The VIL gene CRAWLING ELEPHANT controls maturation and differentiation in tomato via polycomb silencing.

VERNALIZATION INSENSITIVE 3-LIKE (VIL) proteins are PHD-finger proteins that recruit the repressor complex Polycomb Repressive Complex 2 (PRC2) to the promoters of target genes. Most known VIL targets are flowering repressor genes. Here, we show that the tomato VIL gene CRAWLING ELEPHANT (CREL) promotes differentiation throughout plant development by facilitating the trimethylation of Histone H3 on lysine 27 (H3K27me3). We identified the crel mutant in a screen for suppressors of the simple-leaf phenotype of entire (e), a mutant in the AUX/IAA gene ENTIRE/SlIAA9, involved in compound-leaf development in tomato. crel mutants have increased leaf complexity, and suppress the ectopic blade growth of e mutants. In addition, crel mutants are late flowering, and have delayed and aberrant stem, root and flower development. Consistent with a role for CREL in recruiting PRC2, crel mutants show drastically reduced H3K27me3 enrichment at approximately half of the 14,789 sites enriched in wild-type plants, along with upregulation of many underlying genes. Interestingly, this reduction in H3K27me3 across the genome in crel is also associated with gains in H3K27me3 at a smaller number of sites that normally have modest levels of the mark in wild-type plants, suggesting that PRC2 activity is no longer limiting in the absence of CREL. Our results uncover a wide role for CREL in plant and organ differentiation in tomato and suggest that CREL is required for targeting PRC2 activity to, and thus silencing, a specific subset of polycomb targets.

Modulating auxin response stabilizes tomato fruit set.

Fruit formation depends on successful fertilization and is highly sensitive to weather fluctuations that affect pollination. Auxin promotes fruit initiation and growth following fertilization. Class A auxin response factors (Class A ARFs) repress transcription in the absence of auxin and activate transcription in its presence. Here, we explore how multiple members of the ARF family regulate fruit set and fruit growth in tomato (Solanum lycopersicum) and Arabidopsis thaliana, and test whether reduction of SlARF activity improves yield stability in fluctuating temperatures. We found that several tomato Slarf mutant combinations produced seedless parthenocarpic fruits, most notably mutants deficient in SlARF8A and SlARF8B genes. Arabidopsis Atarf8 mutants deficient in the orthologous gene had less complete parthenocarpy than did tomato Slarf8a Slarf8b mutants. Conversely, Atarf6 Atarf8 double mutants had reduced fruit growth after fertilization. AtARF6 and AtARF8 likely switch from repression to activation of fruit growth in response to a fertilization-induced auxin increase in gynoecia. Tomato plants with reduced SlARF8A and SlARF8B gene dosage had substantially higher yield than the wild type under controlled or ambient hot and cold growth conditions. In field trials, partial reduction in the SlARF8 dose increased yield under extreme temperature with minimal pleiotropic effects. The stable yield of the mutant plants resulted from a combination of early onset of fruit set, more fruit-bearing branches and more flowers setting fruits. Thus, ARF8 proteins mediate the control of fruit set, and relieving this control with Slarf8 mutations may be utilized in breeding to increase yield stability in tomato and other crops.

TOR coordinates cytokinin and gibberellin signals mediating development and defense.

Plants constantly perceive and process environmental signals and balance between the energetic demands of growth and defense. Growth arrest upon pathogen attack was previously suggested to result from a redirection of the plants' metabolic resources towards the activation of plant defense. The energy sensor Target of Rapamycin (TOR) kinase is a conserved master coordinator of growth and development in all eukaryotes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms by which TOR may potentially regulate the relationship between these two modalities. The plant hormones cytokinin (CK) and gibberellin (GA) execute various aspects of plant development and defense. The ratio between CK and GA was reported to determine the outcome of developmental programmes. Here, investigating the interplay between TOR-mediated development and TOR-mediated defense in tomato, we found that TOR silencing resulted in rescue of several different aberrant developmental phenotypes, demonstrating that TOR is required for the execution of developmental cues. In parallel, TOR inhibition enhanced immunity in genotypes with a low CK/GA ratio but not in genotypes with a high CK/GA ratio. TOR-inhibition mediated disease resistance was found to depend on developmental status, and was abolished in strongly morphogenetic leaves, while being strongest in mature, differentiated leaves. CK repressed TOR activity, suggesting that CK-mediated immunity may rely on TOR downregulation. At the same time, TOR activity was promoted by GA, and TOR silencing reduced GA sensitivity, indicating that GA signalling requires normal TOR activity. Our results demonstrate that TOR likely acts in concert with CK and GA signalling, executing signalling cues in both defense and development. Thus, differential regulation of TOR or TOR-mediated processes could regulate the required outcome of development-defense prioritisation.

Coordinating the morphogenesis-differentiation balance by tweaking the cytokinin-gibberellin equilibrium.

Morphogenesis and differentiation are important stages in organ development and shape determination. However, how they are balanced and tuned during development is not fully understood. In the compound leaved tomato, an extended morphogenesis phase allows for the initiation of leaflets, resulting in the compound form. Maintaining a prolonged morphogenetic phase in early stages of compound-leaf development in tomato is dependent on delayed activity of several factors that promote differentiation, including the CIN-TCP transcription factor (TF) LA, the MYB TF CLAU and the plant hormone Gibberellin (GA), as well as on the morphogenesis-promoting activity of the plant hormone cytokinin (CK). Here, we investigated the genetic regulation of the morphogenesis-differentiation balance by studying the relationship between LA, CLAU, TKN2, CK and GA. Our genetic and molecular examination suggest that LA is expressed earlier and more broadly than CLAU and determines the developmental context of CLAU activity. Genetic interaction analysis indicates that LA and CLAU likely promote differentiation in parallel genetic pathways. These pathways converge downstream on tuning the balance between CK and GA. Comprehensive transcriptomic analyses support the genetic data and provide insights into the broader molecular basis of differentiation and morphogenesis processes in plants.

[THE MULTIDISCIPLINARY GERIATRIC SURGERY UNIT AT THE CHAIM SHEBA MEDICAL CENTER - FIRST YEAR OF ACTIVITY].

INTRODUCTION: Recently, a Geriatric Surgery Unit (GSU) was established in the Sheba Medical Center. The Unit's aims include: professional assessment of surgical candidates, approval of the surgical plan by a multidisciplinary team discussion (MTD), and meeting the specific needs of the geriatric patient undergoing surgery. METHODS: We describe the establishment of the GSU and preliminary results from the first year of its activity (January-December 2022). The GSU team consisted of a geriatric nurse practitioner (NP), a geriatric physician, surgeons, anesthesiologists and a physiotherapist. Inclusion criteria for GSU assessment/treatment were age>80 years or substantial baseline geriatric morbidity. RESULTS: In 2022, 276 patients were treated by the GSU: 110 underwent elective comprehensive preoperative assessment in the NP clinic and the rest were assessed urgently/semi-electively during their hospitalization. One hundred and fifteen cases (median age 86 (65-98) years) were brought to MTD and considered for elective cholecystectomy (46.1%), colorectal procedures (16.5%), hernia repair (13.9%), hepatobiliary procedures (9.6%) or other surgeries (13.9%); of those, 49 patients (median age 86 (72-98) years) eventually proceeded to surgery, following which the median length of hospital stay (LOS) was 3.5 (1-60) days and the rate of postoperative complications was 46.7%. After discharge, the median duration of follow-up was 2.5 (0-18) months during which 4 patients died. Compared with geriatric patients who underwent cholecystectomy during 2021-2023 without MTD (n=39), in the cases discussed by the MTD, patients (n=17) had a shorter LOS (2.0±0.9 vs. 2.4±2.1 days), less 30-day Emergency Department referrals (12.5% vs. 28.2%) and less 30-day re-admissions (6.2% vs. 15.4%; all p≥0.3). CONCLUSIONS: Geriatric surgical patients require a designated professional approach to meet their unique perioperative needs. The effect of GSUs on perioperative outcomes merits further prospective studies.

Deep Conservation of cis-Element Variants Regulating Plant Hormonal Responses.

Phytohormones regulate many aspects of plant life by activating transcription factors (TFs) that bind sequence-specific response elements (REs) in regulatory regions of target genes. Despite their short length, REs are degenerate, with a core of just 3 to 4 bp. This degeneracy is paradoxical, as it reduces specificity and REs are extremely common in the genome. To study whether RE degeneracy might serve a biological function, we developed an algorithm for the detection of regulatory sequence conservation and applied it to phytohormone REs in 45 angiosperms. Surprisingly, we found that specific RE variants are highly conserved in core hormone response genes. Experimental evidence showed that specific variants act to regulate the magnitude and spatial profile of hormonal response in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum). Our results suggest that hormone-regulated TFs bind a spectrum of REs, each coding for a distinct transcriptional response profile. Our approach has implications for precise genome editing and for rational promoter design.

Leaflet initiation and blade expansion are separable in compound leaf development.

Compound leaves are composed of multiple separate blade units termed leaflets. In tomato (Solanum lycopersicum) compound leaves, auxin promotes both leaflet initiation and blade expansion. However, it is unclear how these two developmental processes interact. With highly variable complexity, tomato compound leaves provide an ideal system to address this question. In this study, we obtained and analyzed mutants of the WUSCHEL-RELATED HOMEOBOX (WOX) family gene SlLAM1 from tomato, whose orthologs in tobacco (Nicotiana sylvestris) and other species are indispensable for blade expansion. We show that SlLAM1 is expressed in the middle and marginal domains of leaves, and is required for blade expansion in leaflets. We demonstrate that sllam1 mutants cause a delay of leaflet initiation and slightly alter the arrangement of first-order leaflets, whereas the overall leaflet number is comparable to that of wild-type leaves. Analysis of the genetic interactions between SlLAM1 and key auxin signaling components revealed an epistatic effect of SlLAM1 in determining the final leaf form. Finally, we show that SlLAM1 is also required for floral organ growth and affects the fertility of gametophytes. Our data suggest that SlLAM1 promotes blade expansion in multiple leaf types, and leaflet initiation can be largely uncoupled from blade expansion during compound leaf morphogenesis.

Coordination of differentiation rate and local patterning in compound-leaf development.

The variability in leaf form in nature is immense. Leaf patterning occurs by differential growth, taking place during a limited window of morphogenetic activity at the leaf marginal meristem. While many regulators have been implicated in the designation of the morphogenetic window and in leaf patterning, how these effectors interact to generate a particular form is still not well understood. We investigated the interaction among different effectors of tomato (Solanum lycopersicum) compound-leaf development, using genetic and molecular analyses. Mutations in the tomato auxin response factor SlARF5/SlMP, which normally promotes leaflet formation, suppressed the increased leaf complexity of mutants with extended morphogenetic window. Impaired activity of the NAC/CUC transcription factor GOBLET (GOB), which specifies leaflet boundaries, also reduced leaf complexity in these backgrounds. Analysis of genetic interactions showed that the patterning factors SlMP, GOB and the MYB transcription factor LYRATE (LYR) coordinately regulate leaf patterning by modulating in parallel different aspects of leaflet formation and shaping. This work places an array of developmental regulators in a morphogenetic context. It reveals how organ-level differentiation rate and local growth are coordinated to sculpture an organ. These concepts are applicable to the coordination of pattering and differentiation in other species and developmental processes.

The Interaction between DELLA and ARF/IAA Mediates Crosstalk between Gibberellin and Auxin Signaling to Control Fruit Initiation in Tomato.

Fruit initiation following fertilization in angiosperms is strictly regulated by phytohormones. In tomato (Solanum lycopersicum), auxin and gibberellin (GA) play central roles in promoting fruit initiation. Without fertilization, elevated GA or auxin signaling can induce parthenocarpy (seedless fruit production). The GA-signaling repressor SlDELLA and auxin-signaling components SlIAA9 and SlARF7 repress parthenocarpy, but the underlying mechanism is unknown. Here, we show that SlDELLA and the SlARF7/SlIAA9 complex mediate crosstalk between GA and auxin pathways to regulate fruit initiation. Yeast-two-hybrid and coimmunoprecipitation assays showed that SlARF7 and additional activator SlARFs interact with SlDELLA and SlIAA9 through distinct domains. SlARF7/SlIAA9 and SlDELLA antagonistically modulate the expression of feedback-regulated genes involved in GA and auxin metabolism, whereas SlARF7/SlIAA9 and SlDELLA coregulate the expression of fruit growth-related genes. Analysis of procera (della), SlARF7 RNAi (with downregulated expression of multiple activator SlARFs), and entire (iaa9) single and double mutants indicated that these genes additively affect parthenocarpy, supporting the notion that the SlARFs/SlIAA9 and SlDELLA interaction plays an important role in regulating fruit initiation. Analysis of the GA-deficient mutant gib1 showed that active GA biosynthesis and signaling are required for auxin-induced fruit initiation. Our study reveals how direct crosstalk between auxin- and GA-signaling components is critical for tomato fruit initiation.

CLAUSA Is a MYB Transcription Factor That Promotes Leaf Differentiation by Attenuating Cytokinin Signaling.

Leaf morphogenesis and differentiation are highly flexible processes, resulting in a large diversity of leaf forms. The development of compound leaves involves an extended morphogenesis stage compared with that of simple leaves, and the tomato (Solanum lycopersicum) mutant clausa (clau) exposes a potential for extended morphogenesis in tomato leaves. Here, we report that the CLAU gene encodes a MYB transcription factor that has evolved a unique role in compound-leaf species to promote an exit from the morphogenetic phase of tomato leaf development. We show that CLAU attenuates cytokinin signaling, and that clau plants have increased cytokinin sensitivity. The results suggest that flexible leaf patterning involves a coordinated interplay between transcription factors and hormones.

Auxin Response Dynamics During Wild-Type and entire Flower Development in Tomato.

The plant hormone auxin is a major regulator of plant development and response to environmental cues. Auxin plays a particularly central role in flower development, but the knowledge of its role of flower development in crop plants with fleshy fruits, such as tomato, is still scarce. Mutations in the Aux/IAA gene ENTIRE/Indole Acetic Acid 9 (E/IAA9) lead to the precocious development of young gynoecia into parthenocarpic fruits. Here, we compared the distribution of the auxin response sensor DR5::VENUS and the auxin efflux transporter PIN1 between the wild type and entire during successive stages of flower and fruit development. Up-regulation of the DR5::VENUS signal in the shoot apical meristem (SAM) was observed upon the transition to flowering, implicating a possible role for auxin in the transition from a vegetative SAM into an inflorescence meristem. DR5::VENUS was expressed in all initiating floral organs. Additionally, DR5::VENUS was highly expressed during gametogenesis, in both male and female organs, and in the developing seeds during embryogenesis. DR5::VENUS is expressed in functional cell layers such as the anther stomium and tapetum, suggesting that auxin plays a role in flower organ development and function. The entire mutation affected DR5::VENUS expression patterns during inflorescence formation and flower organ development, which correlated with phenotypic alterations. We also show dynamic distribution and localization of the auxin transporter PIN1 during flower and fruit organ development. These results emphasize the dynamic auxin response in inflorescence and flower development and suggest multiple roles of auxin in these processes.

Abolishing ARF8A activity promotes disease resistance in tomato.

Auxin response factors (ARFs) are a family of transcription factors that regulate auxin-dependent developmental processes. Class A ARFs function as activators of auxin-responsive gene expression in the presence of auxin, while acting as transcriptional repressors in its absence. Despite extensive research on the functions of ARF transcription factors in plant growth and development, the extent, and mechanisms of their involvement in plant resistance, remain unknown. We have previously reported that mutations in the tomato AUXIN RESPONSE FACTOR8 (ARF8) genes SlARF8A and SlARF8B result in the decoupling of fruit development from pollination and fertilization, leading to partial or full parthenocarpy and increased yield under extreme temperatures. Here, we report that fine-tuning of SlARF8 activity results in increased resistance to fungal and bacterial pathogens. This resistance is mostly preserved under fluctuating temperatures. Thus, fine-tuning SlARF8 activity may be a potent strategy for increasing overall growth and yield.

Genetic dissection of the auxin response network.

The expansion of gene families during evolution, which can generate functional overlap or specialization among their members, is a characteristic feature of signalling pathways in complex organisms. For example, families of transcriptional activators and repressors mediate responses to the plant hormone auxin. Although these regulators were identified more than 20 years ago, their overlapping functions and compensating negative feedbacks have hampered their functional analyses. Studies using loss-of-function approaches in basal land plants and gain-of-function approaches in angiosperms have in part overcome these issues but have still left an incomplete understanding. Here, we propose that renewed emphasis on genetic analysis of multiple mutants and species will shed light on the role of gene families in auxin response. Combining loss-of-function mutations in auxin-response activators and repressors can unravel complex outputs enabled by expanded gene families, such as fine-tuned developmental outcomes and robustness. Similar approaches and concepts may help to analyse other regulatory pathways whose components are also encoded by large gene families.

Characterization of the cytokinin sensor TCSv2 in arabidopsis and tomato.

BACKGROUND: Hormones are crucial to plant life and development. Being able to follow the plants hormonal response to various stimuli and throughout developmental processes is an important and increasingly widespread tool. The phytohormone cytokinin (CK) has crucial roles in the regulation of plant growth and development. RESULTS: Here we describe a version of the CK sensor Two Component signaling Sensor (TCS), referred to as TCSv2. TCSv2 has a different arrangement of binding motifs when compared to previous TCS versions, resulting in increased sensitivity in some examined tissues. Here, we examine the CK responsiveness and distribution pattern of TCSv2 in arabidopsis and tomato. CONCLUSIONS: The increased sensitivity and reported expression pattern of TCSv2 make it an ideal TCS version to study CK response in particular hosts, such as tomato, and particular tissues, such as leaves and flowers.

Multiple Auxin-Response Regulators Enable Stability and Variability in Leaf Development.

Auxin-signal transduction is mediated by the antagonistic activity of transcriptional activators and repressors. Both activators and repressors belong to gene families, but the biological importance of this complexity is not clear. Here, we addressed this question using tomato leaf development as a model by generating and analyzing mutants in multiple auxin-response components. In developing compound tomato leaves, auxin promotes leaflet formation and blade growth, and in the intercalary regions between leaflets, auxin response is inhibited by the Aux/IAA protein ENTIRE (E). e mutants form simple leaves due to ectopic blade growth in the intercalary domain. Using this unique loss-of-function phenotype and genome editing of auxin-response factor (ARF) genes, encoding auxin-response activators, we identified the contribution of specific ARFs to the e phenotype. Mutations in the related ARFs SlMP, SlARF19A, and SlARF19B, but not SlARF7, reduced the leaf blade and suppressed the e phenotype in a dosage-dependent manner that correlated with their relative expression, leading to a continuum of shapes. While single e and slmp mutants affected blade growth in an opposite manner, leaves of e slmp double mutants were similar to those of the wild type. However, the leaf shape of e slmp was more variable than that of the wild type, and it showed increased sensitivity to auxin. Our findings demonstrate that the existence of multiple auxin-response repressors and activators stabilizes the developmental output of auxin and that tuning their activity enables shape variability. The increased complexity of the auxin response therefore balances stability and flexibility in leaf patterning.