A tomato B-box protein regulates plant development and fruit quality through the interaction with PIF4, HY5, and RIN transcription factors.

During the last decade, knowledge about BBX proteins has greatly increased. Genome-wide studies identified the BBX gene family in several ornamental, industry, and food crops; however, reports regarding the role of these genes as regulators of agronomically important traits are scarce. Here, by phenotyping a knockout mutant, we performed a comprehensive functional characterization of the tomato locus Solyc12g089240, hereafter called SlBBX20. The data revealed the encoded protein as a positive regulator of light signaling affecting several physiological processes during the life span of plants. Through inhibition of PHYTOCHROME INTERACTING FACTOR 4 (SlPIF4)-auxin crosstalk, SlBBX20 regulates photomorphogenesis. Later in development, it controls the balance between cell division and expansion to guarantee correct vegetative and reproductive development. In fruits, SlBBX20 is transcriptionally induced by the master transcription factor RIPENING INHIBITOR (SlRIN) and, together with ELONGATED HYPOCOTYL 5 (SlHY5), up-regulates flavonoid biosynthetic genes. Finally, SlBBX20 promotes the accumulation of steroidal glycoalkaloids and attenuates Botrytis cinerea infection. This work clearly demonstrates that BBX proteins are multilayer regulators of plant physiology because they affect not only multiple processes during plant development but they also regulate other genes at the transcriptional and post-translational levels.

Phytol recycling: essential, yet not limiting for tomato fruit tocopherol accumulation under normal growing conditions.

Tocopherols are potent membrane-bound antioxidant molecules that are paramount for plant physiology and also important for human health. In the past years, chlorophyll catabolism was identified as the primary source of phytyl diphosphate for tocopherol synthesis by the action of two enzymes, PHYTOL KINASE (VTE5) and PHYTHYL PHOSPHATE KINASE (VTE6) that are able to recycle the chlorophyll-derived phytol. While VTE5 and VTE6 were proven essential for tocopherol metabolism in tomato fruits, it remains unknown whether they are rate-limiting steps in this pathway. To address this question, transgenic tomato plants expressing AtVTE5 and AtVTE6 in a fruit-specific manner were generated. Although ripe transgenic fruits exhibited higher amounts of tocopherol, phytol recycling revealed a more intimate association with chlorophyll than with tocopherol content. Interestingly, protein-protein interactions assays showed that VTE5 and VTE6 are complexed, channeling free phytol and phytyl-P, thus mitigating their cytotoxic nature. Moreover, the analysis of tocopherol accumulation dynamics in roots, a chlorophyll-devoid organ, revealed VTE5-dependent tocopherol accumulation, hinting at the occurrence of shoot-to-root phytol trafficking. Collectively, these results demonstrate that phytol recycling is essential for tocopherol biosynthesis, even in chlorophyll-devoid organs, yet it is not the rate-limiting step for this pathway under normal growth conditions.

CRYPTOCHROME 1a-mediated blue light perception regulates tomato seed germination via changes in hormonal balance and endosperm-degrading hydrolase dynamics.

MAIN CONCLUSION: Blue light exposure delays tomato seed germination by decreasing endosperm-degrading hydrolase activities, a process regulated by CRY1a-dependent signaling and the hormonal balance between ABA and GA. The germination of tomato seeds (Solanum lycopersicum L.) is tightly controlled by an internal hormonal balance, which is also influenced by environmental factors such as light. In this study, we investigated the blue light (BL)-mediated impacts on physiological, biochemical, and molecular processes during the germination of the blue light photoreceptor CRYPTOCHROME 1a loss-of-function mutant (cry1a) and of the hormonal tomato mutants notabilis (not, deficient in ABA) and procera (pro, displaying a GA-constitutive response). Seeds were germinated in a controlled chamber in the dark and under different intensities of continuous BL (ranging from 1 to 25 µmol m-2 s-1). In general, exposure to BL delayed tomato seed germination in a fluency rate-dependent way due to negative impacts on the activities of endosperm-degrading hydrolases, such as endo-ß-mannanase, ß-mannosidase, and α-galactosidase. However, not and pro mutants presented higher germination speed index (GSI) compared to WT despite the BL influence, associated with higher hydrolase activities, especially evident in pro, indicating that the ABA/GA hormonal balance is important to diminish BL inhibition over tomato germination. The cry1a germination percentage was higher than in WT in the dark but its GSI was lower under BL exposure, suggesting that functional CRY1a is required for BL-dependent germination. BL inhibits the expression of GA-biosynthetic genes, and induces GA-deactivating and ABA-biosynthetic genes. The magnitude of the BL influence over the hormone-related transcriptional profile is also dependent upon CRY1a, highlighting the complex interplay between light and hormonal pathways. These results contribute to a better understanding of BL-induced events behind the photoregulation of tomato seed germination.

Auxin-driven ecophysiological diversification of leaves in domesticated tomato.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalize the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here, we identify the obv gene and the causative mutation, a nonsynonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a 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 obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other pleiotropic effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density, and fruit shape, are controlled by OBV via changes in auxin signaling. 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 subject to breeding selection.

Loss of S-nitrosoglutathione reductase disturbs phytohormone homeostasis and regulates shoot side branching and fruit growth in tomato.

S-Nitrosoglutathione plays a central role in nitric oxide (NO) homeostasis, and S-nitrosoglutathione reductase (GSNOR) regulates the cellular levels of S-nitrosoglutathione across kingdoms. Here, we investigated the role of endogenous NO in shaping shoot architecture and controlling fruit set and growth in tomato (Solanum lycopersicum). SlGSNOR silencing promoted shoot side branching and led to reduced fruit size, negatively impacting fruit yield. Greatly intensified in slgsnor knockout plants, these phenotypical changes were virtually unaffected by SlGSNOR overexpression. Silencing or knocking out of SlGSNOR intensified protein tyrosine nitration and S-nitrosation and led to aberrant auxin production and signaling in leaf primordia and fruit-setting ovaries, besides restricting the shoot basipetal polar auxin transport stream. SlGSNOR deficiency triggered extensive transcriptional reprogramming at early fruit development, reducing pericarp cell proliferation due to restrictions on auxin, gibberellin, and cytokinin production and signaling. Abnormal chloroplast development and carbon metabolism were also detected in early-developing NO-overaccumulating fruits, possibly limiting energy supply and building blocks for fruit growth. These findings provide new insights into the mechanisms by which endogenous NO fine-tunes the delicate hormonal network controlling shoot architecture, fruit set, and post-anthesis fruit development, emphasizing the relevance of NO-auxin interaction for plant development and productivity.

Tomato miR156-targeted SlSBP15 represses shoot branching by modulating hormone dynamics and interacting with GOBLET and BRANCHED1b.

The miRNA156 (miR156)/SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL/SBP) regulatory hub is highly conserved among phylogenetically distinct species, but how it interconnects multiple pathways to converge to common integrators controlling shoot architecture is still unclear. Here, we demonstrated that the miR156/SlSBP15 node modulates tomato shoot branching by connecting multiple phytohormones with classical genetic pathways regulating both axillary bud development and outgrowth. miR156-overexpressing plants (156-OE) displayed high shoot branching, whereas plants overexpressing a miR156-resistant SlSBP15 allele (rSBP15) showed arrested shoot branching. Importantly, the rSBP15 allele was able to partially restore the wild-type shoot branching phenotype in the 156-OE background. rSBP15 plants have tiny axillary buds, and their activation is dependent on shoot apex-derived auxin transport inhibition. Hormonal measurements revealed that indole-3-acetic acid (IAA) and abscisic acid (ABA) concentrations were lower in 156-OE and higher in rSBP15 axillary buds, respectively. Genetic and molecular data indicated that SlSBP15 regulates axillary bud development and outgrowth by inhibiting auxin transport and GOBLET (GOB) activity, and by interacting with tomato BRANCHED1b (SlBRC1b) to control ABA levels within axillary buds. Collectively, our data provide a new mechanism by which the miR156/SPL/SBP hub regulates shoot branching, and suggest that modulating SlSBP15 activity might have potential applications in shaping tomato shoot architecture.

Fruit Physiology through Signaling Processes: Latest Advances and Future Challenges.

Fruits are unique to flowering plants and confer a selective advantage to these species by facilitating seed maturation and dispersal [...].

SlBBX28 positively regulates plant growth and flower number in an auxin-mediated manner in tomato.

KEY MESSAGE: SlBBX28 is a positive regulator of auxin metabolism and signaling, affecting plant growth and flower number in tomato B-box domain-containing proteins (BBXs) comprise a family of transcription factors that regulate several processes, such as photomorphogenesis, flowering, and stress responses. For this reason, attention is being directed toward the functional characterization of these proteins, although knowledge in species other than Arabidopsis thaliana remains scarce. Particularly in the tomato, Solanum lycopersicum, only three out of 31 SlBBX proteins have been functionally characterized to date. To deepen the understanding of the role of these proteins in tomato plant development and yield, SlBBX28, a light-responsive gene, was constitutively silenced, resulting in plants with smaller leaves and fewer flowers per inflorescence. Moreover, SlBBX28 knockdown reduced hypocotyl elongation in darkness-grown tomato. Analyses of auxin content and responsiveness revealed that SlBBX28 promotes auxin-mediated responses. Altogether, the data revealed that SlBBX28 promotes auxin production and signaling, ultimately leading to proper hypocotyl elongation, leaf expansion, and inflorescence development, which are crucial traits determining tomato yield.

A matter of time: regulatory events behind the synchronization of C4 and crassulacean acid metabolism in Portulaca oleracea.

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis.

Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao, interferes with cytokinin metabolism during infection of Micro-Tom tomato and promotes symptom development.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Phytochrome-Dependent Temperature Perception Modulates Isoprenoid Metabolism.

Changes in environmental temperature influence many aspects of plant metabolism; however, the underlying regulatory mechanisms remain poorly understood. In addition to their role in light perception, phytochromes (PHYs) have been recently recognized as temperature sensors affecting plant growth. In particular, in Arabidopsis (Arabidopsis thaliana), high temperature reversibly inactivates PHYB, reducing photomorphogenesis-dependent responses. Here, we show the role of phytochrome-dependent temperature perception in modulating the accumulation of isoprenoid-derived compounds in tomato (Solanum lycopersicum) leaves and fruits. The growth of tomato plants under contrasting temperature regimes revealed that high temperatures resulted in coordinated up-regulation of chlorophyll catabolic genes, impairment of chloroplast biogenesis, and reduction of carotenoid synthesis in leaves in a PHYB1B2-dependent manner. Furthermore, by assessing a triple phyAB1B2 mutant and fruit-specific PHYA- or PHYB2-silenced plants, we demonstrated that biosynthesis of the major tomato fruit carotenoid, lycopene, is sensitive to fruit-localized PHY-dependent temperature perception. The collected data provide compelling evidence concerning the impact of PHY-mediated temperature perception on plastid metabolism in both leaves and fruit, specifically on the accumulation of isoprenoid-derived compounds.

The light and dark sides of nitric oxide: multifaceted roles of nitric oxide in plant responses to light.

Light drives photosynthesis and informs plants about their surroundings. Regarded as a multifunctional signaling molecule in plants, nitric oxide (NO) has been repeatedly demonstrated to interact with light signaling cascades to control plant growth, development and metabolism. During early plant development, light-triggered NO accumulation counteracts negative regulators of photomorphogenesis and modulates the abundance of, and sensitivity to, plant hormones to promote seed germination and de-etiolation. In photosynthetically active tissues, NO is generated at distinct rates under light or dark conditions and acts at multiple target sites within chloroplasts to regulate photosynthetic reactions. Moreover, changes in NO concentrations in response to light stress promote plant defenses against oxidative stress under high light or ultraviolet-B radiation. Here we review the literature on the interaction of NO with the complicated light and hormonal signaling cascades controlling plant photomorphogenesis and light stress responses, focusing on the recently identified molecular partners and action mechanisms of NO in these events. We also discuss the versatile role of NO in regulating both photosynthesis and light-dependent stomatal movements, two key determinants of plant carbon gain. The regulation of nitrate reductase (NR) by light is highlighted as vital to adjust NO production in plants living under natural light conditions.

Multifaceted roles of nitric oxide in tomato fruit ripening: NO-induced metabolic rewiring and consequences for fruit quality traits.

Nitric oxide (NO) has been implicated as part of the ripening regulatory network in fleshy fruits. However, very little is known about the simultaneous action of NO on the network of regulatory events and metabolic reactions behind ripening-related changes in fruit color, taste, aroma and nutritional value. Here, we performed an in-depth characterization of the concomitant changes in tomato (Solanum lycopersicum) fruit transcriptome and metabolome associated with the delayed-ripening phenotype caused by NO supplementation at the pre-climacteric stage. Approximately one-third of the fruit transcriptome was altered in response to NO, including a multilevel down-regulation of ripening regulatory genes, which in turn restricted the production and tissue sensitivity to ethylene. NO also repressed hydrogen peroxide-scavenging enzymes, intensifying nitro-oxidative stress and S-nitrosation and nitration events throughout ripening. Carotenoid, tocopherol, flavonoid and ascorbate biosynthesis were differentially affected by NO, resulting in overaccumulation of ascorbate (25%) and flavonoids (60%), and impaired lycopene production. In contrast, the biosynthesis of compounds related to tomato taste (sugars, organic acids, amino acids) and aroma (volatiles) was slightly affected by NO. Our findings indicate that NO triggers extensive transcriptional and metabolic rewiring at the early ripening stage, modifying tomato antioxidant composition with minimal impact on fruit taste and aroma.

Beyond the limits of photoperception: constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato.

Photoreceptor engineering has recently emerged as a means for improving agronomically beneficial traits in crop species. Despite the central role played by the red/far-red photoreceptor phytochromes (PHYs) in controlling fruit physiology, the applicability of PHY engineering for increasing fleshy fruit nutritional content remains poorly exploited. In this study, we demonstrated that the fruit-specific overexpression of a constitutively active GAF domain Tyr252 -to-His PHYB2 mutant version (PHYB2Y252H ) significantly enhances the accumulation of multiple health-promoting antioxidants in tomato fruits, without negative collateral consequences on vegetative development. Compared with the native PHYB2 overexpression, PHYB2Y252H -overexpressing lines exhibited more extensive increments in transcript abundance of genes associated with fruit plastid development, chlorophyll biosynthesis and metabolic pathways responsible for the accumulation of antioxidant compounds. Accordingly, PHYB2Y252H -overexpressing fruits developed more chloroplasts containing voluminous grana at the green stage and overaccumulated carotenoids, tocopherols, flavonoids and ascorbate in ripe fruits compared with both wild-type and PHYB2-overexpressing lines. The impacts of PHYB2 or PHYB2Y252H overexpression on fruit primary metabolism were limited to a slight promotion in lipid biosynthesis and reduction in sugar accumulation. Altogether, these findings indicate that mutation-based adjustments in PHY properties represent a valuable photobiotechnological tool for tomato biofortification, highlighting the potential of photoreceptor engineering for improving quality traits in fleshy fruits.

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation.

Although biochemically related, C4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C4 . The putative CAM-specific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C4 to C4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C4 crop species.

Downregulation of PHYTOCHROME-INTERACTING FACTOR 4 Influences Plant Development and Fruit Production.

Plant development is highly dependent on the ability to perceive and cope with environmental changes. In this context, PIF proteins are key players in the cellular hub controlling responses to fluctuating light and temperature conditions. Reports in various plant species show that manipulation of the PIF4 level affects important agronomical traits. In tomato (Solanum lycopersicum), SlPIF1a and SlPIF3 regulate fruit nutraceutical composition. However, the wider role of this protein family, and the potential of their manipulation for the improvement of other traits, has not been explored. Here we report the effects of constitutive silencing of tomato SlPIF4 on whole-plant physiology and development. Ripening anticipation and higher carotenoid levels observed in SlPIF4-silenced fruits revealed a redundant role of SlPIF4 in the accumulation of nutraceutical compounds. Furthermore, silencing triggered a significant reduction in plant size, flowering, fruit yield, and fruit size. This phenotype was most likely caused by reduced auxin levels and altered carbon partitioning. Impaired thermomorphogenesis and delayed leaf senescence were also observed in silenced plants, highlighting the functional conservation of PIF4 homologs in angiosperms. Overall, this work improves our understanding of the role of PIF proteins-and light signaling-in metabolic and developmental processes that affect yield and composition of fleshy fruits.

SELF-PRUNING Acts Synergistically with DIAGEOTROPICA to Guide Auxin Responses and Proper Growth Form.

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.

PHYTOCHROME-INTERACTING FACTOR 3 mediates light-dependent induction of tocopherol biosynthesis during tomato fruit ripening.

Tocopherols are important antioxidants exclusively produced in plastids that protect the photosynthetic apparatus from oxidative stress. These compounds with vitamin E activity are also essential dietary nutrients for humans. Although the tocopherol biosynthetic pathway has been elucidated, the mechanisms that regulate tocopherol production and accumulation remain elusive. Here, we investigated the regulatory mechanism underlying tocopherol biosynthesis during ripening in tomato fruits, which are an important source of vitamin E. Our results show that ripening under light conditions increases tocopherol fruit content in a phytochrome-dependent manner by the transcriptional regulation of biosynthetic genes. Moreover, we show that light-controlled expression of the GERANYLGERANYL DIPHOSPHATE REDUCTASE (SlGGDR) gene, responsible for the synthesis of the central tocopherol precursor phytyl diphosphate, is mediated by PHYTOCHROME-INTERACTING FACTOR 3 (SlPIF3). In the absence of light, SlPIF3 physically interacts with the promoter of SlGGDR, down-regulating its expression. By contrast, light activation of phytochromes prevents the interaction between SlPIF3 and the SlGGDR promoter, leading to transcriptional derepression and higher availability of the PDP precursor for tocopherol biosynthesis. The unraveled mechanism provides a new strategy to manipulate fruit metabolism towards improving tomato nutritional quality.

Nitric oxide in the physiology and quality of fleshy fruits.

Fruits are unique to flowering plants and confer a selective advantage as they facilitate seed maturation and dispersal. In fleshy fruits, development and ripening are associated with numerous structural, biochemical, and physiological changes, including modifications in the general appearance, texture, flavor, and aroma, which ultimately convert the immature fruit into a considerably more attractive and palatable structure for seed dispersal by animals. Treatment with exogenous nitric oxide (NO) delays fruit ripening, prevents chilling damage, promotes disease resistance, and enhances the nutritional value. The ripening process is influenced by NO, which operates antagonistically to ethylene, but it also interacts with other regulatory molecules such as abscisic acid, auxin, jasmonic acid, salicylic acid, melatonin, and hydrogen sulfide. NO content progressively declines during fruit ripening, with concomitant increases in protein nitration and nitrosation, two post-translational modifications that are promoted by reactive nitrogen species. Dissecting the intimate interactions of NO with other ripening-associated factors, including reactive oxygen species, antioxidants, and the aforementioned phytohormones, remains a challenging subject of research. In this context, integrative 'omics' and gene-editing approaches may provide additional knowledge of the impact of NO in the regulatory processes involved in controlling physiology and quality traits in both climacteric and non-climacteric fruits.

Manipulation of a Senescence-Associated Gene Improves Fleshy Fruit Yield.

Senescence is the process that marks the end of a leaf's lifespan. As it progresses, the massive macromolecular catabolism dismantles the chloroplasts and, consequently, decreases the photosynthetic capacity of these organs. Thus, senescence manipulation is a strategy to improve plant yield by extending the leaf's photosynthetically active window of time. However, it remains to be addressed if this approach can improve fleshy fruit production and nutritional quality. One way to delay senescence initiation is by regulating key transcription factors (TFs) involved in triggering this process, such as the NAC TF ORESARA1 (ORE1). Here, three senescence-related NAC TFs from tomato (Solanum lycopersicum) were identified, namely SlORE1S02, SlORE1S03, and SlORE1S06. All three genes were shown to be responsive to senescence-inducing stimuli and posttranscriptionally regulated by the microRNA miR164 Moreover, the encoded proteins interacted physically with the chloroplast maintenance-related TF SlGLKs. This characterization led to the selection of a putative tomato ORE1 as target gene for RNA interference knockdown. Transgenic lines showed delayed senescence and enhanced carbon assimilation that, ultimately, increased the number of fruits and their total soluble solid content. Additionally, the fruit nutraceutical composition was enhanced. In conclusion, these data provide robust evidence that the manipulation of leaf senescence is an effective strategy for yield improvement in fleshy fruit-bearing species.