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Multicellular organisms use mobile intercellular signals to generate spatiotemporal patterns of growth and differentiation. These signals, termed morphogens, arise from localized sources and move by diffusion or directional transport to be interpreted at target cells. The classical model for a morphogen is where a substance diffuses from a source to generate a concentration gradient that provides positional information across a field. This concept, presented by Wolpert and popularized as the "French Flag Model," remains highly influential, but other patterning models, which do not rely on morphogen gradients, also exist. Here, we review current evidence for mobile morphogenetic signals in plant root development and how they fit within existing conceptual frameworks for pattern formation. We discuss how the signals are formed, distributed, and interpreted in space and time, emphasizing the regulation of movement on the ability of morphogens to specify patterns. While significant advances have been made in the field since the first identification of mobile morphogenetic factors in plants, key questions remain to be answered, such as how morphogen movement is regulated, how these mechanisms allow scaling in different species, and how morphogens act to enable plant regeneration in response to damage., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co-receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear. We took a gain-of-function approach to study receptor-specific functionality. First, we introduced activating mutations that enforce active ABA-bound receptor conformation. We then transformed Arabidopsis ABA-deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype. Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism. We propose that ABA signaling - from perception to response - flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

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., Competing Interests: Conflict of interest statement. NO and AI are inventors in a Provisional patent application No. 63/267,407, INCREASING YIELD STABILITY IN PLANTS that includes data described in this paper., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Removal of the root system induces the formation of new roots from the remaining shoot. This process is primarily controlled by the phytohormone auxin, which interacts with other signals in a yet unresolved manner. Here, we study the classical tomato mutation rosette (ro), which lacks shoot-borne roots. ro mutants were severely inhibited in formation of wound-induced roots (WiRs) and had reduced auxin transport rates. We mapped ro to the tomato ortholog of the Arabidopsis thaliana BIG and the mammalians UBR4/p600. RO/BIG is a large protein of unknown biochemical function. In A. thaliana, BIG was implicated in regulating auxin transport and calcium homeostasis. We show that exogenous calcium inhibits WiR formation in tomato and A. thaliana ro/big mutants. Exogenous calcium antagonized the root-promoting effects of the auxin indole-3-acetic-acid but not of 2,4-dichlorophenoxyacetic acid, an auxin analog that is not recognized by the polar transport machinery, and accumulation of the auxin transporter PIN-FORMED1 (PIN1) was sensitive to calcium levels in the ro/big mutants. Consistent with a role for calcium in mediating auxin transport, both ro/big mutants and calcium-treated wild-type plants were hypersensitive to treatment with polar auxin transport inhibitors. Subcellular localization of BIG suggests that, like its mammalian ortholog, it is associated with the endoplasmic reticulum. Analysis of subcellular morphology revealed that ro/big mutants exhibited disruption in cytoplasmic streaming. We suggest that RO/BIG maintains auxin flow by stabilizing PIN membrane localization, possibly by attenuating the inhibitory effect of Ca2+ on cytoplasmic streaming., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plants have a broad capacity to regenerate damaged organs. The study of wounding in multiple developmental systems has uncovered many of the molecular properties underlying plants' competence for regeneration at the local cellular level. However, in nature, wounding is rarely localized to one place, and plants need to coordinate regeneration responses at multiple tissues with environmental conditions and their physiological state. Here, we review the evidence for systemic signals that regulate regeneration on a plant-wide level. We focus on the role of auxin and sugars as short- and long-range signals in natural wounding contexts and discuss the varied origin of these signals in different regeneration scenarios. Together, this evidence calls for a broader, system-wide view of plant regeneration competence., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

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., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plants continuously form new organs in different developmental contexts in response to environmental cues. Underground lateral roots initiate from prepatterned cells in the main root, but cells can also bypass the root-shoot trajectory separation and generate shoot-borne roots through an unknown mechanism. We mapped tomato ( Solanum lycopersicum ) shoot-borne root development at single-cell resolution and showed that these roots initiate from phloem-associated cells through a unique transition state. This state requires the activity of a transcription factor that we named SHOOTBORNE ROOTLESS ( SBRL ) . Evolutionary analysis reveals that SBRL 's function and cis regulation are conserved in angiosperms and that it arose as an ancient duplication, with paralogs controlling wound-induced and lateral root initiation. We propose that the activation of a common transition state by context-specific regulators underlies the plasticity of plant root systems.

Since its discovery by F.A.L Clowes, extensive research has been dedicated to identifying the functions of the quiescent center (QC). One of the earliest hypotheses was that it serves a key role in regeneration of the root meristem. Recent works provided support for this hypothesis and began to elucidate the molecular mechanisms underlying this phenomenon. There are two scenarios to consider when assessing the role of the QC in regeneration: one, when the damage leaves the QC intact; and the other, when the QC itself is destroyed. In the first scenario, multiple factors are recruited to activate QC cell division in order to replace damaged cells, but whether the QC has a role in the second scenario is less clear. Both using gene expression studies and following the cell division pattern have shown that the QC is assembled gradually, only to appear as a coherent identity late in regeneration. Similar late emergence of the QC was observed during the de novo formation of the lateral root meristem. These observations can lead to the conclusion that the QC has no role in regeneration. However, activities normally occurring in QC cells, such as local auxin biosynthesis, are still found during regeneration but occur in different cells in the regenerating meristem. Thus, we explore an alternative hypothesis, that following destruction of the QC, QC-related gene activity is temporarily distributed to other cells in the regenerating meristem, and only coalesce into a distinct cell identity when regeneration is complete., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement., Competing Interests: Declaration of interests Z.B.L. is a consultant for and a member of the Scientific Strategy Board of Inari Agriculture, and he is also a named inventor on a number of patents and patent applications directed to related technology that have been exclusively licensed from CSHL to Inari Agriculture., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

The root meristem can regenerate following removal of its stem-cell niche by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized while PIN-mediated transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise temporal pattern.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA ( PLT ) and AINTEGUMENTA ( ANT ) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (© 2019 American Society of Plant Biologists. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The genus Selaginella resides in an early branch of the land plant lineage that possesses a vasculature and roots. The majority of the Selaginella root system is shoot borne and emerges through a distinctive structure known as the rhizophore, the organ identity of which has been a long-debated question. The rhizophore of Selaginella moellendorffii - a model for the lycophytes - shows plasticity to develop into a root or shoot up until 8 d after angle meristem emergence, after which it is committed to root fate. We subsequently use morphology and plasticity to define the stage of rhizophore identity. Transcriptomic analysis of the rhizophore during its plastic stage reveals that, despite some resemblance to the root meristem, rhizophore gene expression patterns are largely distinct from both shoot and root meristems. Based on this transcriptomic analysis and on historical anatomical work, we conclude that the rhizophore is a distinct organ with unique features., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Multicellular organisms develop from a single cell that proliferates to form different cell types with specialized functions. Sixty years ago, Waddington suggested the 'epigenetic landscape' as a useful metaphor for the process. According to this view, cells move through a rugged identity space along genetically encoded trajectories, until arriving at one of the possible final fates. In plants in particular, these trajectories have strong spatial correlates, as cell identity is intimately linked to its relative position within the plant. During regeneration, however, positional signals are severely disrupted and differentiated cells are able to undergo rapid non-canonical identity changes. Moreover, while pluripotent properties have long been ascribed to plant cells, the introduction of induced pluripotent stem cells in animal studies suggests such plasticity may not be unique to plants. As a result, current concepts of differentiation as a gradual and hierarchical process are being reformulated across biological fields. Traditional studies of plant regeneration have placed strong emphasis on the emergence of patterns and tissue organization, and information regarding the events occurring at the level of individual cells is only now beginning to emerge. Here, I review the historical and current concepts of cell identity and identity transitions, and discuss how new views and tools may instruct the future understanding of differentiation and plant regeneration.

Leaves are flat determinate organs derived from indeterminate shoot apical meristems. The presence of a specific leaf meristem is debated, as anatomical features typical of meristems are not present in leaves. Here we demonstrate that multiple NGATHA (NGA) and CINCINNATA-class-TCP (CIN-TCP) transcription factors act redundantly, shortly after leaf initiation, to gradually restrict the activity of a leaf meristem in Arabidopsis thaliana to marginal and basal domains, and that their absence confers persistent marginal growth to leaves, cotyledons and floral organs. Following primordia initiation, the restriction of the broadly acting leaf meristem to the margins is mediated by the juxtaposition of adaxial and abaxial domains and maintained by WOX homeobox transcription factors, whereas other marginal elaboration genes are dispensable for its maintenance. This genetic framework parallels the morphogenetic program of shoot apical meristems and may represent a relic of an ancestral shoot system from which seed plant leaves evolved., Competing Interests: The authors declare that no competing interests exist.

The root meristem has a centrally located group of mitotically quiescent cells, to which current models assign a stem cell organizer function. However, evidence is emerging for decentralized control of stem cell activity, whereby self-renewing behavior emerges from the lack of cell displacement at the border of opposing differentiation gradients. We term this a "stagnation" model due to its reliance on passive mechanics. The position of stem cells is established by two opposing axes that reciprocally control each other's differentiation. Such broad tissue organization programs would allow plants, like some animal systems, to rapidly reconstitute stem cells from non-stem-cell tissues., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Plant roots can regenerate after excision of their tip, including the stem cell niche. To determine which developmental program mediates such repair, we applied a combination of lineage tracing, single-cell RNA sequencing, and marker analysis to test different models of tissue reassembly. We show that multiple cell types can reconstitute stem cells, demonstrating the latent potential of untreated plant cells. The transcriptome of regenerating cells prior to stem cell activation resembles that of an embryonic root progenitor. Regeneration defects are more severe in embryonic than in adult root mutants. Furthermore, the signaling domains of the hormones auxin and cytokinin mirror their embryonic dynamics and manipulation of both hormones alters the position of new tissues and stem cell niche markers. Our findings suggest that plant root regeneration follows, on a larger scale, the developmental stages of embryonic patterning and is guided by spatial information provided by complementary hormone domains., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology.

The definition of cell identity is a central problem in biology. While single-cell RNA-seq provides a wealth of information regarding cell states, better methods are needed to map their identity, especially during developmental transitions. Here, we use repositories of cell type-specific transcriptomes to quantify identities from single-cell RNA-seq profiles, accurately classifying cells from Arabidopsis root tips and human glioblastoma tumors. We apply our approach to single cells captured from regenerating roots following tip excision. Our technique exposes a previously uncharacterized transient collapse of identity distant from the injury site, demonstrating the biological relevance of a quantitative cell identity index.

Plant shoots display indeterminate growth, while their evolutionary decedents, the leaves, are determinate. Determinate leaf growth is conditioned by the CIN-TCP transcription factors, which promote leaf maturation and are negatively regulated by miR319 in leaf primordia. Here we show that CIN-TCPs reduce leaf sensitivity to cytokinin (CK), a phytohormone implicated in inhibition of differentiation in the shoot. We identify the SWI/SNF chromatin remodeling ATPase BRAHMA (BRM) as a genetic mediator of CIN-TCP activities and CK responses. An interactome screen further revealed that SWI/SNF complex components including BRM preferentially interacted with basic-helix-loop-helix (bHLH) transcription factors and the bHLH-related CIN-TCPs. Indeed, TCP4 and BRM interacted in planta. Both TCP4 and BRM bound the promoter of an inhibitor of CK responses, ARR16, and induced its expression. Reconstituting ARR16 levels in leaves with reduced CIN-TCP activity restored normal growth. Thus, CIN-TCP and BRM together promote determinate leaf growth by stage-specific modification of CK responses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In plants, changes in local auxin concentrations can trigger a range of developmental processes as distinct tissues respond differently to the same auxin stimulus. However, little is known about how auxin is interpreted by individual cell types. We performed a transcriptomic analysis of responses to auxin within four distinct tissues of the Arabidopsis thaliana root and demonstrate that different cell types show competence for discrete responses. The majority of auxin-responsive genes displayed a spatial bias in their induction or repression. The novel data set was used to examine how auxin influences tissue-specific transcriptional regulation of cell-identity markers. Additionally, the data were used in combination with spatial expression maps of the root to plot a transcriptomic auxin-response gradient across the apical and basal meristem. The readout revealed a strong correlation for thousands of genes between the relative response to auxin and expression along the longitudinal axis of the root. This data set and comparative analysis provide a transcriptome-level spatial breakdown of the response to auxin within an organ where this hormone mediates many aspects of development.

Arabidopsis TCPs are a family of basic helix loop helix (bHLH)-type transcription factors. Previous studies suggested that antagonistic activities of class I TCPs and class II TCPs are correlated with cell proliferation. We have recently shown that the class I TCPs AtTCP14 and AtTCP15 promote typical cytokinin (CK) responses in Arabidopsis, and proposed that they mediate the effect of CK on cell divisions. To further study the role of AtTCP14 and AtTCP15 in plant development, we expressed them in tomato plants. AtTCP14 and AtTCP15-expressing tomato plants were semi-dwarf, had a reduced apical dominance and developed ectopic meristems on leaflet petioles. CK application to tomato seedlings promoted axillary bud outgrowth and this effect was enhanced in the transgenic AtTCP14 and AtTCP15 overexpressing plants. The results of this study extend our previous suggestion that AtTCP14 and AtTCP15 modulate the plant sensitivity to CK.

O-linked N-acetylglucosamine (O-GlcNAc) modifications regulate the posttranslational fate of target proteins. The Arabidopsis thaliana O-GlcNAc transferase (OGT) SPINDLY (SPY) suppresses gibberellin signaling and promotes cytokinin (CK) responses by unknown mechanisms. Here, we present evidence that two closely related class I TCP transcription factors, TCP14 and TCP15, act with SPY to promote CK responses. TCP14 and TCP15 interacted with SPY in yeast two-hybrid and in vitro pull-down assays and were O-GlcNAc modified in Escherichia coli by the Arabidopsis OGT, SECRET AGENT. Overexpression of TCP14 severely affected plant development in a SPY-dependent manner and stimulated typical CK morphological responses, as well as the expression of the CK-regulated gene RESPONSE REGULATOR5. TCP14 also promoted the transcriptional activity of the CK-induced mitotic factor CYCLIN B1;2. Whereas TCP14-overexpressing plants were hypersensitive to CK, spy and tcp14 tcp15 double mutant leaves and flowers were hyposensitive to the hormone. Reducing CK levels by overexpressing CK OXIDASE/DEHYDROGENASE3 suppressed the TCP14 overexpression phenotypes, and this suppression was reversed when the plants were treated with exogenous CK. Taken together, we suggest that responses of leaves and flowers to CK are mediated by SPY-dependent TCP14 and TCP15 activities.

In seed plants, leaves are born on radial shoots, but unlike shoots, they are determinate dorsiventral organs made of flat lamina. YABBY genes are found only in seed plants and in all cases studied are expressed primarily in lateral organs and in a polar manner. Despite their simple expression, Arabidopsis thaliana plants lacking all YABBY gene activities have a wide range of morphological defects in all lateral organs as well as the shoot apical meristem (SAM). Here, we show that leaves lacking all YABBY activities are initiated as dorsiventral appendages but fail to properly activate lamina programs. In particular, the activation of most CINCINNATA-class TCP genes does not commence, SAM-specific programs are reactivated, and a marginal leaf domain is not established. Altered distribution of auxin signaling and the auxin efflux carrier PIN1, highly reduced venation, initiation of multiple cotyledons, and gradual loss of the SAM accompany these defects. We suggest that YABBY functions were recruited to mold modified shoot systems into flat plant appendages by translating organ polarity into lamina-specific programs that include marginal auxin flow and activation of a maturation schedule directing determinate growth.

The leaves of seed plants evolved from a primitive shoot system and are generated as determinate dorsiventral appendages at the flanks of radial indeterminate shoots. The remarkable variation of leaves has remained a constant source of fascination, and their developmental versatility has provided an advantageous platform to study genetic regulation of subtle, and sometimes transient, morphological changes. Here, we describe how eudicot plants recruited conserved shoot meristematic factors to regulate growth of the basic simple leaf blade and how subsets of these factors are subsequently re-employed to promote and maintain further organogenic potential. By comparing tractable genetic programs of species with different leaf types and evaluating the pros and cons of phylogenetic experimental procedures, we suggest that simple and compound leaves, and, by the same token, leaflets and serrations, are regulated by distinct ontogenetic programs. Finally, florigen, in its capacity as a general growth regulator, is presented as a new upper-tier systemic modulator in the patterning of compound leaves.

The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.

Floral organ identities are specified by a few transcription factors that act as master regulators. Subsequently, specification of organ axes programs the distribution of distinct tissue types within the organs that themselves develop unique identities. The C-class, AGAMOUS-clade MADS box genes are primary promoters of the gynoecium, which is divided into a distal style and a subtending ovary along the apical-basal axis. We show that members of a clade of B3 domain transcription factors, NGATHA1 (NGA1) to NGA4, are expressed distally in all lateral organs, and all four have a redundant and essential role in style development. Loss of all four genes results in gynoecia where style is replaced by valve-like projections and a reduction in style-specific SHATTERPROOF1 (SHP1) expression. In agreement, floral misexpression of NGA1 promotes ectopic style and SHP1 expression. STYLISH1, an auxin biosynthesis inducer, conditionally activated NGA genes, which in turn promoted distal expression of other STY genes in a putative positive feedback loop. Inhibited auxin transport or lack of YABBY1 gene activities resulted in a basally expanded style domain and broader expression of NGA genes. We speculate that early gynoecium factors delimit NGA gene response to an auxin-based signal, elicited by STY gene activity, to restrict the activation of style program to a late and distal carpel domain.

Leaf development has been monitored chiefly by following anatomical markers. Analysis of transcriptome dynamics during leaf maturation revealed multiple expression patterns that rise or fall with age or that display age-specific peaks. These were used to formulate a digital differentiation index (DDI) based on a set of selected markers with informative expression during leaf ontogeny. The leaf-based DDI reliably predicted the developmental state of leaf samples from diverse sources and was independent of mitotic cell division transcripts or propensity of specific cell types. When calibrated by informative root markers, the same algorithm accurately diagnosed dissected root samples. We used the DDI to characterize plants with reduced activities of multiple CINCINNATA (CIN)-TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) growth regulators. These plants had giant curled leaves made up of small cells with abnormal shape, low DDI scores, and low expression of mitosis markers, depicting the primary role of CIN-TCPs as promoters of differentiation. Delayed activity of several CIN-TCPs resulted in abnormally large but flat leaves with regular cells. The application of DDI has therefore portrayed the CIN-TCPs as heterochronic regulators that permit the development of a flexible and robust leaf form through an ordered and protracted maturation schedule.

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.

Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active AtRAC10 fused to the green fluorescent protein. The activated AtRAC10 induced deformation of root hairs and leaf epidermal cells and was primarily localized in Triton X-100-insoluble fractions of the plasma membrane. Actin cytoskeleton reorganization was revealed by creating double transgenic plants expressing activated AtRAC10 and the actin marker YFP-Talin. Plants were further analyzed by membrane staining with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) under different treatments, including the protein trafficking inhibitor brefeldin A or the actin-depolymeryzing agents latrunculin-B (Lat-B) and cytochalasin-D (CD). After drug treatments, activated AtRAC10 did not accumulate in brefeldin A compartments, but rather reduced their number and colocalized with FM4-64-labeled membranes in large intracellular vesicles. Furthermore, endocytosis was compromised in root hairs of activated AtRAC10 transgenic plants. FM4-64 was endocytosed in nontransgenic root hairs treated with the actin-stabilizing drug jasplakinolide. These findings suggest complex regulation of membrane cycling by plant RACs.