Conserved LBL1-ta-siRNA and miR165/166-RLD1/2 modules regulate root development in maize.

Root system architecture and anatomy of monocotyledonous maize is significantly different from dicotyledonous model Arabidopsis The molecular role of non-coding RNA (ncRNA) is poorly understood in maize root development. Here, we address the role of LEAFBLADELESS1 (LBL1), a component of maize trans-acting short-interfering RNA (ta-siRNA), in maize root development. We report that root growth, anatomical patterning, and the number of lateral roots (LRs), monocot-specific crown roots (CRs) and seminal roots (SRs) are significantly affected in lbl1-rgd1 mutant, which is defective in production of ta-siRNA, including tasiR-ARF that targets AUXIN RESPONSE FACTOR3 (ARF3) in maize. Altered accumulation and distribution of auxin, due to differential expression of auxin biosynthesis and transporter genes, created an imbalance in auxin signalling. Altered expression of microRNA165/166 (miR165/166) and its targets, ROLLED1 and ROLLED2 (RLD1/2), contributed to the changes in lbl1-rgd1 root growth and vascular patterning, as was evident by the altered root phenotype of Rld1-O semi-dominant mutant. Thus, LBL1/ta-siRNA module regulates root development, possibly by affecting auxin distribution and signalling, in crosstalk with miR165/166-RLD1/2 module. We further show that ZmLBL1 and its Arabidopsis homologue AtSGS3 proteins are functionally conserved.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

Auxin-mediated regulation of vascular patterning in Arabidopsis thaliana leaves.

The vascular system develops in response to auxin flow as continuous strands of conducting tissues arranged in regular spatial patterns. However, a mechanism governing their regular and repetitive formation remains to be fully elucidated. A model system for studying the vascular pattern formation is the process of leaf vascularization in Arabidopsis. In this paper, we present current knowledge of important factors and their interactions in this process. Additionally, we propose the sequence of events leading to the emergence of continuous vascular strands and point to significant problems that need to be resolved in the future to gain a better understanding of the regulation of the vascular pattern development.

PHABULOSA Mediates an Auxin Signaling Loop to Regulate Vascular Patterning in Arabidopsis.

Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars, and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis (Arabidopsis thaliana), HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels promoting protoxylem at its flanks. It is unclear, however, by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here, we present data suggesting that an important mechanism is their ability to moderate the auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS (MP)/AUXIN RESPONSE FACTOR5, a key factor in vascular formation, and IAA20, encoding an auxin/indole acetic acid protein that is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homolog IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp mutant. Our results provide evidence that HD-ZIP III TFs directly affect the auxin response and mediate a feed-forward loop formed by MP and IAA20 that may focus and stabilize the auxin response during vascular patterning and the differentiation of xylem cell types.

Plant vascular development: from early specification to differentiation.

Vascular tissues in plants are crucial to provide physical support and to transport water, sugars and hormones and other small signalling molecules throughout the plant. Recent genetic and molecular studies have identified interconnections among some of the major signalling networks that regulate plant vascular development. Using Arabidopsis thaliana as a model system, these studies enable the description of vascular development from the earliest tissue specification events during embryogenesis to the differentiation of phloem and xylem tissues. Moreover, we propose a model for how oriented cell divisions give rise to a three-dimensional vascular bundle within the root meristem.

Regulation of FATTY ACID ELONGATION1 expression in embryonic and vascular tissues of Brassica napus.

The expression of the FATTY ACID ELONGATION1 genes was characterised to provide insight into the regulation of very long chain fatty acid (VLCFA) biosynthesis in Brassica napus embryos. Each of the two rapeseed homoeologous genes (Bn-FAE1.1 and Bn-FAE1.2) encoding isozymes of 3-keto-acylCoA synthase, a subunit of the cytoplasmic acyl-CoA elongase complex that controls the production of elongated fatty acids, are expressed predominantly in developing seeds. The proximal regions of the Bn-FAE1.1 and Bn-FAE1.2 promoters possess strong sequence identity suggesting that transcriptional control of expression is mediated by this region which contains putative cis-elements characteristic of those found in the promoters of genes expressed in embryo and endosperm. Histochemical staining of rapeseed lines expressing Bn-FAE1.1 promoter:reporter gene fusions revealed a strong expression in the embryo cotyledon and axis throughout the maturation phase. Quantitative analyses revealed the region, -331 to -149, exerts a major control on cotyledon specific expression and the level of expression. A second region, -640 to -475, acts positively to enhance expression levels and extends expression of Bn-FAE1.1 into the axis and hypocotyl but also acts negatively to repress expression in the root meristem. The expression of the Bn-FAE1.1 gene was not restricted to the seed but was also detected in the vascular tissues of germinating seedlings and mature plants in the fascicular cambium tissue present in roots, stem and leaf petiole. We propose that Bn-FAE1.1 expression in vascular tissue may contribute VLCFA for barrier lipid synthesis and reflects the ancestral function of FAE1 encoded 3-keto-acylCoA synthase.

Building a plant: cell fate specification in the early Arabidopsis embryo.

Embryogenesis is the beginning of plant development, yet the cell fate decisions and patterning steps that occur during this time are reiterated during development to build the post-embryonic architecture. In Arabidopsis, embryogenesis follows a simple and predictable pattern, making it an ideal model with which to understand how cellular and tissue developmental processes are controlled. Here, we review the early stages of Arabidopsis embryogenesis, focusing on the globular stage, during which time stem cells are first specified and all major tissues obtain their identities. We discuss four different aspects of development: the formation of outer versus inner layers; the specification of vascular and ground tissues; the determination of shoot and root domains; and the establishment of the first stem cells.

Initiation of vascular development.

The initiation of vascular development occurs during embryogenesis and the development of lateral organs, such as lateral roots and leaves. Understanding the mechanism underlying the initiation of vascular development has been an important goal of plant biologists. Auxin flow is a crucial factor involved in the initiation of vascular development. In addition, recent studies have identified key factors that regulate the establishment of vascular initial cells in embryos and roots. In this review, we summarize the recent findings in this field and discuss the initiation of vascular development.

Prenatal plumbing--vascular tissue formation in the plant embryo.

The first vascular tissue precursors are specified early during embryogenesis. These precursors give rise to the multi-layered cylinder of hypocotyl and root through controlled, oriented divisions. Concomitant with its growth, the bundle is patterned into xylem and phloem tissues, and intervening procambial cells. These patterns are later maintained during post-embryonic growth and vascular cells will eventually differentiate, displaying characteristic secondary cell wall modifications. Given that the vascular system forms de novo in a simple yet predictable fashion, the embryo provides an excellent model system to study early developmental aspects of vascular tissue formation. However, the benefits of this model are only beginning to be exploited, and most knowledge about the vascular development is derived from growing post-embryonic tissues. Importantly, it is unclear how much of these established post-embryonic mechanisms can be extrapolated to tissue formation during embryogenesis. Here we review concepts established in the model plant Arabidopsis thaliana and focus on recent advances made in understanding embryonic vascular development.

Control of vein patterning by intracellular auxin transport.

The vein networks of plant leaves are among the most spectacular expressions of biological pattern, and the principles controlling their formation have continually inspired artists and scientists. Control of vein patterning by the polar, cell-to-cell transport of the plant signaling molecule auxin--mediated in Arabidopsis primarily by the plasma-membrane-localized PIN1--has long been known. By contrast, the existence of intracellular auxin transport and its contribution to vein patterning are recent discoveries. The endoplasmic-reticulum-localized PIN5, PIN6, and PIN8 of Arabidopsis define an intracellular auxin-transport pathway whose functions in vein patterning overlap with those of PIN1-mediated intercellular auxin transport. The genetic interaction between the components of the intracellular auxin-transport pathway is far from having been resolved. The study of vein patterning provides experimental access to gain such a resolution-a resolution that in turn holds the promise to improve our understanding of one of the most fascinating examples of biological pattern formation.

Auxin-associated initiation of vascular cell differentiation by LONESOME HIGHWAY.

Plant vascular tissues are essential for the existence of land plants. Many studies of transcriptional regulation and cell-cell communication have revealed the process underlying the development of vascular tissues from vascular initial cells. However, the initiation of vascular cell differentiation is still a mystery. Here, we report that LONESOME HIGHWAY (LHW), which encodes a bHLH transcription factor, is expressed in pericycle-vascular mother cells at the globular embryo stage and is required for proper asymmetric cell division to generate vascular initial cells. In addition, ectopic expression of LHW elicits an ectopic auxin response. Moreover, LHW is required for the correct expression patterns of components related to auxin flow, such as PIN-FORMED 1 (PIN1), MONOPTEROS (MP) and ATHB-8, and ATHB-8 partially rescues the vascular defects of lhw. These results suggest that LHW functions as a key regulator to initiate vascular cell differentiation in association with auxin regulation.

incurvata13, a novel allele of AUXIN RESISTANT6, reveals a specific role for auxin and the SCF complex in Arabidopsis embryogenesis, vascular specification, and leaf flatness.

Auxin plays a pivotal role in plant development by modulating the activity of SCF ubiquitin ligase complexes. Here, we positionally cloned Arabidopsis (Arabidopsis thaliana) incurvata13 (icu13), a mutation that causes leaf hyponasty and reduces leaf venation pattern complexity and auxin responsiveness. We found that icu13 is a novel recessive allele of AUXIN RESISTANT6 (AXR6), which encodes CULLIN1, an invariable component of the SCF complex. Consistent with a role for auxin in vascular specification, the vascular defects in the icu13 mutant were accompanied by reduced expression of auxin transport and auxin perception markers in provascular cells. This observation is consistent with the expression pattern of AXR6, which we found to be restricted to vascular precursors and hydathodes in wild-type leaf primordia. AXR1, RELATED TO UBIQUITIN1-CONJUGATING ENZYME1, CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME5A, and CULLIN-ASSOCIATED NEDD8-DISSOCIATED1 participate in the covalent modification of CULLIN1 by RELATED TO UBIQUITIN. Hypomorphic alleles of these genes also display simple venation patterns, and their double mutant combinations with icu13 exhibited a synergistic, rootless phenotype reminiscent of that caused by loss of function of MONOPTEROS (MP), which forms an auxin-signaling module with BODENLOS (BDL). The phenotypes of double mutant combinations of icu13 with either a gain-of-function allele of BDL or a loss-of-function allele of MP were synergistic. In addition, a BDL:green fluorescent protein fusion protein accumulated in icu13, and BDL loss of function or MP overexpression suppressed the phenotype of icu13. Our results demonstrate that the MP-BDL module is required not only for root specification in embryogenesis and vascular postembryonic development but also for leaf flatness.

Deletion of MP/ARF5 domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning.

Combinatorial interactions of AUXIN RESPONSE FACTORs (ARFs) and auxin/indole acetic acid (Aux/IAA) proteins through their common domains III and IV regulate auxin responses, but insight into the functions of individual proteins is still limited. As a new tool to explore this regulatory network, we generated a gain-of-function ARF genotype by eliminating domains III and IV from the functionally well-characterized ARF MONOPTEROS(MP)/ARF5. This truncated version of MP, termed MPΔ, conferred complementing MP activity, but also displayed a number of semi-dominant traits affecting auxin signaling and organ patterning. In MPΔ, the expression levels of many auxin-inducible genes, as well as rooting properties and vascular tissue abundance, were enhanced. Lateral organs were narrow, pointed and filled with parallel veins. This effect was epistatic over the vascular hypotrophy imposed by certain Aux/IAA mutations. Further, in MPΔ leaves, failure to turn off the procambium-selecting gene PIN1 led to the early establishment of oversized central procambial domains and very limited subsequent lateral growth of the leaf lamina. We conclude that MPΔ can selectively uncouple a single ARF from regulation by Aux/IAA proteins and can be used as a genetic tool to probe auxin pathways and explore leaf development.

Putative dual pathway of auxin transport in organogenesis of Arabidopsis.

In Arabidopsis, damage to the superficial acropetal polar auxin transport (PAT) inhibits generative but not vegetative organ initiation. In order to verify whether in a vegetative phase auxin can be transported to the meristem in a different way, the research on wild-type and plants with defective PAT was performed. Distance from the differentiated vascular elements to the shoot apical meristem (SAM) was measured for Arabidopsis cultured in different experimental systems. The influence of this distance on the ability to induce organogenesis as well as transport of the fluorescent dye to the SAM, and the LEAFY gene expression were analyzed. The youngest protoxylem elements were used as a marker of the vascular tissues. The distance of protoxylem to the SAM and organogenesis were interrelated. Organ initiation occurred only when protoxylem was localized near to the SAM. Experimental elongation of internodes in a vegetative rosette caused an increase in the distance between protoxylem and the SAM organogenic zone. Thus, the inhibition of organ initiation took place already during the vegetative phase. The results suggest the presence of at least two pathways of acropetal transport of auxin inducing organogenesis: one superficial way through PAT, and the second, putative one, internal through the vascular system. Possibly, organogenesis is completely blocked only when both these pathways are dysfunctional.

Simultaneous activation of SHR and ATHB8 expression defines switch to preprocambial cell state in Arabidopsis leaf development.

The processes underlying the formation of leaf vascular networks have long captured the attention of developmental biologists, especially because files of elongated vascular-precursor procambial cells seem to differentiate from apparently equivalent, isodiametric ground cells. In Arabidopsis leaves, ground cells that have been specified to vascular fate engage expression of ARABIDOPSIS THALIANA HOMEOBOX8 (ATHB8). While definition of the transcriptional state of ATHB8-expressing ground cells would be particularly informative, no other genes have been identified whose expression is initiated at this stage. Here we show that expression of SHORT-ROOT (SHR) is activated simultaneously with that of ATHB8 in leaf development. Congruence between SHR and ATHB8 expression domains persists under conditions of manipulated vein patterning, suggesting that inception of expression of SHR and ATHB8 identifies transition to a preprocambial cell state that presages vein formation. Our observations further characterize the molecular identity of cells at anatomically inconspicuous stages of leaf vein development.

The immunolocation of XTH1 in embryonic axes during chickpea germination and seedling growth confirms its function in cell elongation and vascular differentiation.

In a previous work, the immunolocation of the chickpea XTH1 (xyloglucan endotransglucosylase/hydrolase 1) protein in the cell walls of epicotyls, radicles, and stems was studied, and a role for this protein in the elongation of vascular cells was suggested. In the present work, the presence and the location of the XTH1 protein in embryonic axes during the first 48 h of seed imbibition, including radicle emergence, were studied. The presence of the XTH1 protein in the cell wall of embryonic axes as early as 3 h after imbibition, before radicle emergence, supports its involvement in germination, and the fact that the protein level increased until 24 h, when the radicle had already emerged, also suggests its participation in the elongation of embryonic axes. The localization of XTH1 clearly indicates that the protein is related to the development of vascular tissue in embryonic axes during the period studied, suggesting that the role of this protein in embryonic axes is the same as that proposed for seedlings and plants.