Defective Spore Development and Differentially Expressed Genes In The Sporophytes of mkn4-2-1, A Triple Deletion Mutant of Physcomitrella patens

Abstract

KNOTTED1-like homeobox (KNOX) genes encode homeodomain transcription factors involved in maintaining the shoot apical meristem of flowering plants. They are widely distributed in land plants where they perform diverse roles. A triple knockout of KNOX genes, mkn4-2-1, in the moss, Physcomitrella patens, produced malformed sporophytes and defective spores with under-developed perine (Singer and Ashton, 2007, Plant Cell Rep, 26: 2039). I hypothesized that moss KNOX homologues (MKN genes) could be the executive regulators of spore wall formation in Physcomitrella, and that the biosynthesis and regulatory networks of spore/pollen grain wall formation might be conserved in plants. The mkn4-2-1 spores shed their walls, and underwent augmented osmolysis. Although defective, the exine in mkn4-2-1 spores is more resistant to alkaline hydrolysis than that of control spores. Mkn4-2-1 produced malformed tetrads and fewer spores per sporophyte. The spores were heterogeneous in size and shape, suggesting defective meiosis of mkn4-2-1 sporocytes. Interestingly, the polymeric and, possibly, lignin-like pigments in the setae of the mutant were alkaline sensitive, suggesting their defective biosynthesis. Based on phenotypic observations and available transcriptomic data, a total of 111 Physcomitrella genes were selected as putative target genes of MKN1, 2 and/or 4, and their differential expression levels in mkn4-2-1 and control sporophytes were investigated by semi-quantitative RT-PCR. Physcomitrella homologues of Arabidopsis genes responsible for sporopollenin deposition (NEF1, NPU), tapetum development (EMS1, TPD1, MS1), and spore wall formation (CUSL) were down-regulated in mkn4-2-1, implicating them in defective spore wall formation. RT-PCR revealed reduced expression of meiosis-specific genes DMC1, ASY1 and SPO11 in mkn4-2-1, providing a ii possible explanation for abnormal tetrad formation, heterogeneous spores and fewer spores per sporophyte. Reduced expression was also found in genes involved in the metabolism of lignin-like materials (CCR, LACL1, LACL2). This study uncovered novel phenotypic differences in spore walls, tetrad formation and sporophytic pigments in the mutant, and identified several differentially expressed genes that may be regulated by MKN1, 2 and/or 4. It also provides insights into the functional evolution of KNOX genes in plants and the regulatory roles of MKN genes in sporophytic ontogeny, sporogenesis and the formation of the spore wall in a moss.