Functional analysis of anther-specific genes essential for pollen exine development and male fertility in tobacco

Abstract

In flowering plants, pollen grains are surrounded by extremely strong outer walls providing solid and firm structure for protecting pollen and species-specific interactions with female stigma. The outer wall of pollen, referred to as exine, is composed of sporopollenin polymer, but the composition and synthesis of sporopollenin remains poorly understood. Previous studies have indicated that several genes such as Fatty Acyl-CoA Synthetase (ACOS5), Polyketide Synthases (PKSA and PKSB), and Tetraketide α-Pyrone Reductase (TKPR1) take part in the biosynthesis of sporopollenin in Arabidopsis thaliana. The existence of ancient biochemical pathways for sporopollenin biosynthesis has been widely proposed but experimental evidence from plant species other than Arabidopsis is not extensively available. In this study, two homologous PKS genes, NtPKS1 and NtPKS2, were found in tobacco (Nicotiana tabacum). Results of RT-PCR and in situ hybridization revealed that NtPKS1 and NtPKS2 are specifically and transiently expressed in tapetal cells during microspore development in tobacco anthers. RNAi plants of NtACOS1 and NtPKS1 were investigated. Comparing with wild-type tobacco (SR1), abnormal pollens, defect exine structure, and male sterility were found in the RNAi lines. Enzymatic assays show that NtPKS1 and NtPKS2 encode anther-specific enzymes using fatty acyl-coenzyme A and p-coumaroyl coenzyme A as substrates to yield tri- and tetra- ketide α-pyrone and bisnoryangonin respectively. In this study, the metabolic steps catalyzed by the anther-specific acyl- CoA synthetase (ACOS), polyketide synthase (PKS), and tetraketide α-pyrone reductase (TKPR) were investigated. Using fatty acids as starting substrates, sequential activities of heterologously-expressed tobacco enzymes NtACOS1, NtPKS1, and NtTKPR1 resulted in the production of reduced tetraketide α- pyrones which propose to contribute to the biosynthesis of sporopollenin precursors in tobacco.