Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis – phylogeny, site‐directed mutagenesis, and expression in nonanther tissues

Abstract

Anther-specific chalcone synthase-like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala-clade and the Val-clade, and HpPKS1 belongs to the Ala-clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium-chain (C 6 ) to very long-chain (C 24 ) fatty acyl-CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl-CoA esters over the analogous unsubstituted fatty acyl-CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl-binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl-CoA, a poor substrate for the wild-type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium-chain to long-chain fatty acyl-CoA substrates. The R202L/D211V double mutant was selective for acyl-CoA with chain lengths of C 16 -C 18 , and showed a diminished preference for the hydroxylated acyl-CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala-clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues.

Type III Polyketide Synthases: Functional Classification and Phylogenomics

“…Only one ASCL gene has been found in each bryophyte or fern genome, whereas each angiosperm genome has two or more ASCL genes that are divided into two distinct sister clades. Two angiosperm ASCLs (PKS‐A and HpPKS1), which are grouped in one of the sister clades, are known to be expressed in non‐anther parts such as leaves, thus indicating that these PKSs gained new functions …”

“…2) Anther-specific CHS-like (ASCL) enzymes are hydroxyalkylpyrone synthases that mainly catalyze the formation of (hydroxylated) tetraketide and triketide pyrones (Lh-4-L, Lh-3-L). [76][77][78][79] The hydroxylated tetraketide pyrone is reduced by tetraketide a-pyrone reductases to produce ap olyhydroxylated pyrone, which is ap robablep recursor of sporopollenin, ac omponento fp ollen wall exine. [4] ASCL genes are specifically expressed in tapetal cells during anther development in several plant species.…”

Genetic and Biochemical Mechanisms of Pollen Wall Development

“…ACOS5, PKSA, PKSB, TKPR1, and CYP704B proteins form a sporopollenin metabolon in the ER to effectively produce lipidic and phenolic precursors for sporopollenin biosynthesis [57]. The function of PKS is conserved in St John's wort (Hypericum perforatum, a herb plant) [58]. Whether this pathway is also conserved in rice or other plants remains unknown.…”

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice

“…With the exception of TKPR2, these enzymes seem to be specific for this pathway, forming a sporopollenin metabolon in Arabidopsis anther tapetal cells that also interacts with membrane cytochrome P450 proteins (Lallemand et al ). This pathway is conserved in tobacco, rice (Wang et al ), moss (Colpitts et al ), Hypericum perforatum (Jepson et al ) and canola (Qin et al ). However, the products of the pathway have yet to be detected in anthers, perhaps because of the additional activities of these enzymes in vivo , or because the products are further modified during pollen exine formation.…”