Knockdown of Arabidopsis RUS4 impacts pollen coat formation by affecting flavonoid and lipid metabolism

The DUF647 family member RUS4 contributes to pollen coat formation in Arabidopsis by modulating transcript levels of key genes for flavonoid and lipid metabolism. RUS4 encodes a member of the broadly conserved DUF647 domain-containing protein family that is distributed across eukaryotes. We have previously shown that artificial microRNA knockdown Arabidopsis RUS4 lines (known as amiR-RUS4) exhibit reduced male fertility due to reduced pollen viability, decreased pollen germination rate, and impaired pollen tube growth. However, amiR-RUS4 plants exhibit a previously uncharacterized phenotype in pollen coat formation, which was investigated in the present study. Scanning electron microscopy and transmission electron microscopy analyses revealed that amiR-RUS4 pollen grains exhibit abnormal pollen wall structure and defective pollen coat deposition. Confocal observation of DPBA and Nile Red double-stained pollen grains indicated less accumulation of lipids and flavonoids in the pollen coat of amiR-RUS4 plants compared with wild type. Quantitative image analysis confirmed significantly lower fluorescence signal intensities of both DPBA and Nile Red in amiR-RUS4 pollen relative to the wild type. Consistently, the expression of flavonol metabolic genes was down-regulated in amiR-RUS4 stamens. Moreover, the transcript abundance of several pollen coat extracellular lipases (EXLs) genes was substantially reduced in amiR-RUS4 flower buds. Similarly, the expression of two 3-ketoacyl-CoA synthase genes KCS5 and KCS6, which are responsible for the biosynthesis of very-long-chain fatty acids in the pollen coat, was also altered in amiR-RUS4 flower buds. Taken together, these results suggest that RUS4 is involved in pollen coat formation in Arabidopsis by modulating the expression of key genes for flavonoid and lipid metabolism. This study advances our understanding of the biological functions of DUF647 family genes during plant pollen development.