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The strong ability of plants to regenerate wounds is exemplified by grafting when two plants are cut and joined together to grow as one. During graft healing, tissues attach, cells proliferate, and the vasculatures connect to form a graft union. The plant hormone auxin plays a central role, and auxin-related mutants perturb grafting success. Here, we investigated the role of individual cell types and their response to auxin during Arabidopsis (Arabidopsis thaliana) graft formation. By employing a cell-specific inducible misexpression system, we blocked auxin response in individual cell types using the bodenlos mutation. We found that auxin signaling in procambial tissues was critical for successful tissue attachment and vascular differentiation. In addition, we found that auxin signaling was required for cell divisions of the procambial cells during graft formation. Loss of function mutants in cambial pathways also perturbed attachment and phloem reconnection. We propose that cambial and procambial tissues drive tissue attachment and vascular differentiation during successful grafting. Our study thus refines our knowledge of graft development and furthers our understanding of the regenerative role of the cambium., 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.)

People have grafted plants since antiquity for propagation, to increase yields, and to improve stress tolerance. This cutting and joining of tissues activates an incredible regenerative ability as different plants fuse and grow as one. For over a hundred years, people have studied the scientific basis for how plants graft. Today, new techniques and a deepening knowledge of the molecular basis for graft formation have allowed a range of previously ungraftable combinations to emerge. Here, we review recent developments in our understanding of graft formation, including the attachment and vascular formation steps. We analyze why plants graft and how biotic and abiotic factors influence successful grafting. We also discuss the ability and inability of plants to graft, and how grafting has transformed both horticulture and fundamental plant science. As our knowledge about plant grafting improves, new combinations and techniques will emerge to allow an expanded use of grafting for horticultural applications and to address fundamental research questions., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

The widespread use of plant grafting enables eudicots and gymnosperms to join with closely related species and grow as one. Gymnosperms have dominated forests for over 200 million years, and despite their economic and ecological relevance, we know little about how they graft. Here we developed a micrografting method in conifers using young tissues that allowed efficient grafting with closely related species and between distantly related genera. Conifer graft junctions rapidly connected vasculature and differentially expressed thousands of genes including auxin and cell-wall-related genes. By comparing these genes to those induced during Arabidopsis thaliana graft formation, we found a common activation of cambium, cell division, phloem and xylem-related genes. A gene regulatory network analysis in Norway spruce (Picea abies) predicted that PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) acted as a core regulator of graft healing. This gene was strongly up-regulated during both spruce and Arabidopsis grafting, and Arabidopsis mutants lacking PAT genes failed to attach tissues or successfully graft. Complementing Arabidopsis PAT mutants with the spruce PAT1 homolog rescued tissue attachment and enhanced callus formation. Together, our data show an ability for young tissues to graft with distantly related species and identifies the PAT gene family as conserved regulators of graft healing and tissue regeneration., (© 2024. The Author(s).)

Parasitic plants are globally prevalent pathogens with important ecological functions but also potentially devastating agricultural consequences. Common to all parasites is the formation of the haustorium which requires parasite organ development and tissue invasion into the host. Both processes involve cell wall modifications. Here, we investigated a role for pectins during haustorium development in the facultative parasitic plant Phtheirospermum japonicum. Using transcriptomics data from infected Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), we identified genes for multiple P. japonicum pectin methylesterases (PMEs) and their inhibitors (PMEIs) whose expression was upregulated by haustoria formation. Changes in PME and PMEI expression were associated with tissue-specific modifications in pectin methylesterification. While de-methylesterified pectins were present in outer haustorial cells, highly methylesterified pectins were present in inner vascular tissues, including the xylem bridge that connects parasite to host. Specifically blocking xylem bridge formation in the haustoria inhibited several PME and PMEI genes from activating. Similarly, inhibiting PME activity using chemicals or by overexpressing PMEI genes delayed haustoria development. Our results suggest a dynamic and tissue-specific regulation of pectin contributes to haustoria initiation and to the establishment of xylem connections between parasite and host., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

In 1957, Skoog and Miller published their seminal work on the effects of hormones upon plant growth. By varying the concentrations of auxin and cytokinin, they observed dramatic differences in shoot and root growth from tobacco stem cultures. Their finding that quantitative differences in hormone concentrations could dramatically alter the fate of developing organs provided a foundation for understanding organ formation and tissue regeneration. Their in vitro assays established plant propagation techniques that were critical for regenerating transgenic plants. Here, I discuss their original paper, what led to their findings and its impact on our understanding of hormone interactions, how plants regenerate and in vitro tissue culture techniques., Competing Interests: The author declares none., (© The Author(s) 2023.)

Developmental programmed cell death (dPCD) controls a plethora of functions in plant growth and reproduction. In the root cap of Arabidopsis (Arabidopsis thaliana), dPCD functions to control organ size in balance with the continuous stem cell activity in the root meristem. Key regulators of root cap dPCD including SOMBRERO/ANAC033 (SMB) belong to the NAC family of transcription factors. Here, we identify the C2H2 zinc finger protein ZINC FINGER OF ARABIDOPSIS THALIANA 14 ZAT14 as part of the gene regulatory network of root cap dPCD acting downstream of SMB. Similar to SMB, ZAT14-inducible misexpression leads to extensive ectopic cell death. Both the canonical EAR motif and a conserved L-box motif of ZAT14 act as transcriptional repression motifs and are required to trigger cell death. While a single zat14 mutant does not show a cell death-related phenotype, a quintuple mutant knocking out 5 related ZAT paralogs shows a delayed onset of dPCD execution in the columella and the adjacent lateral root cap. While ZAT14 is co-expressed with established dPCD-associated genes, it does not activate their expression. Our results suggest that ZAT14 acts as a transcriptional repressor controlling a so far uncharacterized subsection of the dPCD gene regulatory network active in specific root cap tissues., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plants show an unparalleled regenerative capacity, allowing them to survive severe stress conditions, such as injury, herbivory attack, and harsh weather conditions. This potential not only replenishes tissues and restores damaged organs but can also give rise to whole plant bodies. Despite the intertwined nature of development and regeneration, common upstream cues and signaling mechanisms are largely unknown. Here, we demonstrate that in addition to being activators of regeneration, ETHYLENE RESPONSE FACTOR 114 (ERF114) and ERF115 govern developmental growth in the absence of wounding or injury. Increased ERF114 and ERF115 activity enhances auxin sensitivity, which is correlated with enhanced xylem maturation and lateral root formation, whereas their knockout results in a decrease in lateral roots. Moreover, we provide evidence that mechanical cues contribute to ERF114 and ERF115 expression in correlation with BZR1-mediated brassinosteroid signaling under both regenerative and developmental conditions. Antagonistically, cell wall integrity surveillance via mechanosensory FERONIA signaling suppresses their expression under both conditions. Taken together, our data suggest a molecular framework in which cell wall signals and mechanical strains regulate organ development and regenerative responses via ERF114- and ERF115-mediated auxin signaling., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Parasitic plants are globally prevalent pathogens that withdraw nutrients from their host plants using an organ known as the haustorium. The external environment including nutrient availability affects the extent of parasitism and to understand this phenomenon, we investigated the role of nutrients and found that nitrogen is sufficient to repress haustoria formation in the root parasite Phtheirospermum japonicum. Nitrogen increases levels of abscisic acid (ABA) in P. japonicum and prevents the activation of hundreds of genes including cell cycle and xylem development genes. Blocking ABA signaling overcomes nitrogen's inhibitory effects indicating that nitrogen represses haustoria formation by increasing ABA. The effect of nitrogen appears more widespread since nitrogen also inhibits haustoria in the obligate root parasite Striga hermonthica. Together, our data show that nitrogen acts as a haustoria repressing factor and suggests a mechanism whereby parasitic plants use nitrogen availability in the external environment to regulate the extent of parasitism., (© 2022. The Author(s).)

Wound healing is a fundamental property of plants and animals that requires recognition of cellular damage to initiate regeneration. In plants, wounding activates a defense response via the production of jasmonic acid and a regeneration response via the hormone auxin and several ethylene response factor (ERF) and NAC domain-containing protein (ANAC) transcription factors. To better understand how plants recognize damage and initiate healing, we searched for factors upregulated during the horticulturally relevant process of plant grafting and found four related DNA binding with one finger (DOF) transcription factors, HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, whose expression rapidly activated at the Arabidopsis graft junction. Grafting or wounding a quadruple hca2, tmo6, dof2.1, dof6 mutant inhibited vascular and cell-wall-related gene expression. Furthermore, the quadruple dof mutant reduced callus formation, tissue attachment, vascular regeneration, and pectin methylesterification in response to wounding. We also found that activation of DOF gene expression after wounding required auxin, but hormone treatment alone was insufficient for their induction. However, modifying cell walls by enzymatic digestion of cellulose or pectin greatly enhanced TMO6 and HCA2 expression, whereas genetic modifications to the pectin or cellulose matrix using the PECTIN METHYLESTERASE INHIBITOR5 overexpression line or korrigan1 mutant altered TMO6 and HCA2 expression. Changes to the cellulose or pectin matrix were also sufficient to activate the wound-associated ERF115 and ANAC096 transcription factors, suggesting that cell-wall damage represents a common mechanism for wound perception and the promotion of tissue regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing, but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were crucial for this effect, as blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures, and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospermum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Grafting is possible in both animals and plants. Although in animals the process requires surgery and is often associated with rejection of non-self, in plants grafting is widespread, and has been used since antiquity for crop improvement 1 . However, in the monocotyledons, which represent the second largest group of terrestrial plants and include many staple crops, the absence of vascular cambium is thought to preclude grafting 2 . Here we show that the embryonic hypocotyl allows intra- and inter-specific grafting in all three monocotyledon groups: the commelinids, lilioids and alismatids. We show functional graft unions through histology, application of exogenous fluorescent dyes, complementation assays for movement of endogenous hormones, and growth of plants to maturity. Expression profiling identifies genes that unify the molecular response associated with grafting in monocotyledons and dicotyledons, but also gene families that have not previously been associated with tissue union. Fusion of susceptible wheat scions to oat rootstocks confers resistance to the soil-borne pathogen Gaeumannomyces graminis. Collectively, these data overturn the consensus that monocotyledons cannot form graft unions, and identify the hypocotyl (mesocotyl in grasses) as a meristematic tissue that allows this process. We conclude that graft compatibility is a shared ability among seed-bearing plants., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Plants display remarkable abilities to adjust growth and development to environmental conditions, such as the amount of available water. This developmental plasticity is apparent not only in root and shoot growth rates, but also in tissue patterning and cell morphology. 1 , 2 We have previously shown that in response to limited water availability, Arabidopsis thaliana root displays changes in xylem morphology, mediated by the non-cell-autonomous action of abscisic acid, ABA. 2 Here, we show, through analyses of ABA response reporters and tissue-specific suppression of ABA signaling, that xylem cells themselves act as primary signaling centers governing both xylem cell fate and xylem differentiation rate, revealing the cell-autonomous control of multiple aspects of xylem development by ABA. ABA rapidly activates the expression of genes encoding VASCULAR-RELATED NAC DOMAIN (VND) transcription factors. Molecular and genetic analyses revealed that the two ABA-mediated xylem developmental changes are regulated by distinct members of this transcription factor family, with VND2 and VND3 promoting differentiation rate of metaxylem cells, while VND7 promotes the conversion of metaxylem toward protoxylem morphology. This phenomenon shows how different aspects of developmental plasticity can be interlinked, yet genetically separable. Moreover, similarities in phenotypic and molecular responses to ABA in diverse species indicate evolutionary conservation of the ABA-xylem development regulatory network among eudicots. Hence, this study gives molecular insights into how environmental stress modifies plant vascular anatomy and has potential relevance for water use optimization and adaptation to drought conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plant grafting, the ancient practice of cutting and joining different plants, is gaining popularity as an elegant way to generate chimeras that combine desirable traits. Grafting was originally developed in woody species, but the technique has evolved over the past century to now encompass a large number of herbaceous species. The use of plant grafting in science is accelerating in part due to the innovative techniques developed for the model plant Arabidopsis thaliana . Here, we review these developments and discuss the advantages and limitations associated with grafting various Arabidopsis tissues at diverse developmental stages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Bartusch and Melnyk.)

Background: Cotyledon micrografting represents a useful tool for studying the central role of cotyledons during early plant development, especially their interplay with other plant organs with regard to long distance transport. While hypocotyl micrografting methods are well-established, cotyledon micrografting is still inefficient. By optimizing cotyledon micrografting, we aim for higher success rates and increased throughput in the model species Arabidopsis thaliana ., Results: We established a cut and paste cotyledon surgery procedure on a flat and solid but moist surface which improved handling of small seedlings. By applying a specific cutting and joining pattern, throughput was increased up to 40 seedlings per hour. The combination of short-day photoperiods and low light intensities for germination and long days plus high light intensities, elevated temperature and vertical plate positioning after grafting significantly increased 'ligation' efficiency. In particular high temperatures affected success rates favorably. Altogether, we achieved up to 92% grafting success in A. thaliana . Reconnection of vasculature was demonstrated by transport of a vasculature-specific dye across the grafting site. Phloem and xylem reconnection were completed 3-4 and 4-6 days after grafting, respectively, in a temperature-dependent manner. We observed that plants with grafted cotyledons match plants with intact cotyledons in biomass production and rosette development., Conclusions: This cut and paste cotyledon-to-petiole micrografting protocol simplifies the handling of plant seedlings in surgery, increases the number of grafted plants per hour and greatly improves success rates for A. thaliana seedlings. The developed cotyledon micrografting method is also suitable for other plant species of comparable size., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Multicellular organisms rely on the movement of signaling molecules across cells, tissues, and organs to communicate among distal sites. In plants, localized leaf damage activates jasmonic acid (JA)-dependent transcriptional reprogramming in both harmed and unharmed tissues. Although it has been indicated that JA species can translocate from damaged into distal sites, the identity of the mobile compound(s), the tissues through which they translocate, and the effect of their relocation remain unknown. Here, we found that following shoot wounding, the relocation of endogenous jasmonates through the phloem is essential to initiate JA signaling and stunt growth in unharmed roots of Arabidopsis thaliana. By employing grafting experiments and hormone profiling, we uncovered that the hormone precursor cis-12-oxo-phytodienoic acid (OPDA) and its derivatives, but not the bioactive JA-Ile conjugate, translocate from wounded shoots into undamaged roots. Upon root relocation, the mobile precursors cooperatively regulated JA responses through their conversion into JA-Ile and JA signaling activation. Collectively, our findings demonstrate the existence of long-distance translocation of endogenous OPDA and its derivatives, which serve as mobile molecules to coordinate shoot-to-root responses, and highlight the importance of a controlled redistribution of hormone precursors among organs during plant stress acclimation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The capacity of organisms to tune their development in response to environmental cues is pervasive in nature. This phenotypic plasticity is particularly striking in plants, enabled by their modular and continuous development. A good example is the activation of lateral shoot branches in Arabidopsis, which develop from axillary meristems at the base of leaves. The activity and elongation of lateral shoots depends on the integration of many signals both external (e.g. light, nutrient supply) and internal (e.g. the phytohormones auxin, strigolactone and cytokinin). Here, we characterise natural variation in plasticity of shoot branching in response to nitrate supply using two diverse panels of Arabidopsis lines. We find extensive variation in nitrate sensitivity across these lines, suggesting a genetic basis for variation in branching plasticity. High plasticity is associated with extreme branching phenotypes such that lines with the most branches on high nitrate have the fewest under nitrate deficient conditions. Conversely, low plasticity is associated with a constitutively moderate level of branching. Furthermore, variation in plasticity is associated with alternative life histories with the low plasticity lines flowering significantly earlier than high plasticity lines. In Arabidopsis, branching is highly correlated with fruit yield, and thus low plasticity lines produce more fruit than high plasticity lines under nitrate deficient conditions, whereas highly plastic lines produce more fruit under high nitrate conditions. Low and high plasticity, associated with early and late flowering respectively, can therefore be interpreted alternative escape vs mitigate strategies to low N environments. The genetic architecture of these traits appears to be highly complex, with only a small proportion of the estimated genetic variance detected in association mapping., Competing Interests: The authors have declared that no competing interests exist.

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium 1 . Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on 2,3 . Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors 4 -the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

Parasitic plants are widespread pathogens that infect numerous plant species and cause devastating agricultural losses. They efficiently withdraw water, nutrients and sugars from their hosts by fusing tissues and connecting their vasculature to the host vasculature. This ability to parasitize is found in a wide range of species and has evolved at least eleven independent times, suggesting a recurring and flexible developmental strategy. Despite multiple independent origins, a common feature to parasitism is the formation of an invasive organ termed the haustorium. Parasitic plants form haustoria in their stems or roots and use this structure to penetrate host tissues and form vascular connections, often with distantly related species. This ability to join to an unrelated species is remarkable, and together with the economic importance of parasitism, there is a strong need to further understand how parasitic plants infect their hosts. Here, we discuss the developmental basis for plant parasitism, focusing on haustorial initiation, penetration and vascular formation. We also discuss future directions and outstanding questions in this emerging field., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The ability for cut tissues to join and form a chimeric organism is a remarkable property of many plants; however, grafting is poorly characterized at the molecular level. To better understand this process, we monitored genome-wide gene expression changes in grafted Arabidopsis thaliana hypocotyls. We observed a sequential activation of genes associated with cambium, phloem, and xylem formation. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than on the other. This asymmetry correlated with sugar-responsive genes, and we observed an accumulation of starch above the graft junction. This accumulation decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues, indicating that a recognition mechanism was activated independently of functional vascular connections. Auxin, which is transported cell to cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes was expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, intertissue communication, and tissue fusion events., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The shoot apical meristem (SAM) is responsible for the generation of all the aerial parts of plants. Given its critical role, dynamical changes in SAM activity should play a central role in the adaptation of plant architecture to the environment. Using quantitative microscopy, grafting experiments, and genetic perturbations, we connect the plant environment to the SAM by describing the molecular mechanism by which cytokinins signal the level of nutrient availability to the SAM. We show that a systemic signal of cytokinin precursors mediates the adaptation of SAM size and organogenesis rate to the availability of mineral nutrients by modulating the expression of WUSCHEL , a key regulator of stem cell homeostasis. In time-lapse experiments, we further show that this mechanism allows meristems to adapt to rapid changes in nitrate concentration, and thereby modulate their rate of organ production to the availability of mineral nutrients within a few days. Our work sheds light on the role of the stem cell regulatory network by showing that it not only maintains meristem homeostasis but also allows plants to adapt to rapid changes in the environment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCF TIR 1 , an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo , the alf4 mutation destabilizes the CUL1 subunit of the SCF Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCF SLY 1 We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins., (© 2017 The Authors.)

For millennia, people have cut and joined different plant tissues together through a process known as grafting. By creating a chimeric organism, desirable properties from two plants combine to enhance disease resistance, abiotic stress tolerance, vigour or facilitate the asexual propagation of plants. In addition, grafting has been extremely informative in science for studying and identifying the long-distance movement of molecules. Despite its increasing use in horticulture and science, how plants undertake the process of grafting remains elusive. Here, we discuss specifically the role of eight major plant hormones during the wound healing and vascular formation process, two phenomena involved in grafting. We furthermore present the roles of these hormones during graft formation and highlight knowledge gaps and future areas of interest for the field of grafting biology.

Parasitic plants share a common anatomical feature, the haustorium. Haustoria enable both infection and nutrient transfer, which often leads to growth penalties for host plants and yield reduction in crop species. Haustoria also reciprocally transfer substances, such as RNA and proteins, from parasite to host, but the biological relevance for such movement remains unknown. Here, we studied such interspecies transport by using the hemiparasitic plant Phtheirospermum japonicum during infection of Arabidopsis thaliana Tracer experiments revealed a rapid and efficient transfer of carboxyfluorescein diacetate (CFDA) from host to parasite upon formation of vascular connections. In addition, Phtheirospermum induced hypertrophy in host roots at the site of infection, a form of enhanced secondary growth that is commonly observed during various parasitic plant-host interactions. The plant hormone cytokinin is important for secondary growth, and we observed increases in cytokinin and its response during infection in both host and parasite. Phtheirospermum -induced host hypertrophy required cytokinin signaling genes ( AHK3,4 ) but not cytokinin biosynthesis genes ( IPT1,3,5,7) in the host. Furthermore, expression of a cytokinin-degrading enzyme in Phtheirospermum prevented host hypertrophy. Wild-type hosts with hypertrophy were smaller than ahk3,4 mutant hosts resistant to hypertrophy, suggesting hypertrophy improves the efficiency of parasitism. Taken together, these results demonstrate that the interspecies movement of a parasite-derived hormone modified both host root morphology and fitness. Several microbial and animal plant pathogens use cytokinins during infections, highlighting the central role of this growth hormone during the establishment of plant diseases and revealing a common strategy for parasite infections of plants., Competing Interests: The authors declare no conflict of interest.

Contents 1611 I. 1611 II. 1612 III. 1612 IV. 1614 V. 1614 VI. 1614 VII. 1615 VIII. 1616 1616 References 1616 SUMMARY: The plant vasculature transports water, sugars, hormones, RNAs and proteins. Such critical functions need to be protected from attack by pests and pathogens or from damage by wounding. Plants have developed mechanisms to repair vasculature when such protections fail and to even initiate new vascular connections to tissues supporting symbionts. The developmental phenomena underlying vascular repair and rewiring are therefore critical for horticultural grafting, for plant infection and for mutualist associations with rhizosphere microbes. Despite the biological and economic interest, we are only beginning to understand how plants connect and reconnect their vasculature to a wide variety of organisms. Here, I discuss recent work and future prospects for this emerging field., (© 2016 The Author. New Phytologist © 2016 New Phytologist Trust.)

Generating chimeric organisms is an invaluable way to study cell-to-cell movement and non-cell-autonomous actions of molecules. Plant grafting is an ancient method of generating chimeric organisms and recently has been used to study the movement of hormones, proteins, and RNAs. Here, I describe a simple and efficient way to graft Arabidopsis thaliana at the seedling stage to generate plants with roots and shoots of different genotypes. Using this protocol, success rates of over 80 % with up to 80 grafts assembled per hour can be achieved.

Plants have a remarkable ability to regenerate vascular tissue after damage or wounding. A striking example of this phenomenon is the cutting and rejoining of plants during the process of grafting, which humans have used for millennia. Here, I describe how to graft Arabidopsis seedlings and how to monitor the vascular reconnection process during wound healing using fluorescent dyes and fluorescent proteins. Furthermore, I describe how to visualize xylem formation during graft healing. These techniques are useful for studying how the vascular system regenerates and for better understanding the process of plant grafting.

For millennia, people have cut and joined different plants together through a process known as grafting. The severed tissues adhere, the cells divide and the vasculature differentiates through a remarkable process of regeneration between two genetically distinct organisms as they become one. Grafting is becoming increasingly important in horticulture where it provides an efficient means for asexual propagation. Grafting also combines desirable roots and shoots to generate chimeras that are more vigorous, more pathogen resistant and more abiotic stress resistant. Thus, it presents an elegant and efficient way to improve plant productivity in vegetables and trees using traditional techniques. Despite this horticultural importance, we are only beginning to understand how plants regenerate tissues at the graft junction. By understanding grafting better, we can shed light on fundamental regeneration pathways and the basis for self/non-self recognition. We can also better understand why many plants efficiently graft whereas others cannot, with the goal of improving grafting so as to broaden the range of grafted plants to create even more desirable chimeras. Here, I review the latest findings describing how plants graft and provide insight into future directions in this emerging field.

RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism., (© 2015. Published by The Company of Biologists Ltd.)

Auxin produced by an active primary shoot apex is transported down the main stem and inhibits the growth of the axillary buds below it, contributing to apical dominance. Here we use Arabidopsis thaliana cytokinin (CK) biosynthetic and signalling mutants to probe the role of CK in this process. It is well established that bud outgrowth is promoted by CK, and that CK synthesis is inhibited by auxin, leading to the hypothesis that release from apical dominance relies on an increased supply of CK to buds. Our data confirm that decapitation induces the expression of at least one ISOPENTENYLTRANSFERASE (IPT) CK biosynthetic gene in the stem. We further show that transcript abundance of a clade of the CK-responsive type-A Arabidopsis response regulator (ARR) genes increases in buds following CK supply, and that, contrary to their typical action as inhibitors of CK signalling, these genes are required for CK-mediated bud activation. However, analysis of the relevant arr and ipt multiple mutants demonstrates that defects in bud CK response do not affect auxin-mediated bud inhibition, and increased IPT transcript levels are not needed for bud release following decapitation. Instead, our data suggest that CK acts to overcome auxin-mediated bud inhibition, allowing buds to escape apical dominance under favourable conditions, such as high nitrate availability., (© 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant grafting is a biologically important phenomenon involving the physical joining of two plants to generate a chimeric organism. It is widely practiced in horticulture and used in science to study the long-distance movement of molecules. Despite its widespread use, the mechanism of graft formation and vascular reconnection is not well understood. Here, we study the dynamics and mechanisms of vascular regeneration in Arabidopsis thaliana during graft formation when the vascular strands are severed and reconnected. We demonstrate a temporal separation between tissue attachment, phloem connection, root growth, and xylem connection. By analyzing cell division patterns and hormone responses at the graft junction, we found that tissues initially show an asymmetry in cell division, cell differentiation, and gene expression and, through contact with the opposing tissue, lose this asymmetry and reform the vascular connection. In addition, we identified genes involved in vascular reconnection at the graft junction and demonstrate that these auxin response genes are required below the graft junction. We propose an inter-tissue communication process that occurs at the graft junction and promotes vascular connection by tissue-specific auxin responses involving ABERRANT LATERAL ROOT FORMATION 4 (ALF4). Our study has implications for phenomena where forming vascular connections are important including graft formation, parasitic plant infection, and wound healing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Since ancient times, people have cut and joined together plants of different varieties or species so they would grow as a single plant - a process known as grafting (Figures 1 and 2). References to grafting appear in the Bible, ancient Greek and ancient Chinese texts, indicating that grafting was practised in Europe, the Middle East and Asia by at least the 5(th) century BCE. It is unknown where or how grafting was first discovered, but it is likely that natural grafting, the process by which two plants touch and fuse limbs or roots in the absence of human interference (Figure 3), influenced people's thinking. Such natural grafts are generally uncommon, but are seen in certain species, including English ivy. Parasitic plants, such as mistletoe, that grow and feed on often unrelated species may have also contributed to the development of grafting as a technique, as people would have observed mistletoe growing on trees such as apples or poplars., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

RNA silencing in flowering plants generates a signal that moves between cells and through the phloem [1, 2]. Nucleotide sequence specificity of the signal is conferred by 21, 22, and 24 nucleotide (nt) sRNAs that are generated by Dicer-like (DCL) proteins [3]. In the recipient cells these sRNAs bind to Argonaute (AGO) effectors of silencing and the 21 nt sRNAs mediate posttranscriptional regulation (PTGS) via mRNA cleavage [4] whereas the 24 nt sRNAs are associated with RNA-dependent DNA methylation (RdDM) [5] that may underlie transcriptional gene silencing (TGS). Intriguingly, genes involved in TGS are required for graft-transmissible gene silencing associated with PTGS [6]. However, some of the same genes were also required for spread of a PTGS silencing signal out of the veins of Arabidopsis [7], and grafting tests failed to demonstrate direct transmission of TGS signals [8-10]. It seemed likely, therefore, that mobile silencing is associated only with PTGS. To address this possibility, we grafted TGS-inducing wild-type Arabidopsis and a mutant that is compromised in 24 nt sRNA production onto a wild-type reporter line. The 21-24 nt sRNAs from the TGS construct were transmitted across a graft union but only the 24 nt sRNAs directed RdDM and TGS of a transgene promoter in meristematic cells. These data extend the significance of an RNA silencing signal to embrace epigenetics and transcriptional gene silencing and support the hypothesis that these signals transmit information to meristematic cells where they initiate persistent epigenetic changes that may influence growth, development, and heritable phenotypes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing or RNA interference (RNAi) and, in plants and nematodes, it is associated with the production of a mobile signal that can travel from cell-to-cell and over long distances. The sequence-specific nature of systemic RNA silencing indicates that a nucleic acid is a component of the signalling complex. Recent work has shed light on the mobile RNA species, the genes involved in the production and transport of the signal. This review discusses the advances in systemic RNAi and presents the current challenges and questions in this rapidly evolving field.

JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.

A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA, we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using mutants that block small RNA (sRNA) biogenesis in either source or recipient tissue, we found that transgene-derived sRNA as well as a substantial proportion of the endogenous sRNA had moved across the graft union, and we provide evidence that 24-nucleotide mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defense and responses to external stimuli that have persistent effects in plants.

To understand how DNA sequence is translated to phenotype we must understand the epigenetic features that regulate gene expression. Recent research illuminates the complex interactions between DNA methylation, small RNAs, silencing of transposable elements, and genomic imprinting in the Arabidopsis (Arabidopsis thaliana) seed. These studies suggest that transposable elements reactivated in specific cells of the gametophyte and seed might enhance silencing of transposable elements in the germline and embryo. By sacrificing genomic integrity these cells might make an epigenetic rather than genetic contribution to the progeny. This research could have implications for interspecies hybridization, the evolution of genomic imprinting, and epigenetic communication from plant to progeny.

Background: Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened., Results: Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies., Conclusion: Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health.