Cloning and functional characterization of the OBSCURAVENOSA gene in tomato (Solanum lycopersicum L.)

Abstract

Heterobaric and homobaric leaves are found in many plant families and have a remarkable distribution in nature. What differentiates these two types of leaves is the presence or absence of bundle sheath extensions (BSEs), a structural trait which produces different physiological responses. Leaves with BSEs (heterobaric) have greater hydraulic integration and greater photosynthetic performance, on the other hand, leaves without BSEs (homobaric) have uniform photosynthesis along the blade and probably an improved mechanism to control water loss under stress conditions. Tomatoes currently cultivated were selected after long years of crop breeding. Originally, tomato is a heterobaric crop, however, most field cultivars have a spontaneous recessive mutation known as obscuravenosa (obv) and, as a consequence, have lost the BSEs. For several years, this mutation was unconsciously selected in conjunction with other traits of agronomic interest. This suggests that the variation between homobaric and heterobaric characters has agronomic value in tomato. The first step to better understand the impacts of BSE at the plant level and to be able to manipulate it within the tomato crop and in other crops is knowing the genes involved in its formation. For this, we use tomato as a model to elucidate the molecular bases that control the development of BSEs. Using publicly available resources combined with in silico analysis, we identified a strong candidate gene for the obv mutation. Solyc05g054030 has a SNP (A→G) in the third exon, which leads to the exchange of a histidine residue for arginine at position 135 of the protein. The gene encodes a zinc finger transcription factor C2H2 type. We have shown through predictions that the OBV protein has three classic zinc finger domains, which allow interaction with DNA. In the obv mutant, the exchange of histidine for arginine caused the loss of a zinc finger motif, which may have led to the interruption of the protein functionality. We complemented the obv mutant with the functional OBV allele recovering the clear vein phenotype . OBV further regulates the leaf insertion angle, leaf serration, vein density and fruit shape. OBV appears to coordinate the development of BSEs through an auxin-mediated mechanism, specifically by changes in some members involved in auxin signaling (ARFs and Aux/IAAs). We have identified a link between OBV and AUXIN RESPONSE FACTOR 4 (ARF4). The findings reported here will give support for identification of other components linked to the molecular pathway that directs the formation of BSEs in leaves. Keywords: Bundle Sheath Extension. Heterobaric leaf. Molecular cloning. Auxin.

Functional characterization of plant specific Indeterminate Domain (IDD) transcription factors in tomato (Solanum lycopersicum L.)

“…Heterobaric leaves contains Bundle sheath extensions in the leaves which provide mechanical strength. SlIDD5 mutants failed to produce bundle sheath extension cells (homobaric leaves) 78 . Increased chlorophyll content has been observed in obv mutants, such as M82 and CRISPR/ Cas9 mutants of Micro-Tom, where the absence of BSE allowed chloroplast development in leaf veins and reduced water conductivity 79,80 .…”