplasmic reticulum of the anther tapetal cells. The sporopollenin polymer is the major constituent of exine, the outer pollen wall. Recently fatty acid derivatives have been shown to be the precursors of sporopollenin building units. ACYL-COA SYNTHETASE, POLYKETIDE SYNTHASE A (PKSA) and PKSB, TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 have been demonstrated to be involved in sporopollenin biosynthesis in Arabidopsis (Arabidopsis thaliana). Here all these sporopollenin biosynthetic enzymes but TKPR2 have been immunolocalized to endoplasmic reticulum of anther tapetal cells. Pull-down experiments demonstrated that tagged recombinant proteins interacted to form complexes whose constituents were characterized by immunoblotting. In vivo protein interactions were evidenced by yeast (Saccharomyces cerevisiae) two-hybrid analysis and by fluorescence lifetime imaging microscopy/Förster resonance energy transfer studies in transgenic Nicotiana benthamiana, which were used to test the possibility that the enzymes interact to form a biosynthetic metabolon. Various pairs of proteins fused to two distinct fluorochromes were coexpressed in N. benthamiana leaf tissues and fluorescence lifetime imaging microscopy/Förster resonance energy transfer measurements demonstrated that proteins interacted pairwise in planta. Taken together, these results suggest the existence of a sporopollenin metabolon.

[1]  R. Sederoff,et al.  Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis , 2011, Proceedings of the National Academy of Sciences.

[2]  L. Sumner,et al.  A Large-Scale Genetic Screen in Arabidopsis to Identify Genes Involved in Pollen Exine Production1[C][W][OA] , 2011, Plant Physiology.

[3]  L. Schreiber,et al.  Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis1[C][W][OA] , 2011, Plant Physiology.

[4]  T. Gadella,et al.  Förster resonance energy transfer demonstrates a flavonoid metabolon in living plant cells that displays competitive interactions between enzymes , 2011, FEBS letters.

[5]  T. Ariizumi,et al.  Genetic regulation of sporopollenin synthesis and pollen exine development. , 2011, Annual review of plant biology.

[6]  C. Douglas,et al.  LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B Encode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis thaliana[C][W][OA] , 2010, Plant Cell.

[7]  C. Douglas,et al.  Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W] , 2010, Plant Cell.

[8]  C. Douglas,et al.  ATP-Binding Cassette Transporter G26 Is Required for Male Fertility and Pollen Exine Formation in Arabidopsis1[W][OA] , 2010, Plant Physiology.

[9]  Dabing Zhang,et al.  OsC6, Encoding a Lipid Transfer Protein, Is Required for Postmeiotic Anther Development In Rice1[W][OA] , 2010, Plant Physiology.

[10]  S. Ishiguro,et al.  The Arabidopsis FLAKY POLLEN1 gene encodes a 3-hydroxy-3-methylglutaryl-coenzyme A synthase required for development of tapetum-specific organelles and fertility of pollen grains. , 2010, Plant & cell physiology.

[11]  D. Huhman,et al.  LAP5 and LAP6 Encode Anther-Specific Proteins with Similarity to Chalcone Synthase Essential for Pollen Exine Development in Arabidopsis1[W][OA] , 2010, Plant Physiology.

[12]  B. Møller,et al.  CYP704B1 Is a Long-Chain Fatty Acid ω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis1[W][OA] , 2009, Plant Physiology.

[13]  L. Sumner,et al.  LAP3, a novel plant protein required for pollen development, is essential for proper exine formation , 2009, Sexual Plant Reproduction.

[14]  Sung Soo Kim,et al.  A Novel Fatty Acyl-CoA Synthetase Is Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis[C][W] , 2009, The Plant Cell Online.

[15]  W. Yip,et al.  An anther-specific dihydroflavonol 4-reductase-like gene (DRL1) is essential for male fertility in Arabidopsis. , 2009, The New phytologist.

[16]  A. Huang,et al.  Analyses of Advanced Rice Anther Transcriptomes Reveal Global Tapetum Secretory Functions and Potential Proteins for Lipid Exine Formation1[W][OA] , 2008, Plant Physiology.

[17]  I. Abe,et al.  Structure function analysis of novel type III polyketide synthases from Arabidopsis thaliana. , 2008, Biological & pharmaceutical bulletin.

[18]  S. Ishiguro,et al.  Identification of kaonashi Mutants Showing Abnormal Pollen Exine Structure in Arabidopsis thaliana , 2008, Plant & cell physiology.

[19]  R. Pepperkok,et al.  Membrane Dynamics in the Early Secretory Pathway , 2007 .

[20]  B. Møller,et al.  CYP703 Is an Ancient Cytochrome P450 in Land Plants Catalyzing in-Chain Hydroxylation of Lauric Acid to Provide Building Blocks for Sporopollenin Synthesis in Pollen[W] , 2007, The Plant Cell Online.

[21]  J. Skvarla,et al.  Pollen wall development in flowering plants. , 2007, The New phytologist.

[22]  A. Huang,et al.  Tapetosomes in Brassica Tapetum Accumulate Endoplasmic Reticulum–Derived Flavonoids and Alkanes for Delivery to the Pollen Surface[W] , 2007, The Plant Cell Online.

[23]  A. Huang,et al.  Lipid-rich tapetosomes in Brassica tapetum are composed of oleosin-coated oil droplets and vesicles, both assembled in and then detached from the endoplasmic reticulum. , 2005, The Plant journal : for cell and molecular biology.

[24]  H. Dickinson,et al.  Stamen Structure and Function , 2004, The Plant Cell Online.

[25]  J. Lambert,et al.  Acetylation and Silylation of Piperidine Solubilized Sporopollenin from Pollen of Typha angustifolia L. , 2003, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[26]  Qun Xia,et al.  Arabidopsis AtGPAT1, a Member of the Membrane-Bound Glycerol-3-Phosphate Acyltransferase Gene Family, Is Essential for Tapetum Differentiation and Male Fertility Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.012427. , 2003, The Plant Cell Online.

[27]  H. Owen,et al.  DEX1, a novel plant protein, is required for exine pattern formation during pollen development in Arabidopsis. , 2001, Plant physiology.

[28]  B. Winkel-Shirley,et al.  Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  B. Winkel-Shirley,et al.  Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways , 1999 .

[30]  D. Murphy,et al.  Composition and role of tapetal lipid bodies in the biogenesis of the pollen coat of Brassica napus , 1999, Planta.

[31]  A. Huang,et al.  Steryl esters in the elaioplasts of the tapetum in developing Brassica anthers and their recovery on the pollen surface , 1999, Lipids.

[32]  E. Domínguez,et al.  Pollen sporopollenin: degradation and structural elucidation , 1999, Sexual Plant Reproduction.

[33]  P. M. Sanders,et al.  Anther developmental defects in Arabidopsis thaliana male-sterile mutants , 1999, Sexual Plant Reproduction.

[34]  P. Laporte,et al.  The loculus content and tapetum during pollen development in Lilium , 1998, Sexual Plant Reproduction.

[35]  D. Murphy,et al.  Biogenesis and function of the lipidic structures of pollen grains , 1998, Sexual Plant Reproduction.

[36]  Tzann-Wei Wang,et al.  Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K. Kalantidis,et al.  The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. , 1997, The Plant journal : for cell and molecular biology.

[38]  D. Murphy,et al.  Intra- and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus. , 1997, The Plant journal : for cell and molecular biology.

[39]  G. Hannon,et al.  Plant cells contain a novel member of the retinoblastoma family of growth regulatory proteins. , 1996, The EMBO journal.

[40]  H. Owen,et al.  Ultrastructure of microsporogenesis and microgametogenesis inArabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae) , 1995, Protoplasma.

[41]  J. Bowman,et al.  Early flower development in Arabidopsis. , 1990, The Plant cell.

[42]  A. Zobel,et al.  Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G. Wagner,et al.  Metabolic pathways as enzyme complexes: evidence for the synthesis of phenylpropanoids and flavonoids on membrane associated enzyme complexes. , 1985, Archives of biochemistry and biophysics.

[44]  J. Tukey,et al.  Variations of Box Plots , 1978 .

[45]  M. Davidson,et al.  Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. , 2012, Methods in enzymology.

[46]  M. Suh,et al.  An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen. , 2011, The Plant journal : for cell and molecular biology.

[47]  D. Ye,et al.  WBC27, an adenosine tri-phosphate-binding cassette protein, controls pollen wall formation and patterning in Arabidopsis. , 2011, Journal of integrative plant biology.

[48]  C. Douglas,et al.  ATP-Binding Cassette Transporter G26 Is Required for Male Fertility and Pollen Exine Formation in Arabidopsis , 2010 .

[49]  H. Kindl,et al.  Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution , 2004, Planta.

[50]  M. Cresti,et al.  Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features , 2004, Sexual Plant Reproduction.

[51]  J. Lambert,et al.  Continuous Decomposition of Sporopollenin from Pollen of Typha angustifolia L. by Acidic Methanolysis , 2002, Zeitschrift fur Naturforschung. C, Journal of biosciences.