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Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

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The majority of the several hundred thousand specialized metabolites produced by plants function in defense against insects and other herbivores. Despite this diversity, identical metabolites or structurally distinct metabolites hitting the same targets in herbivorous animals have evolved repeatedly. This convergent evolution may reflect the constraints of plant primary metabolism in providing metabolic precursors, as well as the limited number of readily accessible targets in animals. These restrictions may make it uncommon for plants to develop completely novel toxic and deterrent metabolites, despite the ongoing evolution of resistance mechanisms in insect herbivores. Defensive compounds that are unique to individual genera or species often have long biosynthetic pathways that may complicate the repeated evolution of these metabolites in different plant species.

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Despite decades of extensive study, the role of cuticular lipids in sustaining plant fitness is far from being understood. We utilized genome-edited tree tobacco (Nicotiana glauca) to investigate the significance of different classes of epicuticular wax in abiotic stress such as cuticular water loss, drought, and light response. We generated mutants displaying a range of wax compositions. Four wax mutants and one cutin mutant were extensively investigated for alterations in their response to abiotic factors. Although the mutations led to elevated cuticular water loss, the wax mutants did not display elevated transpiration or reduced growth under nonstressed conditions. However, under drought, plants lacking alkanes were unable to reduce their transpiration, leading to leaf death, impaired recovery, and stem cracking. By contrast, plants deficient in fatty alcohols exhibited elevated drought tolerance, which was part of a larger trend of plant phenotypes not clustering by a glossy/glaucous appearance in the parameters examined in this study. We conclude that although alkanes have little effect on whole N. glauca transpiration and biomass gain under normal, nonstressed conditions, they are essential during drought responses, since they enable plants to seal their cuticle upon stomatal closure, thereby reducing leaf death and facilitating a speedy recovery.

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In a recent paper in Nature, Edith Heard from the European Molecular Biology Laboratory (EMBL) suggested that molecular biologists should 'reconnect with nature' by diversifying sampling locations. Although this approach has its own benefits, we suggest that advanced methods should rather be used to take hypothesis-based experiments to nature, thereby supplying a much-needed context for experimentation under controlled conditions. Following the CRISPR (clustered regularly interspaced short palindromic repeats) revolution, this approach has become accessible to many research groups. For the past several years we have developed the groundwork and initiated such experimentation. This included the assembly of a mobile laboratory on a four-wheel drive truck and examining genome-edited metabolic mutants in wild tree tobacco (Nicotiana glauca), grown in nature. Our findings included both targeted answers to focused questions, but also surprising results that could only be reached while working in natural settings.

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Plants transmit their experiences of environmental conditions to their progeny through epigenetic inheritance, improving their progeny's fitness under prevailing conditions. Though ABA is known to regulate epigenetic-modification genes, no strong phenotypic link between those genes and intergenerational "memory" has been shown. Previously, we demonstrated that mesophyll insensitivity to ABA (FBPase::abi1-1{fa} transgenic plants) results in a range of developmental phenotypes, including early growth vigor and early flowering (i.e., stress-escape behavior). Here, we show that null plants, used as controls (segregates of FBPase::abi1 that are homozygote descendants of a heterozygous transgenic plant, but do not contain the transformed abi1-1 gene) phenotypically resembled their FBPase::abi1-1 parents. However, in germination and early seedling development assays, null segregants resembled WT plants. These FBPase::abi1-1 null segregants mesophyll-related phenotypes were reproducible and stable for at least three generations. These results suggest that the heritability of stress response is linked to ABA's epigenetic regulatory effect through ABI1 and mesophyll-related traits. The discrepancy between the epigenetic heritability of seed and mesophyll-related traits is an example of the complexity of epigenetic regulation, which is both gene and process-specific, and may be attributed to the fine-tuning of tradeoffs between flowering time, growth rate and levels of risk that allow annual plants to optimize their fitness in uncertain environments.

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Abscisic acid (ABA) levels increase significantly in plants under stress conditions, and ABA is thought to serve as a key stress-response regulator. However, the direct effect of ABA on photosynthesis and the effect of mesophyll ABA on yield under both well-watered and drought conditions are still the subject of debate. Here, we examined this issue using transgenic Arabidopsis (Arabidopsis thaliana) plants carrying a dominant ABA-signaling inhibitor under the control of a mesophyll-specific promoter (FBPase::abi1-1, abbreviated to fa). Under normal conditions, fa plants displayed slightly higher stomatal conductance and carbon assimilation than wild-type plants; however, these parameters were comparable following ABA treatment. These observations suggest that ABA does not directly inhibit photosynthesis in the short term. The fa plants also exhibited a variety of altered phenotypes under optimal conditions, including more vigorous initial growth, earlier flowering, smaller flowers, and delayed chlorophyll degradation. Furthermore, under optimal conditions, fa plant seed production was less than a third of that observed for the wild type. However, under drought conditions, wild-type and fa seed yields were similar due to a significant reduction in wild-type seed and no reduction in fa seed. These findings suggest that endogenous basal ABA inhibits a stress-escape response under nonstressed conditions, allowing plants to accumulate biomass and maximize yield. The lack of a correlation between flowering time and plant biomass combined with delayed chlorophyll degradation suggests that this stress-escape behavior is regulated independently and upstream of other ABA-induced effects such as rapid growth and flowering.

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Abscisic acid (ABA) is known to be involved in stomatal closure. However, its role in stomatal response to rapid increases in the vapor pressure deficit (VPD) is unclear. To study this issue, we generated guard cell-specific ABA-insensitive Arabidopsis plants (guard-cell specific abi1-1; GCabi). Under non-stressed conditions, the stomatal conductance (gs) and apertures of GCabi plants were greater than those of control plants. This supports guard-cell ABA role as limiting steady-state stomatal aperture under non-stressful conditions. When there was a rapid increase in VPD (0.15 to 1 kPa), the gs and stomatal apertures of GCabi decreased in a manner similar that observed in the WT control, but different from that observed in WT plants treated with fusicoccin. Low VPD increased the size of the stomatal apertures of the WT, but not of GCabi. We conclude that guard-cell ABA does not play a significant role in the initial, rapid stomatal closure that occurs in response to an increase in VPD, but is important for stomatal adaptation to ambient VPD. We propose a biphasic angiosperm VPD-sensing model that includes an initial ABA-independent phase and a subsequent ABA-dependent steady-state phase in which stomatal behavior is optimized for ambient VPD conditions.

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The WUSCHEL homeobox transcription factor is required to specify stem-cell identity at the shoot apical meristem and its ectopic expression is sufficient to induce de novo shoot meristem formation. Yet, the manner by which WUS promotes stem-cell fate is not yet fully understood. In the present research we address this question by inducing WUS function outside of its domain. We show that activation of WUS function in the root inhibits the responses to exogenous auxin and suppresses the initiation and growth of lateral roots. Using time lapse movies to follow the cell-cycle marker CYCB1;1::GFP, we also show that activation of WUS function suppresses cell division and cell elongation. In addition, activation of WUS represses the auxin-induced expression of the PLETHORA1 root identity gene and promotes shoot fate. Shoot apical meristem formation requires a high cytokinin-to-auxin ratio. Our findings provide evidence for the manner by which WUS specifies stem-cell identity: by affecting auxin responses, by reducing the cell mitotic activity and by repressing other developmental pathways. At the meristem, the stem-cells which are characterized by low division rate are surrounded by the highly proliferative meristematic cells. Our results also provide a model for WUS establishing the differential mitotic rates between two cell populations at the minute structure of the meristem.

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Abscisic acid is found in a wide variety of organisms. In the plant kingdom, ABA's role in mediating responses to abiotic stress has been conserved and enhanced throughout evolution. The emergence of plants to terrestrial environments required the development of mechanisms to cope with ongoing and severe abiotic stress such as drought and rapid changes in humidity and temperature. The common understanding is that terrestrial plants evolved strategies ranging from desiccation-tolerance mechanisms (mosses) to drought tolerance (CAM plants), to better exploit different ecological niches. In between these divergent water regulation strategies, ABA plays a significant role in managing plants' adaptation to new environments by optimizing water-use efficiency (WUE) under particular environmental conditions. ABA plays some very different roles in the regulation of WUE. ABA's role in the regulation of guard cells and transpiration has yielded a wide variety of WUE-regulation mechanisms, ranging from no sensitivity (ferns) to low sensitivity (anisohydric behavior) to hypersensitivity to ABA (isohydric behavior and putatively CAM plants). ABA also plays a role in the regulation of non-stomatal, biochemical mechanisms of WUE regulation. In angiosperms, this includes the control of osmotic adjustment and morphological changes, including changes in leaf size, stomatal density, stomatal size and root development. Under severe stress, ABA also appears to initiate leaf senescence via transcriptional regulation, to directly inhibit photosynthesis.

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Increasing worldwide demand for food, feed and fuel presents a challenge in light of limited resources and climatic challenges. Breeding for stress tolerance and drought tolerance, in particular, is one the most challenging tasks facing breeders. The comparative screening of immense numbers of plant and gene candidates and their interactions with the environment represents a major bottleneck in this process. We suggest four key components to be considered in pre-field screens (phenotyping) for complex traits under drought conditions: (i) where, when and under which conditions to phenotype; (ii) which traits to phenotype; (iii) how to phenotype (which method); and (iv) how to translate collected data into knowledge that can be used to make practical decisions. We describe some common pitfalls, including inadequate phenotyping methods, incorrect terminology and the inappropriate use of non-relevant traits as markers for drought tolerance. We also suggest the use of more non-imaging, physiology-based, high-throughput phenotyping systems, which, used in combination with soil-plant-atmosphere continuum (SPAC) measurements and fitting models of plant responses to continuous and fluctuating environmental conditions, should be further investigated in order to serve as a phenotyping tool to better understand and characterise plant stress response. In the future, we assume that many of today's phenotyping challenges will be solved by technology and automation, leaving us with the main challenge of translating large amounts of accumulated data into meaningful knowledge and decision making tools.