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.

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.

CLASS-II KNOX genes coordinate spatial and temporal ripening in tomato.

Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.

Sucrose promotes stem branching through cytokinin.

Shoot branching is an important aspect of plant architecture because it substantially affects plant biology and agricultural performance. Sugars play an important role in the induction of shoot branching in several species, including potato (Solanum tuberosum L.). However, the mechanism by which sugars affect shoot branching remains mostly unknown. In the present study, we addressed this question using sugar-mediated induction of bud outgrowth in potato stems under etiolated conditions. Our results indicate that sucrose feeding to detached stems promotes the accumulation of cytokinin (CK), as well as the expression of vacuolar invertase (VInv), an enzyme that contributes to sugar sink strength. These effects of sucrose were suppressed by CK synthesis and perception inhibitors, while CK supplied to detached stems induced bud outgrowth and VInv activity in the absence of sucrose. CK-induced bud outgrowth was suppressed in vinv mutants, which we generated by genome editing. Altogether, our results identify a branching-promoting module, and suggest that sugar-induced lateral bud outgrowth is in part promoted by the induction of CK-mediated VInv activity.

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.

The KNOXI Transcription Factor SHOOT MERISTEMLESS Regulates Floral Fate in Arabidopsis.

Plants have evolved a unique and conserved developmental program that enables the conversion of leaves into floral organs. Elegant genetic and molecular work has identified key regulators of flower meristem identity. However, further understanding of flower meristem specification has been hampered by redundancy and by pleiotropic effects. The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is a well-characterized regulator of shoot apical meristem maintenance. Arabidopsis thaliana stm loss-of-function mutants arrest shortly after germination; therefore, the knowledge on later roles of STM in later processes, including flower development, is limited. Here, we uncover a role for STM in the specification of flower meristem identity. Silencing STM in the APETALA1 (AP1) expression domain in the ap1-4 mutant background resulted in a leafy-flower phenotype, and an intermediate stm-2 allele enhanced the flower meristem identity phenotype of ap1-4 Transcriptional profiling of STM perturbation suggested that STM activity affects multiple floral fate genes, among them the F-box protein-encoding gene UNUSUAL FLORAL ORGANS (UFO). In agreement with this notion, stm-2 enhanced the ufo-2 floral fate phenotype, and ectopic UFO expression rescued the leafy flowers in genetic backgrounds with compromised AP1 and STM activities. This work suggests a genetic mechanism that underlies the activity of STM in the specification of flower meristem identity.

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.

Etiolated Stem Branching Is a Result of Systemic Signaling Associated with Sucrose Level.

The potato (Solanum tuberosum) tuber is a swollen stem. Sprouts growing from the tuber nodes represent loss of apical dominance and branching. Long cold storage induces loss of tuber apical dominance and results in secondary branching. Here, we show that a similar branching pattern can be induced by short heat treatment of the tubers. Detached sprouts were induced to branch by the heat treatment only when attached to a parenchyma cylinder. Grafting experiments showed that the scion branches only when grafted onto heat- or cold-treated tuber parenchyma, suggesting that the branching signal is transmitted systemically from the bud-base parenchyma to the grafted stem. Exogenous supply of sucrose (Suc), glucose, or fructose solution to detached sprouts induced branching in a dose-responsive manner, and an increase in Suc level was observed in tuber parenchyma upon branching induction, suggesting a role for elevated parenchyma sugars in the regulation of branching. However, sugar analysis of the apex and node after grafting showed no distinct differences in sugar levels between branching and nonbranching stems. Vacuolar invertase is a key enzyme in determining the level of Suc and its cleavage products, glucose and fructose, in potato parenchyma. Silencing of the vacuolar invertase-encoding gene led to increased tuber branching in combination with branching-inducing treatments. These results suggest that Suc in the parenchyma induces branching through signaling and not by excess mobilization from the parenchyma to the stem.

Hormones in tomato leaf development.

Leaf development serves as a model for plant developmental flexibility. Flexible balancing of morphogenesis and differentiation during leaf development results in a large diversity of leaf forms, both between different species and within the same species. This diversity is particularly evident in compound leaves. Hormones are prominent regulators of leaf development. Here we discuss some of the roles of plant hormones and the cross-talk between different hormones in tomato compound-leaf development.

Auxin-mediated lamina growth in tomato leaves is restricted by two parallel mechanisms.

In the development of tomato compound leaves, local auxin maxima points, separated by the expression of the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs), which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In wild-type leaf primordia, the miR160-targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160-targeted ARFs SlARF10A, SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160-targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to ensure leaflet separation in tomato leaf margins.

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.

Leaf development and morphogenesis.

The development of plant leaves follows a common basic program that is flexible and is adjusted according to species, developmental stage and environmental circumstances. Leaves initiate from the flanks of the shoot apical meristem and develop into flat structures of variable sizes and forms. This process is regulated by plant hormones, transcriptional regulators and mechanical properties of the tissue. Here, we review recent advances in the understanding of how these factors modulate leaf development to yield a substantial diversity of leaf forms. We discuss these issues in the context of leaf initiation, the balance between morphogenesis and differentiation, and patterning of the leaf margin.

CLAUSA restricts tomato leaf morphogenesis and GOBLET expression.

Leaf morphogenesis and differentiation are highly flexible processes. The development of compound leaves is characterized by an extended morphogenesis stage compared with that of simple leaves. The tomato mutant clausa (clau) possesses extremely elaborate compound leaves. Here we show that this elaboration is generated by further extension of the morphogenetic window, partly via the activity of ectopic meristems present on clau leaves. Further, we propose that CLAU might negatively affect expression of the NAM/CUC gene GOBLET (GOB), an important modulator of compound-leaf development, as GOB expression is elevated in clau mutants and reducing GOB expression suppresses the clau phenotype. Expression of GOB is also elevated in the compound leaf mutant lyrate (lyr), and the remarkable enhancement of the clau phenotype by lyr suggests that clau and lyr affect leaf development and GOB in different pathways.

A role for APETALA1/fruitfull transcription factors in tomato leaf development.

Flexible maturation rates underlie part of the diversity of leaf shape, and tomato (Solanum lycopersicum) leaves are compound due to prolonged organogenic activity of the leaf margin. The CINCINNATA-teosinte branched1, cycloidea, PCF (CIN-TCP) transcription factor lanceolate (LA) restricts this organogenic activity and promotes maturation. Here, we show that tomato APETALA1/fruitfull (AP1/FUL) MADS box genes are involved in tomato leaf development and are repressed by LA. AP1/FUL expression is correlated negatively with LA activity and positively with the organogenic activity of the leaf margin. LA binds to the promoters of the AP1/FUL genes MBP20 and TM4. Overexpression of MBP20 suppressed the simple-leaf phenotype resulting from upregulation of LA activity or from downregulation of class I knotted like homeobox (KNOXI) activity. Overexpression of a dominant-negative form of MBP20 led to leaf simplification and partly suppressed the increased leaf complexity of plants with reduced LA activity or increased KNOXI activity. Tomato plants overexpressing miR319, a negative regulator of several CIN-TCP genes including LA, flower with fewer leaves via an SFT-dependent pathway, suggesting that miR319-sensitive CIN-TCPs delay flowering in tomato. These results identify a role for AP1/FUL genes in vegetative development and show that leaf and plant maturation are regulated via partially independent mechanisms.

Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato.

Plant leaves show pronounced plasticity of size and form. In the classical, partially dominant mutation Lanceolate (La), the large compound leaves of tomato (Solanum lycopersicum) are converted into small simple ones. We show that LA encodes a transcription factor from the TCP family containing an miR319-binding site. Five independent La isolates are gain-of-function alleles that result from point mutations within the miR319-binding site and confer partial resistance of the La transcripts to microRNA (miRNA)-directed inhibition. The reduced sensitivity to miRNA regulation leads to elevated LA expression in very young La leaf primordia and to precocious differentiation of leaf margins. In contrast, downregulation of several LA-like genes using ectopic expression of miR319 resulted in larger leaflets and continuous growth of leaf margins. Our results imply that regulation of LA by miR319 defines a flexible window of morphogenetic competence along the developing leaf margin that is required for leaf elaboration.

BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis.

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor-like cytoplasmic kinase sub-family (RLCK-XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T-DNA insertion lines for eight BSK genes (BSK1-BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24-epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR-responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3-like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3-like kinases.