LAP5 and LAP6 Encode Anther-Specific Proteins with Similarity to Chalcone Synthase Essential for Pollen Exine Development in Arabidopsis

Abstract

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.

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

“…We now significantly extend previous studies (Dobritsa et al, 2010) to show that the plant-specific CHS-like type III PKSs LAP6/PKSA and LAP5/PKSB are coexpressed with ACOS5, are specifically and transiently expressed in tapetum cells, are required for exine formation and development, and function at specific stages of microspore development. Furthermore, we show that LAP6/PKSA and LAP5/PKSB encode enzymes that catalyze the condensation of hydroxy fatty acyl-CoA esters synthesized by 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.…”

“…Since the Arabidopsis PKSA and PKSB genes encode PKSs with similar in vitro activities (Mizuuchi et al, 2008;Dobritsa et al, 2010), biochemical redundancy between PKSA and PKSB was a strong possibility. To test this, we generated two independent homozygous double mutants, pksa-1 pksb-2 and pksa-1 pksb-3, identified within F2 populations derived from crossing the corresponding homozygous pksa and pksb lines.…”

“…While this work was in progress, another group reported that plants homozygous for pksa or pksb loss-of-function alleles have morphological defects in exine structure but were fertile (Dobritsa et al, 2010). To examine pollen morphology of pksa and pksb mutants in more detail and at greater resolution, pollen grains from pksa-1, pksa-2, pksb-1, and pksb-2 homozygotes were obtained and compared with those from wild-type plants.…”

“…DRL1/TKPR1 encodes a reductase similar to DIHYDROFLAVONOL 4-REDUCTASE, involved in flavonoid metabolism, and plays a pivotal role in sporopollenin precursor biosynthesis as discussed in the companion article (Grienenberger et al, 2010). Recently, it was reported that the enzymes closely related to chalcone synthase (CHS) encoded by At1g02050 (LESS ADHESIVE POLLEN [LAP6]/POLYKETIDE SYNTHASE A [PKSA]) and At4g34850 (LAP5/PKSB) catalyze the sequential condensation of a starter acyl-CoA substrate with malonyl-CoA molecules to produce alkylpyrones in vitro (Mizuuchi et al, 2008;Dobritsa et al, 2010) and that the corresponding enzymes are involved in exine formation (Dobritsa et al, 2010).…”

Two ATP Binding Cassette G (ABCG) Transporters, OsABCG26 and OsABCG15, Collaboratively Regulate Rice Male Reproduction

“…Sporopollenin is extremely resistant to physical, biological, and chemical degradation, therefore, it plays a critical role in providing protection for pollen grains from abiotic and biotic stresses (Dobritsa et al, 2010;Ariizumi and Toriyama, 2011). The chemical nature and assembly mechanism of sporopollenin still remain largely unclear.…”