Parasitic modulation of host development by ubiquitin-independent protein degradation.

Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.

Information Integration and Communication in Plant Growth Regulation.

Plants are equipped with the capacity to respond to a large number of diverse signals, both internal ones and those emanating from the environment, that are critical to their survival and adaption as sessile organisms. These signals need to be integrated through highly structured intracellular networks to ensure coherent cellular responses, and in addition, spatiotemporal actions of hormones and peptides both orchestrate local cell differentiation and coordinate growth and physiology over long distances. Further, signal interactions and signaling outputs vary significantly with developmental context. This review discusses our current understanding of the integrated intracellular and intercellular signaling networks that control plant growth.

The Arabidopsis SGN3/GSO1 receptor kinase integrates soil nitrogen status into shoot development.

The Casparian strip is a barrier in the endodermal cell walls of plants that allows the selective uptake of nutrients and water. In the model plant Arabidopsis thaliana, its development and establishment are under the control of a receptor-ligand mechanism termed the Schengen pathway. This pathway facilitates barrier formation and activates downstream compensatory responses in case of dysfunction. However, due to a very tight functional association with the Casparian strip, other potential signaling functions of the Schengen pathway remain obscure. In this work, we created a MYB36-dependent synthetic positive feedback loop that drives Casparian strip formation independently of Schengen-induced signaling. We evaluated this by subjecting plants in which the Schengen pathway has been uncoupled from barrier formation, as well as a number of established barrier-mutant plants, to agar-based and soil conditions that mimic agricultural settings. Under the latter conditions, the Schengen pathway is necessary for the establishment of nitrogen-deficiency responses in shoots. These data highlight Schengen signaling as an essential hub for the adaptive integration of signaling from the rhizosphere to aboveground tissues.

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

The AGAMOUS-LIKE 16-GENERAL REGULATORY FACTOR 1 module regulates axillary bud outgrowth via catabolism of abscisic acid in cucumber.

Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16-CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.

Transcription factor FveMYB117a inhibits axillary bud outgrowth by regulating cytokinin homeostasis in woodland strawberry.

Shoot branching affects plant architecture. In strawberry (Fragaria L.), short branches (crowns) develop from dormant axillary buds to form inflorescences and flowers. While this developmental transition contributes greatly to perenniality and yield in strawberry, its regulatory mechanism remains unclear and understudied. In the woodland strawberry (Fragaria vesca), we identified and characterized 2 independent mutants showing more crowns. Both mutant alleles reside in FveMYB117a, a R2R3-MYB transcription factor gene highly expressed in shoot apical meristems, axillary buds, and young leaves. Transcriptome analysis revealed that the expression of several cytokinin pathway genes was altered in the fvemyb117a mutant. Consistently, active cytokinins were significantly increased in the axillary buds of the fvemyb117a mutant. Exogenous application of cytokinin enhanced crown outgrowth in the wild type, whereas the cytokinin inhibitors suppressed crown outgrowth in the fvemyb117a mutant. FveMYB117a binds directly to the promoters of the cytokinin homeostasis genes FveIPT2 encoding an isopentenyltransferase and FveCKX1 encoding a cytokinin oxidase to regulate their expression. Conversely, the type-B Arabidopsis response regulators FveARR1 and FveARR2b can directly inhibit the expression of FveMYB117a, indicative of a negative feedback regulation. In conclusion, we identified FveMYB117a as a key repressor of crown outgrowth by inhibiting cytokinin accumulation and provide a mechanistic basis for bud fate transition in an herbaceous perennial plant.

Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.

The presence of diffuse morphogen gradients in tissues supports a view in which growth is locally homogenous. Here we challenge this view: we used a high-resolution quantitative approach to reveal significant growth variability among neighboring cells in the shoot apical meristem, the plant stem cell niche. This variability was strongly decreased in a mutant impaired in the microtubule-severing protein katanin. Major shape defects in the mutant could be related to a local decrease in growth heterogeneity. We show that katanin is required for the cell's competence to respond to the mechanical forces generated by growth. This provides the basis for a model in which microtubule dynamics allow the cell to respond efficiently to mechanical forces. This in turn can amplify local growth-rate gradients, yielding more heterogeneous growth and supporting morphogenesis.

Transcriptional regulation of strigolactone signalling in Arabidopsis.

Plant hormones known as strigolactones control plant development and interactions between host plants and symbiotic fungi or parasitic weeds1-4. In Arabidopsis thaliana and rice, the proteins DWARF14 (D14), MORE AXILLARY GROWTH 2 (MAX2), SUPPRESSOR OF MAX2-LIKE 6, 7 and 8 (SMXL6, SMXL7 and SMXL8) and their orthologues form a complex upon strigolactone perception and play a central part in strigolactone signalling5-10. However, whether and how strigolactones activate downstream transcription remains largely unknown. Here we use a synthetic strigolactone to identify 401 strigolactone-responsive genes in Arabidopsis, and show that these plant hormones regulate shoot branching, leaf shape and anthocyanin accumulation mainly through transcriptional activation of the BRANCHED 1, TCP DOMAIN PROTEIN 1 and PRODUCTION OF ANTHOCYANIN PIGMENT 1 genes. We find that SMXL6 targets 729 genes in the Arabidopsis genome and represses the transcription of SMXL6, SMXL7 and SMXL8 by binding directly to their promoters, showing that SMXL6 serves as an autoregulated transcription factor to maintain the homeostasis of strigolactone signalling. These findings reveal an unanticipated mechanism through which a transcriptional repressor of hormone signalling can directly recognize DNA and regulate transcription in higher plants.

Hormonal interactions in the regulation of plant development.

Plants exhibit a unique developmental flexibility to ever-changing environmental conditions. To achieve their profound adaptability, plants are able to maintain permanent stem cell populations and form new organs during the entire plant life cycle. Signaling substances, called plant hormones, such as auxin, cytokinin, abscisic acid, brassinosteroid, ethylene, gibberellin, jasmonic acid, and strigolactone, govern and coordinate these developmental processes. Physiological and genetic studies have dissected the molecular components of signal perception and transduction of the individual hormonal pathways. However, over recent years it has become evident that hormones do not act only in a linear pathway. Hormonal pathways are interconnected by a complex network of interactions and feedback circuits that determines the final outcome of the individual hormone actions. This raises questions about the molecular mechanisms underlying hormonal cross talk and about how these hormonal networks are established, maintained, and modulated throughout plant development.

A dynamic WUSCHEL/Layer 1 interplay directs shoot apical meristem formation during regeneration in tobacco.

De novo shoot apical meristem (SAM) organogenesis during regeneration in tissue culture has been investigated for several decades, but the precise mechanisms governing early-stage cell fate specification remain elusive. In contrast to SAM establishment during embryogenesis, in vitro SAM formation occurs without positional cues and is characterized by autonomous initiation of cellular patterning. Here, we report on the initial stages of SAM organogenesis and on the molecular mechanisms that orchestrate gene patterning to establish SAM homeostasis. We found that SAM organogenesis in tobacco calli starts with protuberance formation followed by the formation of an intact L1 layer covering the nascent protuberance. We also exposed a complex interdependent relationship between L1 and WUS expression and revealed that any disruption in this interplay compromises shoot formation. Silencing WUS in nascent protuberances prevented L1 formation and caused the disorganization of the outer cell layers exhibiting both anticlinal and periclinal divisions, suggesting WUS plays a critical role in the proper establishment and organization of L1 during SAM organogenesis. We further discovered that silencing TONNEAU1 prevents the exclusive occurrence of anticlinal divisions in the outermost layer of the protuberances and suppresses the acquisition of L1 cellular identity and L1 formation, ultimately impeding SAM formation and regeneration. This study provides a novel molecular framework for the characterization of a WUS/L1 interplay that mediates SAM formation during regeneration.

At the crossroads: strigolactones mediate changes in cytokinin synthesis and signalling in response to nitrogen limitation.

Strigolactones (SLs) are key regulators of shoot growth and responses to environmental stimuli. Numerous studies have indicated that nitrogen (N) limitation induces SL biosynthesis, suggesting that SLs may play a pivotal role in coordinating systemic responses to N availability, but this idea has not been clearly demonstrated. Here, we generated triple knockout mutants in the SL synthesis gene TaDWARF17 (TaD17) in bread wheat and investigated their phenotypic and transcriptional responses under N limitation, aiming to elucidate the role of SLs in the adaptation to N limitation. Tad17 mutants display typical SL mutant phenotypes, and fail to adapt their shoot growth appropriately to N. Despite exhibiting an increased tillering phenotype, Tad17 mutants continued to respond to N limitation by reducing tiller number, suggesting that SLs are not the sole regulators of tillering in response to N availability. RNA-seq analysis of basal nodes revealed that the loss of D17 significantly altered the transcriptional response of N-responsive genes, including changes in the expression profiles of key N response master regulators. Crucially, our findings suggest that SLs are required for the transcriptional downregulation of cytokinin (CK) synthesis and signalling in response to N limitation. Collectively, our results suggest that SLs are essential for the appropriate morphological and transcriptional adaptation to N limitation in wheat, and that the repressive effect of SLs on shoot growth is partly mediated by their repression of CK synthesis.

Development of a mobile, high-throughput, and low-cost image-based plant growth phenotyping system.

Nondestructive plant phenotyping forms a key technique for unraveling molecular processes underlying plant development and response to the environment. While the emergence of high-throughput phenotyping facilities can further our understanding of plant development and stress responses, their high costs greatly hinder scientific progress. To democratize high-throughput plant phenotyping, we developed sets of low-cost image- and weight-based devices to monitor plant shoot growth and evapotranspiration. We paired these devices to a suite of computational pipelines for integrated and straightforward data analysis. The developed tools were validated for their suitability for large genetic screens by evaluating a cowpea (Vigna unguiculata) diversity panel for responses to drought stress. The observed natural variation was used as an input for a genome-wide association study, from which we identified nine genetic loci that might contribute to cowpea drought resilience during early vegetative development. The homologs of the candidate genes were identified in Arabidopsis (Arabidopsis thaliana) and subsequently evaluated for their involvement in drought stress by using available T-DNA insertion mutant lines. These results demonstrate the varied applicability of this low-cost phenotyping system. In the future, we foresee these setups facilitating the identification of genetic components of growth, plant architecture, and stress tolerance across a wide variety of plant species.

Defying gravity: WEEP promotes negative gravitropism in peach trees by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and are a resource for understanding how plants regulate posture control. The peach (Prunus persica) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Little is known about the function of WEEP despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach trees do not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster lateral root gravitropic response. This suggests that WEEP moderates root gravitropism and is essential to establishing the set-point angle of lateral roots from the gravity vector. Additionally, size exclusion chromatography indicated that WEEP proteins self-oligomerize, like other proteins with sterile alpha motif domains. Collectively, our results from weeping peach provide insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Phytohormonal regulation determines the organization pattern of shoot aerenchyma in greater duckweed (Spirodela polyrhiza).

Airspace or aerenchyma is crucial for plant development and acclimation to stresses such as hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades are known to regulate aerenchyma formation in stems and roots under hypoxic conditions, the precise mechanisms remain unclear. Moreover, the cellular dynamics underlying airspace formation in shoots are poorly understood. We investigated the stage-dependent structural dynamics of shoot aerenchyma in greater duckweed (Spirodela polyrhiza), a fast-growing aquatic herb with well-developed aerenchyma in its floating fronds. Using X-ray micro-computed tomography and histological analysis, we showed that the spatial framework of aerenchyma is established before frond volume increases, driven by cell division and expansion. The substomatal cavity connecting aerenchyma to stomata formed via programmed cell death (PCD) and was closely associated with guard cell development. Additionally, transcriptome analysis and pharmacological studies revealed that the organization of aerenchyma in greater duckweed is determined by the interplay between PCD and proliferation. This balance is governed by spatiotemporal regulation of phytohormone signaling involving ethylene, abscisic acid, and salicylic acid. Overall, our study reveals the structural dynamics and phytohormonal regulation underlying aerenchyma development in duckweed, improving our understanding of how plants establish distinct architectural arrangements. These insights hold the potential for wide-ranging application, not only in comprehending aerenchyma formation across various plant species but also in understanding how airspaces are formed within the leaves of terrestrial plants.

Transcription factor OsWRKY72 controls rice leaf angle by regulating LAZY1-mediated shoot gravitropism.

Leaf angle is a major trait of ideal architecture, which is considered to influence rice (Oryza sativa) cultivation and grain yield. Although a few mutants with altered rice leaf inclination angles have been reported, the underlying molecular mechanism remains unclear. In this study, we showed that a WRKY transcription factor gene, OsWRKY72, was highly expressed in the leaf sheath and lamina joint. Phenotypic analyses showed that oswrky72 mutants had smaller leaf angles than the wild type, while OsWRKY72 overexpression lines exhibited an increased leaf angle. This observation suggests that OsWRKY72 functions as a positive regulator, promoting the enlargement of the leaf angle. Our bioinformatics analysis identified LAZY1 as the downstream gene of OsWRKY72. Electrophoretic mobility shift assays and dual-luciferase analysis revealed that OsWRKY72 directly inhibited LAZY1 by binding to its promoter. Moreover, knocking out OsWRKY72 enhanced shoot gravitropism, which contrasted with the phenotype of lazy1 plants. These results imply that OsWRKY72 regulates the leaf angle through gravitropism by reducing the expression of LAZY1. In addition, OsWRKY72 could directly regulate the expression of other leaf angle-related genes such as FLOWERING LOCUS T-LIKE 12 (OsFTL12) and WALL-ASSOCIATED KINASE 11 (OsWAK11). Our study indicates that OsWRKY72 contributes positively to the expansion of the leaf angle by interfering with shoot gravitropism in rice.

Control of root nodule formation ensures sufficient shoot water availability in Lotus japonicus.

Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of nonstructural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability.

PECTIN ACETYLESTERASE12 regulates shoot branching via acetic acid and auxin accumulation in alfalfa shoots.

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa.

MIR396-GRF/GIF enhances in planta shoot regeneration of Dendrobium catenatum.

Recent studies on co-transformation of the growth regulator, TaGRF4-GIF1 chimera (Growth Regulating Factor 4-GRF Interacting Factor 1), in cultivated wheat varieties (Triticum aestivum), showed improved regeneration efficiency, marking a significant breakthrough. Here, a simple and reproducible protocol using the GRF4-GIF1 chimera was established and tested in the medicinal orchid Dendrobium catenatum, a monocot orchid species. TaGRF4-GIF1 from T. aestivum and DcGRF4-GIF1 from D. catenatum were reconstructed, with the chimeras significantly enhancing the regeneration efficiency of D. catenatum through in planta transformation. Further, mutating the microRNA396 (miR396) target sites in TaGRF4 and DcGRF4 improved regeneration efficiency. The target mimicry version of miR396 (MIM396) not only boosted shoot regeneration but also enhanced plant growth. Our methods revealed a powerful tool for the enhanced regeneration and genetic transformation of D. catenatum.

Transcriptome analysis and functional validation reveal the novel role of LhCYCL in axillary bud development in hybrid Liriodendron.

Shoot branching significantly influences yield and timber quality in woody plants, with hybrid Liriodendron being particularly valuable due to its rapid growth. However, understanding of the mechanisms governing shoot branching in hybrid Liriodendron remains limited. In this study, we systematically examined axillary bud development using morphological and anatomical approaches and selected four distinct developmental stages for an extensive transcriptome analysis. A total of 9,449 differentially expressed genes have been identified, many of which are involved in plant hormone signal transduction pathways. Additionally, we identified several transcription factors downregulated during early axillary bud development, including a noteworthy gene annotated as CYC-like from the TCP TF family, which emerged as a strong candidate for modulating axillary bud development. Quantitative real-time polymerase chain reaction results confirmed the highest expression levels of LhCYCL in hybrid Liriodendron axillary buds, while histochemical ß-glucuronidase staining suggested its potential role in Arabidopsis thaliana leaf axil development. Ectopic expression of LhCYCL in A. thaliana led to an increase of branches and a decrease of plant height, accompanied by altered expression of genes involved in the plant hormone signaling pathways. This indicates the involvement of LhCYCL in regulating shoot branching through plant hormone signaling pathways. In summary, our results emphasize the pivotal role played by LhCYCL in shoot branching, offering insights into the function of the CYC-like gene and establishing a robust foundation for further investigations into the molecular mechanisms governing axillary bud development in hybrid Liriodendron.

Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns.

In plants, aerial organs originate continuously from stem cells in the center of the shoot apical meristem. Descendants of stem cells in the subepidermal layer are progenitors of germ cells, giving rise to male and female gametes. In these cells, mutations, including insertions of transposable elements or viruses, must be avoided to preserve genome integrity across generations. To investigate the molecular characteristics of stem cells in Arabidopsis, we isolated their nuclei and analyzed stage-specific gene expression and DNA methylation in plants of different ages. Stem cell expression signatures are largely defined by developmental stage but include a core set of stem cell-specific genes, among which are genes implicated in epigenetic silencing. Transiently increased expression of transposable elements in meristems prior to flower induction correlates with increasing CHG methylation during development and decreased CHH methylation, before stem cells enter the reproductive lineage. These results suggest that epigenetic reprogramming may occur at an early stage in this lineage and could contribute to genome protection in stem cells during germline development.