Meristem Function Research Articles

Machine Learning Approaches for Prediction of Expansin Gene Family in Indica Rice

Expansin refers to a family of proteins present in the plant cell wall which has important roles in plant cell growth, emergence of root hairs, meristem function and other developmental processes. A major constraint to rice production is submergence of rice by flash flooding. In our earlier study, we had identified 21 novel sequences related to expansin gene families in the genome of indica rice using genome-wide analysis. Development of a tool for the prediction of these expansin genes using computational approaches might significantly enhance rice gene annotation. ExpansinPred, a novel computational method based on radial basis function (RBF) and support vector machines (SVMs) for prediction of α-expansins (EXPA) and β-expansins (EXPB), is presented in this work. Two large families of expansin genes have been discovered in plants, namely EXPA and EXPB. The experimental data are curated from NCBI and include 24 EXPA and 20 EXPB, of indica rice, after redundancy elimination. The proper window length for a potential expansin was optimized as 4 for EXPA and EXPB with prediction accuracies 100 % each for both classifiers for RBF classifier. For SVM, the window length was optimized as 3 for EXPA and 4 for EXPB with prediction accuracies 90 and 100 %, respectively. To evaluate the prediction performance of ExpansinPred, cross-validation, independent dataset validation and jackknife validation were carried out. ExpansinPred was also compared with four more algorithms namely Naive Bayes, sequential minimal optimization, J48 and random forest. To further prove that species-specific predictor is much better than general tool, ExpansinPred was compared with an All-plant tool and also with plants other than rice as test set. The different statistical analyses carried out demonstrated that the proposed algorithm is a useful computational tool for rice genome annotation, specifically for predicting expansin gene family, and can benefit rice research community.

Characterization and Evolution of Conserved MicroRNA through Duplication Events in Date Palm (Phoenix dactylifera)

MicroRNAs (miRNAs) are important regulators of gene expression at the post-transcriptional level in a wide range of species. Highly conserved miRNAs regulate ancestral transcription factors common to all plants, and control important basic processes such as cell division and meristem function. We selected 21 conserved miRNA families to analyze the distribution and maintenance of miRNAs. Recently, the first genome sequence in Palmaceae was released: date palm (Phoenix dactylifera). We conducted a systematic miRNA analysis in date palm, computationally identifying and characterizing the distribution and duplication of conserved miRNAs in this species compared to other published plant genomes. A total of 81 miRNAs belonging to 18 miRNA families were identified in date palm. The majority of miRNAs in date palm and seven other well-studied plant species were located in intergenic regions and located 4 to 5 kb away from the nearest protein-coding genes. Sequence comparison showed that 67% of date palm miRNA members were present in duplicated segments, and that 135 pairs of miRNA-containing segments were duplicated in Arabidopsis, tomato, orange, rice, apple, poplar and soybean with a high similarity of non coding sequences between duplicated segments, indicating genomic duplication was a major force for expansion of conserved miRNAs. Duplicated miRNA pairs in date palm showed divergence in pre-miRNA sequence and in number of promoters, implying that these duplicated pairs may have undergone divergent evolution. Comparisons between date palm and the seven other plant species for the gain/loss of miR167 loci in an ancient segment shared between monocots and dicots suggested that these conserved miRNAs were highly influenced by and diverged as a result of genomic duplication events.

TALE and Shape: How to Make a Leaf Different.

The Three Amino acid Loop Extension (TALE) proteins constitute an ancestral superclass of homeodomain transcription factors conserved in animals, plants and fungi. In plants they comprise two classes, KNOTTED1-LIKE homeobox (KNOX) and BEL1-like homeobox (BLH or BELL, hereafter referred to as BLH), which are involved in shoot apical meristem (SAM) function, as well as in the determination and morphological development of leaves, stems and inflorescences. Selective protein-protein interactions between KNOXs and BLHs affect heterodimer subcellular localization and target affinity. KNOXs exert their roles by maintaining a proper balance between undifferentiated and differentiated cell state through the modulation of multiple hormonal pathways. A pivotal function of KNOX in evolutionary diversification of leaf morphology has been assessed. In the SAM of both simple- and compound-leafed seed species, downregulation of most class 1 KNOX (KNOX1) genes marks the sites of leaf primordia initiation. However, KNOX1 expression is re-established during leaf primordia development of compound-leafed species to maintain transient indeterminacy and morphogenetic activity at the leaf margins. Despite the increasing knowledge available about KNOX1 protein function in plant development, a comprehensive view on their downstream effectors remains elusive. This review highlights the role of TALE proteins in leaf initiation and morphological plasticity with a focus on recent advances in the identification of downstream target genes and pathways.

Core pathways controlling shoot meristem maintenance

Essential to the function of shoot meristems in plants to act as sites of continuous organ and tissue formation is the ability of cells within the meristem to remain undifferentiated and proliferate indefinitely. These are characteristics of the stem cells within meristems that are critical for their growth properties. Stem cells are found in tight association with the stem cell niche-those cells that signal to maintain stem cells. Shoot meristems are unique among stem cell systems in that the stem cell niche is a constantly changing population of recent daughter stem cells. Recent progress from Arabidopsis and other systems have uncovered a large number of genes with defined roles in meristem structure and maintenance. This review will focus on well-studied pathways that represent signaling between the stem cells and the niche, that prevent ectopic differentiation of stem cells, that regulate the chromatin status of stem cell factors, and that reveal intersection of hormone signaling and meristem maintenance.

The ArabidopsisTUMOR PRONE5Gene Encodes an Acetylornithine Aminotransferase Required for Arginine Biosynthesis and Root Meristem Maintenance in Blue Light

Arginine is an essential amino acid necessary for protein synthesis and is also a nitrogen storage compound. The genes encoding the enzymes of arginine biosynthesis in plants are not well characterized and have mainly been predicted from homologies to bacterial and fungal genes. We report the cloning and characterization of the TUMOR PRONE5 (TUP5) gene of Arabidopsis (Arabidopsis thaliana) encoding an acetylornithine aminotransferase (ACOAT), catalyzing the fourth step of arginine biosynthesis. The free arginine content was strongly reduced in the chemically induced recessive mutant tup5-1, root growth was restored by supplementation with arginine and its metabolic precursors, and a yeast (Saccharomyces cerevisiae) ACOAT mutant was complemented by TUP5. Two null alleles of TUP5 caused a reduced viability of gametes and embryo lethality, possibly caused by insufficient Arg supply from maternal tissue. TUP5 expression is positively regulated by light, and a TUP5-green fluorescent protein was localized in chloroplasts. tup5-1 has a unique light-dependent short root phenotype. Roots of light-grown tup5-1 seedlings switch from indeterminate growth to determinate growth with arresting cell production and an exhausted root apical meristem. The inhibitory activity was specific for blue light, and the inhibiting light was perceived by the root. Thus, tup5-1 reveals a novel role of amino acids and blue light in regulating root meristem function.

Emerging Role of the Ubiquitin Proteasome System in the Control of Shoot Apical Meristem FunctionF

The shoot apical meristem (SAM) is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant's life. A plethora of different families of transcription factors (TFs) play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system (UPS). However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors.

Systems Analysis of Shoot Apical Meristem Growth and Development: Integrating Hormonal and Mechanical Signaling

The shoot apical meristem (SAM) is a small population of stem cells that continuously generates organs and tissues. This review covers our current understanding of organ initiation by the SAM in Arabidopsis thaliana. Meristem function and maintenance involves two major hormones, cytokinins and auxins. Cytokinins appear to play a major role in meristem maintenance and in controlling meristematic properties, such as cell proliferation. Self-organizing transport processes, which are still only partially understood, lead to the patterned accumulation of auxin at particular positions, where organs will grow out. A major downstream target of auxin-mediated growth regulation is the cell wall, which is a determinant for both growth rates and growth distribution, but feedbacks with metabolism and the synthetic capacity of the cytoplasm are crucial as well. Recent work has also pointed at a potential role of mechanical signals in growth coordination, but the precise mechanisms at work remain to be elucidated.

Overexpression of Arabidopsis Plasmodesmata Germin-Like Proteins Disrupts Root Growth and Development

In plants, a population of non-cell-autonomous proteins (NCAPs), including numerous transcription factors, move cell to cell through plasmodesmata (PD). In many cases, the intercellular trafficking of these NCAPs is regulated by their interaction with specific PD components. To gain further insight into the functions of this NCAP pathway, coimmunoprecipitation experiments were performed on a tobacco (Nicotiana tabacum) plasmodesmal-enriched cell wall protein preparation using as bait the NCAP, pumpkin (Cucurbita maxima) PHLOEM PROTEIN16 (Cm-PP16). A Cm-PP16 interaction partner, Nt-PLASMODESMAL GERMIN-LIKE PROTEIN1 (Nt-PDGLP1) was identified and shown to be a PD-located component. Arabidopsis thaliana putative orthologs, PDGLP1 and PDGLP2, were identified; expression studies indicated that, postgermination, these proteins were preferentially expressed in the root system. The PDGLP1 signal peptide was shown to function in localization to the PD by a novel mechanism involving the endoplasmic reticulum-Golgi secretory pathway. Overexpression of various tagged versions altered root meristem function, leading to reduced primary root but enhanced lateral root growth. This effect on root growth was corrected with an inability of these chimeric proteins to form stable PD-localized complexes. PDGLP1 and PDGLP2 appear to be involved in regulating primary root growth by controlling phloem-mediated allocation of resources between the primary and lateral root meristems.

Genome-wide Analysis of Ovate Family Proteins in Arabidopsis

Arabidopsis thaliana ovate family proteins (AtOFPs) is a newly found plant-specific protein family interacting with TALE (3-aa loop extension homeodomain proteins) homeodomain proteins in Arabidopsis. Here, based on bioinformatic analysis, we found that Arabidopsis genome actually encoded 17 OVATE domain-containing proteins. One of them, AtOFP19, has not been previously identified. Based on their amino acid sequence similarity, AtOFPs proteins can be divided into two groups. Most of the AtOFPs were located in nuclear, four of them were presented in chloroplast and the remaining two members appeared in cytoplasmic. A genomewide microarray based gene expression analysis involving 47 stages of vegetative and reproductive development revealed that AtOFPs have diverse expression patterns. Investigation of proteins interaction showed that nine AtOFPs only interacted with TALE homeodomain proteins, which are fundamental regulators of plant meristem function and leaf development. Our work could provide important leads toward functional genomics studies of ovate family proteins, which may be involved in a previously unrecognized control mechanism in plant development.

Arabidopsis JAGGED LATERAL ORGANS Acts with ASYMMETRIC LEAVES2 to Coordinate KNOX and PIN Expression in Shoot and Root Meristems

Organ initiation requires the specification of a group of founder cells at the flanks of the shoot apical meristem and the creation of a functional boundary that separates the incipient primordia from the remainder of the meristem. Organ development is closely linked to the downregulation of class I KNOTTED1 LIKE HOMEOBOX (KNOX) genes and accumulation of auxin at sites of primordia initiation. Here, we show that Arabidopsis thaliana JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, is required for coordinated organ development in shoot and floral meristems. Loss of JLO function results in ectopic expression of the KNOX genes SHOOT MERISTEMLESS and BREVIPEDICELLUS (BP), indicating that JLO acts to restrict KNOX expression. JLO acts in a trimeric protein complex with ASYMMETRIC LEAVES2 (AS2), another LBD protein, and AS1 to suppress BP expression in lateral organs. In addition to its role in KNOX regulation, we identified a role for AS2 in regulating PINFORMED (PIN) expression and auxin transport from embryogenesis onwards together with JLO. We propose that different JLO and AS2 protein complexes, possibly also comprising other LBD proteins, coordinate auxin distribution and meristem function through the regulation of KNOX and PIN expression during Arabidopsis development.

Three Arabidopsis AIL/PLT genes act in combination to regulate shoot apical meristem function

The shoot apical meristem, a small dome-shaped structure at the shoot apex, is responsible for the initiation of all post-embryonic shoot organs. Pluripotent stem cells within the meristem replenish themselves and provide daughter cells that become incorporated into lateral organ primordia around the meristem periphery. We have identified three novel regulators of shoot apical meristem activity in Arabidopsis thaliana that encode related AIL/PLT transcription factors: AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE6 (AIL6)/PLETHORA3 (PLT3) and AINTEGUMENTA-LIKE7 (AIL7)/PLETHORA7 (PLT7). Loss of these genes results in plants that initiate only a few leaves prior to termination of shoot apical meristem activity. In 7-day-old ant ail6 ail7 seedlings, we observed reduced cell division in the meristem region, differentiation of meristematic cells and altered expression of the meristem regulators WUSCHEL (WUS), CLAVATA3 (CLV3) and SHOOT MERISTEMLESS (STM). Genetic experiments suggest that these three AIL genes do not act specifically in either the WUS/CLV or STM pathway regulating meristem function. Furthermore, these studies indicate that ANT, AIL6 and AIL7 have distinct functions within the meristem rather than acting in a strictly redundant manner. Our study thus identifies three new genes whose distinct functions are together required for continuous shoot apical meristem function.

ABERRANT SPIKELET AND PANICLE1, encoding a TOPLESS‐related transcriptional co‐repressor, is involved in the regulation of meristem fate in rice

Post-embryonic development depends on the activity of meristems in plants, and thus control of cell fate in the meristem is crucial to plant development and its architecture. In grasses such as rice and maize, the fate of reproductive meristems changes from indeterminate meristems, such as inflorescence and branch meristems, to determinate meristems, such as the spikelet meristem. Here we analyzed a recessive mutant of rice, aberrant spikelet and panicle1 (asp1), that showed pleiotropic phenotypes such as a disorganized branching pattern, aberrant spikelet morphology, and disarrangement of phyllotaxy. Close examination revealed that regulation of meristem fate was compromised in asp1: degeneration of the inflorescence meristem was delayed, transition from the branch meristem to the spikelet meristem was accelerated, and stem cell maintenance in both the branch meristem and the spikelet meristem was compromised. The genetic program was also disturbed in terms of spikelet development. Gene isolation revealed that ASP1 encodes a transcriptional co-repressor that is related to TOPLESS (TPL) in Arabidopsis and RAMOSA ENHANCER LOCUS2 (REL2) in maize. It is likely that the pleiotropic defects are associated with de-repression of multiple genes related to meristem function in the asp1 mutant. The asp1 mutant also showed de-repression of axillary bud growth and disturbed phyllotaxy in the vegetative phase, suggesting that the function of this gene is closely associated with auxin action. Consistent with these observations and the molecular function of Arabidopsis TPL, auxin signaling was also compromised in the rice asp1 mutant. Taken together, these results indicate that ASP1 regulates various aspects of developmental processes and physiological responses as a transcriptional co-repressor in rice.

POPCORNFunctions in the Auxin Pathway to Regulate Embryonic Body Plan and Meristem Organization inArabidopsis

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.

Antagonistic Interaction of BLADE-ON-PETIOLE1 and 2 with BREVIPEDICELLUS and PENNYWISE Regulates Arabidopsis Inflorescence Architecture

The transition to flowering in many plant species, including Arabidopsis (Arabidopsis thaliana), is marked by the elongation of internodes to make an inflorescence upon which lateral branches and flowers are arranged in a characteristic pattern. Inflorescence patterning relies in part on the activities of two three-amino-acid loop-extension homeodomain transcription factors: BREVIPEDICELLUS (BP) and PENNYWISE (PNY) whose interacting products also promote meristem function. We examine here the genetic interactions between BP-PNY whose expression is up-regulated in stems at the floral transition, and the lateral organ boundary genes BLADE-ON-PETIOLE1 (BOP1) and BOP2, whose expression is restricted to pedicel axils. Our data show that bp and pny inflorescence defects are caused by BOP1/2 gain of function in stems and pedicels. Compatible with this, inactivation of BOP1/2 rescues these defects. BOP expression domains are differentially enlarged in bp and pny mutants, corresponding to the distinctive patterns of growth restriction in these mutants leading to compacted internodes and clustered or downward-oriented fruits. Our data indicate that BOP1/2 are positive regulators of KNOTTED1-LIKE FROM ARABIDOPSIS THALIANA6 expression and that growth restriction in BOP1/2 gain-of-function plants requires KNOTTED1-LIKE FROM ARABIDOPSIS THALIANA6. Antagonism between BOP1/2 and BP is explained in part by their reciprocal regulation of gene expression, as evidenced by the identification of lignin biosynthetic genes that are repressed by BP and activated by BOP1/2 in stems. These data reveal BOP1/2 gain of function as the basis of bp and pny inflorescence defects and reveal how antagonism between BOP1/2 and BP-PNY contributes to inflorescence patterning in a model plant species.

Bypassing Transcription: A Shortcut in Cytokinin-Auxin Interactions

In this issue of Developmental Cell, Marhavý etal. (2011) uncover a transcription-independent molecular mechanism of interaction between auxin and cytokinin in the regulation of plant meristem function. By modulating endocytic trafficking of PIN1, cytokinin controls auxin flux and, therefore, auxin gradients.

Type‐A response regulators are required for proper root apical meristem function through post‐transcriptional regulation of PIN auxin efflux carriers

The phytohormones cytokinin and auxin regulate a diverse array of plant processes, often acting together to modulate growth and development. Although much has been learned with regard to how each of these hormones act individually, we are just beginning to understand how these signals interact to achieve an integrated response. Previous studies indicated that exogenous cytokinin has an effect on the transcription of several PIN efflux carriers. Here we show that disruption of type-A Arabidopsis response regulators (ARRs), which are negative regulators of cytokinin signalling, alters the levels of PIN proteins and results in increased sensitivity to N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport. Disruption of eight of the 10 type-A ARR genes affects root development by altering the size of the apical meristem. Furthermore, we show that the effect of cytokinin on PIN abundance occurs primarily at the post-transcriptional level. Alterations of PIN levels in the type-A ARR mutants result in changes in the distribution of auxin in root tips as measured by a DR5::GFP reporter, and an altered pattern of cell division and differentiation in the stem cell niche in the root apical meristem. Together, these data indicate that cytokinin, acting through the type-A ARRs, alters the level of several PIN efflux carriers, and thus regulates the distribution of auxin within the root tip.

Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach

BackgroundPhenotype-driven forward genetic experiments are powerful approaches for linking phenotypes to genomic elements but they still involve a laborious positional cloning process. Although sequencing of complete genomes now becomes available, discriminating causal mutations from the enormous amounts of background variation remains a major challenge.MethodTo improve this, we developed a universal two-step approach, named 'fast forward genetics', which combines traditional bulk segregant techniques with targeted genomic enrichment and next-generation sequencing technologyResultsAs a proof of principle we successfully applied this approach to two Arabidopsis mutants and identified a novel factor required for stem cell activity.ConclusionWe demonstrated that the 'fast forward genetics' procedure efficiently identifies a small number of testable candidate mutations. As the approach is independent of genome size, it can be applied to any model system of interest. Furthermore, we show that experiments can be multiplexed and easily scaled for the identification of multiple individual mutants in a single sequencing run.

Auxin, cytokinin and the control of shoot branching

It has been known for many decades that auxin inhibits the activation of axillary buds, and hence shoot branching, while cytokinin has the opposite effect. However, the modes of action of these two hormones in branching control is still a matter of debate, and their mechanisms of interaction are equally unresolved. Here we review the evidence for various hypotheses that have been put forward to explain how auxin and cytokinin influence axillary bud activity. In particular we discuss the roles of auxin and cytokinin in regulating each other's synthesis, the cell cycle, meristem function and auxin transport, each of which could affect branching. These different mechanisms have implications for the main site of hormone action, ranging from systemic action throughout the plant, to local action at the node or in the bud meristem or leaves. The alternative models have specific predictions, and our increasing understanding of the molecular basis for hormone transport and signalling, cell cycle control and meristem biology is providing new tools to enable these predictions to be tested.

The Function of the CLE Peptides in Plant Development and Plant-Microbe Interactions.

The CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE) peptides consist of 12 or 13 amino acids, including hydroxylated proline residues that may or may not contain sugar modifications, and function in a non-cell-autonomous fashion. The CLE gene was first reported in Zea mays (maize) as an endosperm-specific gene, ESR, in 1997 (Opsahl-Ferstad et al., 1997). CLE genes encode secreted peptides that function in the extracellular space as intercellular signaling molecules and bind to cellular surface receptor-like proteins to transmit a signal. CLE peptides regulate various physiological and developmental processes and its signaling pathway are conserved in diverse land plants. Recent CLE functional studies have pointed to their significance in regulating meristematic activity in plant meristems, through the CLE-receptor kinase-WOX signaling node. CLV3 and CLE40 are responsible for maintenance of shoot apical meristem (SAM) and root apical meristem (RAM) function, regulating homeodomain transcription factors, WUSCHEL (WUS) and WUSCHEL-related homeobox 5 (WOX5), respectively. CLE and WOX form an interconnected and self-correcting feedback loop to provide robustness to stem cell homeostasis. CLE peptides are required for certain plant-microbe interactions, such as those that occur during legume symbiosis and phytopathogenic nematode infection. Understanding the molecular properties of CLE peptides may provide insight into plant cell-cell communication, and therefore also into plant-microbe interactions.

ThePopulusClass III HD ZIP,popREVOLUTA, Influences Cambium Initiation and Patterning of Woody Stems

The secondary growth of a woody stem requires the formation of a vascular cambium at an appropriate position and proper patterning of the vascular tissues derived from the cambium. Class III homeodomain-leucine zipper (HD ZIP) transcription factors have been implicated in polarity determination and patterning in lateral organs and primary vascular tissues and in the initiation and function of shoot apical meristems. We report here the functional characterization of a Populus class III HD ZIP gene, popREVOLUTA (PRE), that demonstrates another role for class III HD ZIPs in regulating the development of cambia and secondary vascular tissues. PRE is orthologous to Arabidopsis (Arabidopsis thaliana) REVOLUTA and is expressed in both the shoot apical meristem and in the cambial zone and secondary vascular tissues. Transgenic Populus expressing a microRNA-resistant form of PRE presents unstable phenotypic abnormalities affecting both primary and secondary growth. Surprisingly, phenotypic changes include abnormal formation of cambia within cortical parenchyma that can produce secondary vascular tissues in reverse polarity. Genes misexpressed in PRE mutants include transcription factors and auxin-related genes previously implicated in class III HD ZIP functions during primary growth. Together, these results suggest that PRE plays a fundamental role in the initiation of the cambium and in regulating the patterning of secondary vascular tissues.