Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

Water availability is the main factor that explains current patterns of palm abundance. However, the interaction between water stress and increasing atmospheric CO 2 concentrations caused by climatic change and its effects on palm physiology remain poorly known. Macauba palm is a widespread Neotropical species commonly found in ecosystems subjected to seasonal drought and has potential use in oil production. The present work investigated the influence of increased CO 2 concentrations on photosynthetic responses to drought in macauba palm plants. Exposure to increased CO 2 concentrations led to up-regulation of photosynthesis through higher stomatal conductance and improved light and water use efficiency. Macauba palm plants under water stress, irrespective of CO 2 concentration, were able to maintain constant levels of proline and chlorophyll, while preventing oxidative damage. Plants grown at higher CO 2 concentrations were more capable of recovering from drought due to higher Rubisco carboxylation rate (Vc max) and electron transport rate (J max), which prevented a reduction in total dry mass at the end of the stress period. Stomatal control of photosynthesis, coupled with the prevention of severe damage under stress, would allow efficient biomass production by the macauba palm under future scenarios of climate change.

Changes in atmospheric CO 2 levels are accompanied by ecological interactions involving other environmental factors, such as drought, which can severely alter the water balance of plants, thereby influencing sap flow, gas exchange, and plant growth. The objective of this study was to evaluate the water use economy, leaf gas exchange, and growth of young plants of the species Lafoensia pacari subjected to high concentrations of atmospheric CO 2 ([CO 2 ]) and soil water deficit. Increased vapor pressure deficit was observed from 1200 to 1800 hours. Plants under elevated [CO 2 ] exhibited increased leaf photosynthesis, resulting in improved growth. Specific leaf area was lower in plants under high [CO 2 ], mainly after soil water deficit treatment. Daily irrigated plants growing under ambient [CO 2 ] had higher stem sap flow velocity (cm h-1) and hourly sap flow (kg h-1), mainly during the hottest hours of the day, than plants under high [CO 2 ]. Improved water use by plants growing under high [CO 2 ] may result in increased availability of water in the soil, partially offsetting future drought events and extending the growth period.

Fire is an abiotic disturbance that regulates vegetation structure and biodiversity. Some plant species have adapted to fi re prone environments by evolving protective structures. Acrocomia aculeata (macaw palm) is widely distributed throughout tropical America, and is found in environments continuously infl uenced by anthropogenic fi re. We aimed to determine whether the fruit characteristics of A. aculeata enable seeds to resist the eff ects of fi re and also the consequent eff ects of fi re on fruit biometric traits and embryo viability. After a fi re event in a region of pastureforest transition, we marked 30 individuals of A. aculeata. Th e trees were separated by UPGMA analysis into 5 groups according to fi re exposure, ranging from trees with no exposure to trees with fruit completely exposed to fi re. Fruit exposure to high temperatures led to lower values in fruit fresh weight, length, density, and processable mass.Fire had no signifi cant eff ect on seed biometric variables, because of the structures of the fruit, including its lignifi ed endocarp and its insulating and mucilaginous mesocarp. Th ese structures helped to maintain the embryos viability by preventing oxidative damage. In conclusion, the fruit structure of the macaw palm may facilitate seed persistence, even when subject to increasingly frequent fi re events.

1ARF4-as plants displayed a leaf curl phenotype, a low stomatal conductance coupled with an 2 2 increase in leaf relative water content and ABA content under normal and stressful conditions. 3This increase in ABA content was correlated with the activation of ABA biosynthesis genes 2 4 and the repression of ABA catabolism genes. cat1, Cu/ZnSOD and mdhar genes were up-2 5 regulated in ARF4-as plants suggesting that ARF4-as mutant is more tolerant to salt and water 2 6 stress.The data presented in this work strongly support the involvement of ARF4 as a key 2 7 actor in tomato tolerance to salt and drought stresses. 2 8

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

The development of crop varieties capable of maintaining satisfactory yields under stressful conditions such as drought is an important step towards ensuring adequate food production in the future. In this context, natural genetic variation in tomato can be allied with modern techniques such as the production of introgression lines, as well as mutant and transgenic organisms in the search for varieties more resistant to water deficit. Here, we show that S. pennellii introgression lines IL2-5, IL4-3, and IL2-5/4-3 exhibit increased leaf succulence, as well as significant changes in leaf thickness and stomatal density. Together, these leaf traits contributed to the maintenance of leaf water status, which improved photosynthetic performance and plant resilience when subjected to drought conditions. In this work, we also demonstrated the physiological and hydraulic changes caused by an allelic variant of the OBSCURAVENOSA (OBV) gene. In addition to controlling the development of vascular bundle sheath extensions (BSE), this gene also resulted in significant changes in leaf insertion angle, leaf margin serration, venation density, and fruit shape. We found that BSE development is strongly linked functionally to the auxin signaling network involving AUXIN RESPONSE FACTOR 4 (ARF4). Lastly, we show that loss of function of ARF4 alters leaf structure, resulting in a phenotype with severe leaf curling and low stomatal conductance. Loss of ARF4 function increased water and abscisic acid content in leaves, resulting in significant improvements in tomato plant resistance to salt and osmotic stress. Our data provide evidence that anatomical and morphological changes in leaves, whether from natural genetic variation or genetically modified organisms, can help to better understand the process of resistance to abiotic stress, such as drought and salinity. Thus, we suggest that mapping and identifying the genes responsible for the leaf traits demonstrated here may help in the creation of future varieties that are more resistant to water deficit. Keywords: Solanum pennellii. Introgression lines. Auxin. CRISPR-Cas9. Bundle sheath extensions. Drought stress.