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Allelic variation in the CETS (CENTRORADIALIS, TERMINAL FLOWER 1, SELF PRUNING) gene family has been shown to control agronomically important traits in many crops. CETS genes encode phosphatidylethanolamine binding proteins (PEBPs) that have a central role in the control of flowering time as florigenic and anti-florigenic signals. The great expansion of CETS genes in many species suggests that the functions of this family go beyond flowering. Here, we characterized the tomato SELF PRUNING 3C (SP3C) gene, and show that besides acting as a flowering repressor it also regulates seed germination and modulates root architecture. We show that loss of SP3C function in CRISPR/Cas9-generated mutant lines increases root length with lower root side branching. Higher SP3C expression in transgenic lines promotes the opposite effects in roots, represses seed germination and also improves tolerance to water stress in seedlings. These discoveries provide new insights into the role of SP paralogs in agronomically relevant traits, and support future exploration of the involvement of CETS genes in abiotic stress responses.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalize the sub-stomatal cavity, whereas homobaric leaves do not. In tomato (Solanum lycopersicum), BSE development is controlled by the OBSCURAVENOSA (OBV) locus. The obv mutant lacks BSEs, whereas leaves carrying the wild-type allele have BSEs. Here, we identify the obv gene and the causative mutation, a non-synonymous amino acid change. This mutation exists as a rare polymorphism in the natural range of wild tomatoes but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The mutation disrupts a C2H2 zinc finger motif in the OBV protein, resulting in the absence of BSEs in leaves and alterations to leaf function: Photosynthetic assimilation rate and leaf hydraulic conductance are both reduced in obv. Here, we show that both of these and other pleiotropic effects, including changes in leaf insertion angle, leaf margin serration, minor vein density, and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR 4 (ARF4) also results in defective BSE development, revealing an additional component of a genetic module controlling aspects of leaf development important for ecological adaptation and for breeding selection.

SummaryThe study of crop diversification has focussed mainly on the genetic changes underlying traits favoured by humans. However, the passage from natural habitats to agronomic settings probably operated changes beyond those comprising the classical domestication syndrome. A deeper understanding of these traits and their genetic signature would be valuable to inform conventional crop breeding and de novo domestication of crop wild relatives. Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalise the sub-stomatal cavity whereas homobaric leaves do not; BSE development is known to be controlled by the OBSCURAVENOSA (OBV) locus and the obv mutant lacks BSEs whereas leaves carrying the wild type allele have BSEs. Here we identify the OBV gene and the causative mutation, a non-synonymous amino acid change. This mutation exists as a rare balanced polymorphism in the natural range of wild tomatoes, but has increased in frequency in domesticated tomatoes suggesting that the latter diversified into heterobaric and homobaric leaf types. The mutation disrupts a C2H2 zinc finger motif in the OBV protein, resulting in the absence of BSEs in leaves and here we show that this and other pleiotropic effects, including changes in leaf insertion angle, leaf margin serration, minor vein density and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR (ARF4) also results in defective BSE development, revealing an additional component of a novel genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selections.One sentence summaryDistribution of heterobaric and homobaric leaves is controlled by natural variation in an auxin-related transcription factor

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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.