Investigating leaf development in moss (P. patens) using Tnt1 insertional "short-leaf (shlf )" and targeted knockout "slender-leaf" mutants

Abstract

Leaves are specialized photosynthetic organs that greatly influence the fitness of the plant. Due to extensive selection pressure on leaf size and shape, multiple convergent evolutions resulted in development of phyllids, microphylls and megaphylls in land plants. Phyllids are uni-layered early leaf-like organs that appeared in bryophytes itself, arranged spirally on a gametophore. There are scarce information about development and the genetic network that govern such leaf like structures in bryophyte. To understand how phyllids development differs from microphylls and megaphylls in land plants, we chose Physcomitrella patens, a bryophyte model organism. We developed a Tnt1 retrotransposon mediated insertional mutagenesis protocol and carried out a forward genetic screen. High gene preference of Tnt1 transposition activity resulted in isolation of many interesting developmental phenotypes. Short-leaf mutant produced two times shorter leaves compared to wild type among many other phenotypes. Using whole genome sequencing and candidate gene over-expression and knockdown approaches, we have identified Tnt1 insertion sites and the causal mutation for short-leaf phenotype. Characterization of the mutant locus is in progress. In our parallel reverse genetics approach, we chose SCARECROW(SCR) gene which is essential for a critical asymmetric cell division pattern in Arabidopsis root. Our Ppscr3 knock out lines produced slender leaves compared to wild type. Histological analysis revealed an interesting alternation in a well-orchestrated cell division pattern that possibly the cause for slender leaf phenotype. Our study would shed light on the differences in development of simple, early leaf-like organs and sophisticated micro- and megaphylls in land plants.