TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation via the WOX4 Homeobox Gene in Arabidopsis

The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell–cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.     

Stem cell function during plant vascular development

While many regulatory mechanisms controlling the development and function of root and shoot apical meristems have been revealed, our knowledge of similar processes in lateral meristems, including the vascular cambium, is still limited. Our understanding of even the anatomy and development of lateral meristems (procambium or vascular cambium) is still relatively incomplete, let alone their genetic regulation. Research into this particular tissue type has been mostly hindered by a lack of suitable molecular markers, as well as the fact that thus far very few mutants affecting plant secondary development have been described. The development of suitable molecular markers is a high priority in order to help define the anatomy, especially the location and identity of cambial stem cells and the developmental phases and molecular regulatory mechanisms of the cambial zone. To date, most of the advances have been obtained by studying the role of the major plant hormones in vascular development. Thus far auxin, cytokinin, gibberellin and ethylene have been implicated in regulating the maintenance and activity of cambial stem cells; the most logical question in research would be how these hormones interact during the various phases of cambial development.     

The role of auxin stored in scots pine trunk during spring activation of cambial activity

In a 9-year-old pine girdled during the winter cambial activity was observed below the girdle in the next spring. This indicates that cambial activity was initiated without auxin produced in the spring by buds. The auxin produced in apical shoots successively flows down the stem, where as a result of periodic restriction in transport it remains over the winter till the next year. This auxin of apical origin but locally stored over the winter in the stem is responsible for the activation of cambium before the new flow of auxin produced in the apical meristems arrives. Calculations based on seasonal changes in auxin levels can explain both, earlier spring activation of cambium in the crown and the temporary cambial divisions below the girdle, without assumption of direct auxin synthesis in the lateral meristems.     

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.     

NO VEIN Mediates Auxin-Dependent Specification and Patterning in the Arabidopsis Embryo,Shoot, and Root

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type–specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type–specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.     

Lateral meristems of higher plants: Phytohormonal and genetic control

Lateral meristems (pericycle, procambium and cambium, phellogen) are positioned in parallel to the lateral surface of the organ, where they are present, and produce concentric layers of undifferentiated cells. Primary lateral meristems, procambium and pericycle, arise during embryogenesis; secondary lateral meristems, cambium and phellogen, — during post embryonic development. Pericycle is most pluripotent plant meristem, as it may give rise to a variety of other types of meristems: lateral meristems (cambium, phellogen), apical meristems of lateral roots, and also shoot meristems during plant in vitro regeneration. Procambium and cambium developing from it give rise to the vascular tissues of the stems and roots, ensuring their thickening. The review considers the genetic control of lateral meristem development and the role of phytohormones in the control of their activities.     

Flux-based transport enhancement as a plausible unifying mechanism for auxin transport in meristem development

Plants continuously generate new organs through the activity of populations of stem cells called meristems. The shoot apical meristem initiates leaves, flowers, and lateral meristems in highly ordered, spiralled, or whorled patterns via a process called phyllotaxis. It is commonly accepted that the active transport of the plant hormone auxin plays a major role in this process. Current hypotheses propose that cellular hormone transporters of the PIN family would create local auxin maxima at precise positions, which in turn would lead to organ initiation. To explain how auxin transporters could create hormone fluxes to distinct regions within the plant, different concepts have been proposed. A major hypothesis, canalization, proposes that the auxin transporters act by amplifying and stabilizing existing fluxes, which could be initiated, for example, by local diffusion. This convincingly explains the organised auxin fluxes during vein formation, but for the shoot apical meristem a second hypothesis was proposed, where the hormone would be systematically transported towards the areas with the highest concentrations. This implies the coexistence of two radically different mechanisms for PIN allocation in the membrane, one based on flux sensing and the other on local concentration sensing. Because these patterning processes require the interaction of hundreds of cells, it is impossible to estimate on a purely intuitive basis if a particular scenario is plausible or not. Therefore, computational modelling provides a powerful means to test this type of complex hypothesis. Here, using a dedicated computer simulation tool, we show that a flux-based polarization hypothesis is able to explain auxin transport at the shoot meristem as well, thus providing a unifying concept for the control of auxin distribution in the plant. Further experiments are now required to distinguish between flux-based polarization and other hypotheses.     

Multiple MONOPTEROS-dependent pathways are involved in leaf initiation

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN-FORMED1 (PIN) genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Because PIN expression is regulated by auxin response factor activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here, we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In the mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size, harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem, respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wild-type meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.     

Role of CLE41 Peptide in the Development of Root Storage Parenchyma in Species of the Genus <Emphasis Type="Italic">Raphanus</Emphasis> L.

CLE peptides (CLAVATA3/ENDOSPERM SURROUNDING REGION) are signal molecules or plant peptide hormones that play an important role in regulation of development of various meristems governing the expression of WOX (WUSCHEL-RELATED HOMEOBOX) genes. In particular, CLE peptides belonging to a small TDIF (Tracheary Element Differentiation Inhibitory Factor) group are responsible for the operation of gene WOX4 controlling the development of cambium and the conducting system. We looked into the role of CLE41 peptide from the TDIF group in the development of storage root in two species of the genus Raphanus: cultivated radish (Raphanus sativus var. radicula Pers.) that is a popular root crop with a storage root and its ancestor wild radish (Raphanus raphanistrum L.) where storage parenchyma of the root is poorly developed. It was shown that overexpression of gene RsCLE41 and plant treatment with exogenous peptide CLE41 influenced the development of cambium and xylem in the roots of R. sativus and R. raphanistrum and affected expression of the genes from different groups. One could say that peptide CLE41 activates expression of the genes whose homologues in arabidopsis play a key role in the maintenance of cambium (RsWOX4, RsWOX14, RsHAM4, and RsCYCD3). In the storage root of radish, peptide CLE41 activates proliferation of cambium cells reducing the amount of one of the xylem’s elements (lignified parenchyma). The obtained results point to an important role of CLE41 in the development of storage root in radish.     

WUSCHEL-RELATED HOMEOBOX4 Is Involved in Meristem Maintenance and Is Negatively Regulated by the CLE Gene FCP1 in Rice

The shoot apical meristem is the ultimate source of the cells that constitute the entire aboveground portion of the plant body. In Arabidopsis thaliana, meristem maintenance is regulated by the negative feedback loop of WUSCHEL-CLAVATA (WUS-CLV). Although CLV-like genes, such as FLORAL ORGAN NUMBER1 (FON1) and FON2, have been shown to be involved in maintenance of the reproductive meristems in rice (Oryza sativa), current understanding of meristem maintenance remains insufficient. In this article, we demonstrate that the FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2 genes encoding proteins with similar CLE domains are involved in negative regulation of meristem maintenance in the vegetative phase. In addition, we found that WUSCHEL-RELATED HOMEOBOX4 (WOX4) promotes the undifferentiated state of the meristem in rice and that WOX4 function is associated with cytokinin action. Consistent with similarities in the shoot apical meristem phenotypes caused by overexpression of FCP1 and downregulation of WOX4, expression of WOX4 was negatively regulated by FCP1 (FCP2). Thus, FCP1/2 and WOX4 are likely to be involved in maintenance of the vegetative meristem in rice.     

Between Xylem and Phloem: The Genetic Control of Cambial Activity in Plants

Abstract: Post-embryonic development is controlled by two types of meristems: apical and lateral. There has been considerable progress recently in understanding the function of root and shoot apical meristems at the molecular level. Knowledge of analogous processes in the lateral, or secondary, meristems, i.e. the vascular cambium or cork cambium, is, however, rudimentary. This is despite the fact that much of the diversity in the plant kingdom is based on the differential functions of these meristems, emphasizing the importance of lateral meristems in the development of different plant forms. The vascular cambium is particularly important for woody plants, but it also plays an important role during the development of various herbaceous species, such as Arabidopsis thaliana. In this review, we focus on the two basic functions of cambial activity: cell proliferation and pattern formation.     

The Arabidopsis D-type cyclin CYCD2;1 and the inhibitor ICK2/KRP2 modulate auxin-induced lateral root formation

The integration of cell division in root growth and development requires mediation of developmental and physiological signals through regulation of cyclin-dependent kinase activity. Cells within the pericycle form de novo lateral root meristems, and D-type cyclins (CYCD), as regulators of the G₁-to-S phase cell cycle transition, are anticipated to play a role. Here, we show that the D-type cyclin protein CYCD2;1 is nuclear in Arabidopsis thaliana root cells, with the highest concentration in apical and lateral meristems. Loss of CYCD2;1 has a marginal effect on unstimulated lateral root density, but CYCD2;1 is rate-limiting for the response to low levels of exogenous auxin. However, while CYCD2;1 expression requires sucrose, it does not respond to auxin. The protein Inhibitor-Interactor of CDK/Kip Related Protein2 (ICK2/KRP2), which interacts with CYCD2;1, inhibits lateral root formation, and ick2/krp2 mutants show increased lateral root density. ICK2/KRP2 can modulate the nuclear levels of CYCD2;1, and since auxin reduces ICK2/KRP2 protein levels, it affects both activity and cellular distribution of CYCD2;1. Hence, as ICK2/KRP2 levels decrease, the increase in lateral root density depends on CYCD2;1, irrespective of ICK2/CYCD2;1 nuclear localization. We propose that ICK2/KRP2 restrains root ramification by maintaining CYCD2;1 inactive and that this modulates pericycle responses to auxin fluctuations.     

The Stem Cell Niche in Leaf Axils Is Established by Auxin and Cytokinin in Arabidopsis

Plants differ from most animals in their ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems harboring new stem cell niches. In seed plants, this is achieved by axillary meristems, which are established in the axil of each leaf base and develop into lateral branches. Here, we describe the initial processes of Arabidopsis thaliana axillary meristem initiation. Using reporter gene expression analysis, we find that axillary meristems initiate from leaf axil cells with low auxin through stereotypical stages. Consistent with this, ectopic overproduction of auxin in the leaf axil efficiently inhibits axillary meristem initiation. Furthermore, our results demonstrate that auxin efflux is required for the leaf axil auxin minimum and axillary meristem initiation. After lowering of auxin levels, a subsequent cytokinin signaling pulse is observed prior to axillary meristem initiation. Genetic analysis suggests that cytokinin perception and signaling are both required for axillary meristem initiation. Finally, we show that cytokinin overproduction in the leaf axil partially rescue axillary meristem initiation-deficient mutants. These results define a mechanistic framework for understanding axillary meristem initiation.     

Participation of Auxin Transport in the Early Response of the <i>Arabidopsis</i> Root System to Inoculation with <i>Azospirillum brasilense</i>

The potential of Plant Growth Promoting Rhizobacteria (PGPR) has been demonstrated in the case of plant inoculation with bacteria of the genus Azospirillum which improves yield. A. brasilense produces a wide variety of molecules, including the natural auxin indole-3-acetic acid (IAA), as well as other phytoregulators. However, several studies have suggested that auxin induces changes in plant development during their interaction with the bacteria. The effects of A. brasilense Sp245 on the development of Arabidopsis thaliana root were investigated to help explain the molecular basis of the interaction. The results obtained showed a decrease in primary root length from the first day and remained so throughout the exposure, accompanied by a stimulation of initiation and maturation of lateral root primordia and an increase of lateral roots. An enhanced auxin response was evident in the vascular tissue and lateral root meristems of inoculated plants. However, after five days of bacterization, the response disappeared in the primary root meristems. The role of polar auxin transport (PAT) in auxins relocation involved the PGP1, AXR4-1, and BEN2 proteins, which apparently mediated A. brasilense-induced root branching of Arabidopsis seedlings.     

Auxin at the Shoot Apical Meristem

Plants continuously generate new tissues and organs through the activity of populations of undifferentiated stem cells, called meristems. Here, we discuss the so-called shoot apical meristem (SAM), which generates all the aerial parts of the plant. It has been known for many years that auxin plays a central role in the functioning of this meristem. Auxin is not homogeneously distributed at the SAM and it is thought that this distribution is interpreted in terms of differential gene expression and patterned growth. In this context, auxin transporters of the PIN and AUX families, creating auxin maxima and minima, are crucial regulators. However, auxin transport is not the only factor involved. Auxin biosynthesis genes also show specific, patterned activities, and local auxin synthesis appears to be essential for meristem function as well. In addition, auxin perception and signal transduction defining the competence of cells to react to auxin, add further complexity to the issue. To unravel this intricate signaling network at the SAM, systems biology approaches, involving not only molecular genetics but also live imaging and computational modeling, have become increasingly important.Plants continuously generate new tissues and organs through the activity of populations of undifferentiated stem cells, called meristems. Because meristems can modulate their activity, they provide the developmental flexibility that allows plants to adapt their development in reaction to the environment (reviews: Lyndon 1998; Traas and Doonan 2001; Aida and Tasaka 2006; Sablowski 2007).Distinct meristems exist. Apical meristems, positioned at the tip of the shoots and roots, initiate aerial and underground organs, respectively. Along the stems and roots, more diffuse secondary meristems are responsible for secondary thickening of these structures.The plant hormone auxin plays an instrumental role in meristem biology and we discuss here its role in a particular meristem, the shoot apical meristem (SAM) that generates all the aerial organs including the floral meristems (Fig. 1A–C). In this context, we limit ourselves to the meristems in angiosperm that have been studied in most detail.Open in a separate windowFigure 1.The shoot apical meristem of Arabidopsis thaliana. (A) Aerial part of a wild-type plant of the Columbia ecotype (Col-0). The SAM is responsible for the production of rosette leaves and, after floral transition, for the production of the stem, cauline leaves, lateral meristems, and flowers of the inflorescence. (B) Details of the tip of the inflorescence, showing the highly organized positioning of flowers around the main axis (a spiral). (C) A dissected inflorescence meristem. Older flowers have been removed to expose the meristem surrounded by young floral buds. (D) Longitudinal section of an inflorescence meristem showing the layered organization (L1, L2, and L3 cell layers). L1 and L2 are also called the tunica and L3 to the corpus. The functional zones are also represented. At the meristem summit the central zone (CZ) contains the stem cells, whereas primordia are initiated in the peripheral zone (PZ). The rib zone (RZ) produces the internal part of the stem.