Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.

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.

Root Regeneration Triggers an Embryo-like Sequence Guided by Hormonal Interactions.

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.

Mobile signals, patterning, and positional information in root development.

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.

Constitutive activation of ABA receptors in Arabidopsis reveals unique regulatory circuitries.

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.

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.

Mapping of the Classical Mutation rosette Highlights a Role for Calcium in Wound-Induced Rooting.

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.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

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.

Systemic control of plant regeneration and wound repair.

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.

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.

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.

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 quiescent center and root regeneration.

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.

A Conceptual Framework for Cell Identity Transitions in Plants.

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.

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.

The Arabidopsis O-linked N-acetylglucosamine transferase SPINDLY interacts with class I TCPs to facilitate cytokinin responses in leaves and flowers.

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.

A map of cell type-specific auxin responses.

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.

Morphogenesis of simple and compound leaves: a critical review.

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.

Differentiating Arabidopsis shoots from leaves by combined YABBY 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.