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

Abstract

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 a-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.

Abundant Type III Lipid Transfer Proteins in Arabidopsis Tapetum Are Secreted to the Locule and Become a Constituent of the Pollen Exine

“…Pollen exine is a polymer collectively termed sporopollenin, which consists of unbranched and branched aliphatic and aromatic monomers that are hydroxylated or oxygenated (Ariizumi and Toriyama, 2011;Kim and Douglas, 2013;Lallemand et al, 2013). These monomers are synthesized by ER-associated or cytosolic enzymes in the tapetum.…”

“…Because the tapetum is secretory in nature, the tapetum LTPs, which are also found in the anther locule, are considered to function to transfer lipid materials from the tapetum to maturing microspores. However, this function has not been defined, even though the pollen exine consists of lipid monomers and some of their synthetic enzymes are associated with the ER (Ariizumi and Toriyama, 2011;Kim and Douglas, 2013;Lallemand et al, 2013).…”

IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation

“…However, the mechanism underlying the conversion from hydroxyacids to dicarboxylic acids in the reproductive organs is little known. It is believed that modifications of fatty acids from the plastid occur in the endoplasmic reticulum, which contributes to anther cuticle and pollen wall development (Kunst and Samuels, 2003;Lallemand et al, 2013). In our work, we cloned a novel maize malesterile gene, IPE1, putatively encoding a member of the GMC oxidoreductase superfamily.…”

MYB107 and MYB9 Homologs Regulate Suberin Deposition in Angiosperms

“…A selected number of genes are provided in Table 2 and include those involved in both aliphatic and aromatic metabolic pathways associated with suberin formation. Genes potentially linked with suberin biosynthesis found to be downregulated in both myb9 and myb107 seeds include those encoding SUS (the putative suberin synthase); an SGNH hydrolase-type esterase superfamily protein (likely involved in lipid metabolic processes; Li-Beisson et al, 2013); CASP-LIKE PROTEIN 1B2 (CASPL1B2, showing strong homology to CASP proteins involved in casparian strip metabolism); LACCASE3 (a member of the laccase family of which LACCASE4 has been demonstrated to be involved in lignin metabolism; Zhao et al, 2013;Schuetz et al, 2014); DIHYDROFLAVONOL 4-REDUCTASE-LIKE1 (DFR-like1; involved in phenylpropanoid metabolism and previously linked to pollen wall development and seed production (Lallemand et al, 2013); and LUPEOL SYNTHASE5 (LUP5; a multifunctional triterpene synthase; Ebizuka et al, 2003). While genes downregulated in myb107 seeds (but not myb9 seeds) encode GPAT5 and ASFT (known to act in suberin formation); 3-KETOACYL-COA SYNTHASE17 (KCS17; a very-long-chain fatty acid synthase); 4-COUMARATE:COA LIGASE5 (4CL5; involved in phenylpropanoid metabolism); and GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED LIPID PROTEIN TRANSFER5 (LTPG5; a homolog to the lipid transporter LTPG1; Debono et al, 2009;Edstam and Edqvist, 2014).…”