Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis  

Abstract

The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield a-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/ dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide a-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE a-PYRONE REDUC-TASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated a-pyrone compounds that were previously unknown to be sporopollenin precursors.

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

“…Since the P450 hydroxylases are likely ER localized, they may form metabolons with the ER-associated PKSs to form alkyl-a-pyrones. Consistent with this, the TKPR1 enzyme that appears to act downstream of PKSA and PKSB is also ER localized (Grienenberger et al, 2010), suggesting the presence of an ER-localized pathway for alkyla-pyrone sporopollenin precursor biosynthesis.…”

“…In the majority of anthers observed, defective microspore development was first observed at stage 9 (Figure 8), consistent with the timing of transient PKSA and PKSB expression (Figure 3) and protein accumulation (Figure 4) in the tapetum and the timing of exine formation (Blackmore et al, 2007). High-resolution TEM images of stage 9 wild-type and double mutant anthers (Figure 9) showed that mutant microspores completely lacked exine, which was replaced by an amorphous material similar to that we observed in other mutants defective in sporopollenin biosynthesis (acos5, abcg26, and drl1/tkpr1; de Azevedo Souza et al, 2009;Grienenberger et al, 2010;Quilichini et al, 2010). No abnormalities in tapetum cells were observed, and anther epidermal wall cutin deposition was similar to that in wild-type plants.…”

“…For example, reduction of keto functions after elongation of the chain by PKSA/PKSB would prevent the formation of the a-pyrone ring, and alkyl phloroglucinols could be formed as sporopollenin natural products. However, the strong substrate preferences for tapetumexpressed reductases DRL1/TKPR1 and TKPR2, involved in sporopollenin biosynthesis, for the in vitro tetraketide a-pyrone product (Grienenberger et al, 2010) support the hypothesis that these are true PKSA/PKSB-derived natural products.…”

“…Information on such components may come from enzymes encoded by genes that are coregulated with ACOS5 and other genes involved in sporopollenin biosynthesis. Among such coregulated genes is Arabidopsis DIHYDROFLAVONOL 4-REDUCTASE LIKE1 (DRL1)/ TETRAKETIDE a-PYRONE REDUCTASE (TKPR1) (Grienenberger et al, 2010), which is required for male fertility and exine formation (Tang et al, 2009). 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).…”

“…Among such coregulated genes is Arabidopsis DIHYDROFLAVONOL 4-REDUCTASE LIKE1 (DRL1)/ TETRAKETIDE a-PYRONE REDUCTASE (TKPR1) (Grienenberger et al, 2010), which is required for male fertility and exine formation (Tang et al, 2009). 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).…”