LAP6/POLYKETIDE SYNTHASE AandLAP5/POLYKETIDE SYNTHASE BEncode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis inArabidopsis thaliana

Sung Soo Kim,Carl J Douglas,Clarice De Azevedo Souza,Daniel Krahn,Etienne Grienenberger,Che C Colpitts,Dae-Yeon Suh,Dimitri Heintz,Sun Young Kim,Benjamin Lallemand,Pierrette Geoffroy,Erich Kombrink,Thierry Heitz,Michel Legrand,Markus Kaiser

doi:10.1105/tpc.110.080028

Abstract

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Highlights

Microsporogenesis and pollen development are complex processes that take place during flower development, starting with4046 The Plant Cell locules and are released by anther dehiscence (Sanders et al, 1999).A critical event during pollen maturation is the formation of the pollen surface structure
We show that LAP6/POLYKETIDE SYNTHASE A (PKSA) and LAP5/PKSB encode enzymes that catalyze the condensation of hydroxy fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) with malonyl-CoAs to yield triketide and tetraketide a-pyrones and that PKSA has a strong in vitro preference for medium-chain hydroxy fatty acyl-CoAs that, based on in vitro data, may be preferentially synthesized by ACOS5
Two of the most promising potential candidates were genes annotated as encoding plant-specific type III polyketide synthases (PKSs), LAP6/PKSA (At1g02050) and LAP5/PKSB (At4g34850), which were previously reported to generate triketide and tetraketide a-pyrone compounds using fatty acyl-CoAs as a starter substrates (Mizuuchi et al, 2008) and were strongly coexpressed with ACOS5 (r2 for LAP6/PKSA of 0.94; r2 for LAP5/PKSB of 0.99)

Summary

Introduction

Microsporogenesis and pollen development are complex processes that take place during flower development, starting with4046 The Plant Cell locules and are released by anther dehiscence (Sanders et al, 1999).A critical event during pollen maturation is the formation of the pollen surface structure. The major pollen wall components are a microspore-derived cellulosic primexine synthesized by the developing haploid microspores themselves at the tetrad stage (Blackmore et al, 2007), a thick exine deposited on the outer surface of the primexine largely after the release of free microspores, and a pectocellulosic intine produced by developing microspores and male gametophytes (Blackmore et al, 2007). In contrast with the primexine and intine, the exine is maternally derived, and exine constituents are produced in the sporophytic tapetum cell layer (Piffanelli et al, 1998) These exine constituents are secreted into the locules and incorporated into the exine by polymerization on the surface of the primexine (Piffanelli et al, 1998; Scott et al, 2004; Ma, 2005), where it often assumes a characteristic reticulate pattern, consisting of baculae and tecta. Pollen coat components accumulate in tapetum cells and are released into locules during the course of tapetum cell degeneration (Ma, 2005)

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