Transcriptional control of hydrogen peroxide homeostasis regulates ground tissue patterning in the Arabidopsis root.

In multicellular organisms, including higher plants, asymmetric cell divisions (ACDs) play a crucial role in generating distinct cell types. The Arabidopsis root ground tissue initially has two layers: endodermis (inside) and cortex (outside). In the mature root, the endodermis undergoes additional ACDs to produce the endodermis itself and the middle cortex (MC), located between the endodermis and the pre-existing cortex. In the Arabidopsis root, gibberellic acid (GA) deficiency and hydrogen peroxide (H2O2) precociously induced more frequent ACDs in the endodermis for MC formation. Thus, these findings suggest that GA and H2O2 play roles in regulating the timing and extent of MC formation. However, details of the molecular interaction between GA signaling and H2O2 homeostasis remain elusive. In this study, we identified the PEROXIDASE 34 (PRX34) gene, which encodes a class III peroxidase, as a molecular link to elucidate the interconnected regulatory network involved in H2O2- and GA-mediated MC formation. Under normal conditions, prx34 showed a reduced frequency of MC formation, whereas the occurrence of MC in prx34 was restored to nearly WT levels in the presence of H2O2. Our results suggest that PRX34 plays a role in H2O2-mediated MC production. Furthermore, we provide evidence that SCARECROW-LIKE 3 (SCL3) regulates H2O2 homeostasis by controlling transcription of PRX34 during root ground tissue maturation. Taken together, our findings provide new insights into how H2O2 homeostasis is achieved by SCL3 to ensure correct radial tissue patterning in the Arabidopsis root.

Putative pectate lyase PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport in Arabidopsis.

In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE-LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve-element-specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long-distance transport as the plant grows-a defect that is aggravated when combined with another sieve-element-specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the subcellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan, thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.

A root phloem pole cell atlas reveals common transcriptional states in protophloem-adjacent cells.

Single-cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here we use a second-generation single-cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks common to protophloem sieve element (PSE)-adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF (PEAR) genes and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development.

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

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.

Mobile PEAR transcription factors integrate positional cues to prime cambial growth.

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 cambium1. 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 on2,3. 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 factors4-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.

Phloem differentiation: an integrative model for cell specification.

Plant vasculature consists of two major conductive cell types, xylem tracheary elements and phloem sieve elements (SEs). Both cell types undergo a highly specialized differentiation process. The root meristem of Arabidopsis displays a stereotypical anatomy in which the central vasculature is surrounded by concentric layers of outer tissues. Each cell file is derived from stem cells located in the root tip. A series of formative and proliferative divisions take place in the meristem; these are followed by cell expansion and differentiation. Protophloem differentiation is unique in being complete only 20-25 cells away from the first stem cell, and during the differentiation process the cells lose several organelles, including the nucleus, while the remaining organelles are rearranged. Defects in SE development have been shown to result in impaired auxin transport and response and therefore systemically affect root growth. Although a few genes have been demonstrated to function in phloem development, detailed analyses and a comprehensive understanding of sieve element development (i.e. how often the stem cells divide, how frequently enucleation takes place, and how SE development is coordinated between cell division and differentiation on a molecular level) are still lacking. Advanced live-imaging techniques which enable prolonged time-lapse captures of root tip growth as well as single-cell transcriptomic analysis of the 20-25 cells in the SE file could help resolve these questions. In addition, understanding the interplay between the PLETHORA (PLT) gradient, which is known to govern the root zonation, and phloem development within the root meristem could shed light on the rapidity of SE differentiation and its importance to the meristem.

Differentiation of conductive cells: a matter of life and death.

Two major conducting tissues in plants, phloem and xylem, are composed of highly specialized cell types adapted to long distance transport. Sieve elements (SEs) in the phloem display a thick cell wall, callose-rich sieve plates and low cytoplasmic density. SE differentiation is driven by selective autolysis combined with enucleation, after which the plasma membrane and some organelles are retained. By contrast, differentiation of xylem tracheary elements (TEs) involves complete clearance of the cellular components by programmed cell death followed by autolysis of the protoplast; this is accompanied by extensive deposition of lignin and cellulose in the cell wall. Emerging molecular data on TE and SE differentiation indicate a central role for NAC and MYB type transcription factors in both processes.

Interplay between ABA and GA Modulates the Timing of Asymmetric Cell Divisions in the Arabidopsis Root Ground Tissue.

In multicellular organisms, controlling the timing and extent of asymmetric cell divisions (ACDs) is crucial for correct patterning. During post-embryonic root development in Arabidopsis thaliana, ground tissue (GT) maturation involves an additional ACD of the endodermis, which generates two different tissues: the endodermis (inner) and the middle cortex (outer). It has been reported that the abscisic acid (ABA) and gibberellin (GA) pathways are involved in middle cortex (MC) formation. However, the molecular mechanisms underlying the interaction between ABA and GA during GT maturation remain largely unknown. Through transcriptome analyses, we identified a previously uncharacterized C2H2-type zinc finger gene, whose expression is regulated by GA and ABA, thus named GAZ (GA- AND ABA-RESPONSIVE ZINC FINGER). Seedlings ectopically overexpressing GAZ (GAZ-OX) were sensitive to ABA and GA during MC formation, whereas GAZ-SRDX and RNAi seedlings displayed opposite phenotypes. In addition, our results indicated that GAZ was involved in the transcriptional regulation of ABA and GA homeostasis. In agreement with previous studies that ABA and GA coordinate to control the timing of MC formation, we also confirmed the unique interplay between ABA and GA and identified factors and regulatory networks bridging the two hormone pathways during GT maturation of the Arabidopsis root.

Phloem development: current knowledge and future perspectives.

Phloem, as a major tissue mediating long-distance communication, has been an object of extensive research ever since its structure was first reported in 1837. Functional phloem consists of sieve elements (SEs) and companion cells (CCs). While SEs are enucleated conducting cells in the phloem, CCs are cells with intact cellular components and are known to support the functioning of SEs. CCs are closely linked to SEs by symplastic connections mediated by plasmodesmata (PD). Sieve elements are notoriously sensitive to manipulation, which has hampered efforts to investigate their structure using microscopy or histology; phloem thus remains a mysterious tissue almost 200 yr after its discovery. Nevertheless, consistent efforts have overcome many of the technical barriers and generated considerable amounts of data about the structure and function of phloem. Advances in the 1950s and 1960s significantly improved our understanding of phloem anatomy and function. A major function of the phloem is to establish symplastic connections throughout the plant body, delivering nutrients and various signaling molecules, which play pivotal roles in growth and development. Despite the importance of phloem, details about the molecular mechanisms responsible for the establishment and maintenance of phloem continuity remain elusive.

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

Plant Vascular Biology 2013: vascular trafficking.

About 200 researchers from around the world attended the Third International Conference on Plant Vascular Biology (PVB 2013) held in July 2013 at the Rantapuisto Conference Center, in Helsinki, Finland (http://www.pvb2013.org). The plant vascular system, which connects every organ in the mature plant, continues to attract the interest of researchers representing a wide range of disciplines, including development, physiology, systems biology, and computational biology. At the meeting, participants discussed the latest research advances in vascular development, long- and short-distance vascular transport and long-distance signalling in plant defence, in addition to providing a context for how these studies intersect with each other. The meeting provided an opportunity for researchers working across a broad range of fields to share ideas and to discuss future directions in the expanding field of vascular biology. In this report, the latest advances in understanding the mechanism of vascular trafficking presented at the meeting have been summarized.

Analysis of Arabidopsis glucose insensitive growth mutants reveals the involvement of the plastidial copper transporter PAA1 in glucose-induced intracellular signaling.

Sugars play important roles in many aspects of plant growth and development, acting as both energy sources and signaling molecules. With the successful use of genetic approaches, the molecular components involved in sugar signaling have been identified and their regulatory roles in the pathways have been elucidated. Here, we describe novel mutants of Arabidopsis (Arabidopsis thaliana), named glucose insensitive growth (gig), identified by their insensitivity to high-glucose (Glc)-induced growth inhibition. The gig mutant displayed retarded growth under normal growth conditions and also showed alterations in the expression of Glc-responsive genes under high-Glc conditions. Our molecular identification reveals that GIG encodes the plastidial copper (Cu) transporter PAA1 (for P(1B)-type ATPase 1). Interestingly, double mutant analysis indicated that in high Glc, gig is epistatic to both hexokinase1 (hxk1) and aba insensitive4 (abi4), major regulators in sugar and retrograde signaling. Under high-Glc conditions, the addition of Cu had no effect on the recovery of gig/paa1 to the wild type, whereas exogenous Cu feeding could suppress its phenotype under normal growth conditions. The expression of GIG/PAA1 was also altered by mutations in the nuclear factors HXK1, ABI3, and ABI4 in high Glc. Furthermore, a transient expression assay revealed the interaction between ABI4 and the GIG/PAA1 promoter, suggesting that ABI4 actively regulates the transcription of GIG/PAA1, likely binding to the CCAC/ACGT core element of the GIG/PAA1 promoter. Our findings indicate that the plastidial Cu transporter PAA1, which is essential for plastid function and/or activity, plays an important role in bidirectional communication between the plastid and the nucleus in high Glc.

Funneling of gibberellin signaling by the GRAS transcription regulator scarecrow-like 3 in the Arabidopsis root.

During plant development, because no cell movement takes place, control of the timing and extent of cell division and coordination of the direction and extent of cell expansion are particularly important for growth and development. The plant hormone gibberellins (GAs) play key roles in the control of these developmental processes. However, little is known about the molecular components that integrate the generic GA signaling into a specific cell/tissue to coordinate cell division and cell expansion. Here we report that scarecrow-like 3 (SCL3), a GRAS protein, acts as a positive regulator to integrate and maintain a functional GA pathway by attenuating the DELLA repressors in the root endodermis. The tissue-specific maintenance of GA signaling in the root endodermis plays distinct roles along the longitudinal root axis. While in the elongation/differentiation zone (EDZ), the endodermis-confined GA pathway by SCL3 controls primarily coordination of root cell elongation; in the meristem zone (MZ) SCL3 in conjunction with the short-root/scarecrow (SHR/SCR) pathway controls GA-modulated ground tissue maturation. Our findings highlight the regulatory network of the GRAS transcription regulators (SCL3, DELLAs, and SHR/SCR) in the root endodermis, shedding light on how GA homeostasis is achieved and how the maintenance of GA signaling controls developmental processes in roots.

Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis.

The diterpenoid phytohormone gibberellin (GA) controls diverse developmental processes throughout the plant life cycle. DELLA proteins are master growth repressors that function immediately downstream of the GA receptor to inhibit GA signaling. By doing so, DELLAs also play pivotal roles as integrators of internal developmental signals from multiple hormone pathways and external cues. DELLAs are likely nuclear transcriptional regulators, which interact with other transcription factors to modulate expression of GA-responsive genes. DELLAs are also involved in maintaining GA homeostasis through feedback up-regulating expression of GA biosynthesis and receptor genes. However, the molecular mechanisms by which DELLAs restrict growth and development are largely unknown. This study reveals an important step of the mechanism. Previous microarray studies identified scarecrow-like 3 (SCL3) as a direct target gene of DELLA in Arabidopsis seedlings. SCL3 expression is induced by DELLA and repressed by GA. Unexpectedly, a scl3 null mutant displays reduced GA responses and elevated expression of GA biosynthesis genes during seed germination and seedling growth, indicating that SCL3 functions as a positive regulator of GA signaling. SCL3 seems to act as an attenuator of DELLA proteins. Transient expression, ChIP, and co-IP studies show that SCL3 autoregulates its own transcription by directly interacting with DELLA. Our data further show that SCL3 and DELLA antagonize each other in controlling both downstream GA responses and upstream GA biosynthetic genes. This work is beginning to shed light on how this complex regulatory network achieves GA homeostasis and controls GA-mediated growth and development in the plant.

Characterization of SHORT-ROOT function in the Arabidopsis root vascular system.

Development of the vascular tissues is a dynamic process that integrates extrinsic and intrinsic factors to control vascular tissue formation throughout the plant life cycle. During vascular tissue formation in Arabidopsis roots, radial and longitudinal signals, including nuclear factors and plant hormones, control the developmental processes involved in the specification, differentiation, and maintenance of the correct cell types. SHR, a GRAS transcription factor, has been known to regulate the specification of the stem cell niche and ground tissue identity in the root meristem in a non-cell-autonomous manner. However, the role of SHR in the root vasculature is relatively overlooked, despite localization of its mRNA and protein in the stele. Here, we investigated the role of SHR in the vascular system of the primary root using a reverse genetic approach and detailed phenotypic analysis. A novel, loss-of-function null mutant, shr-6, was isolated in the Columbia background, and vascular patterning was characterized in detail. Our results reveal that shr mutants have developmental defects in both protophloem and protoxylem elements. Our study also suggests that SHR plays a central role in the root vascular system to control patterning processes, possibly regulated by longitudinal and radial signals.

Large-scale analysis of the GRAS gene family in Arabidopsis thaliana.

GRAS proteins belong to a plant-specific transcription factor family. Currently, 33 GRAS members including a putative expressed pseudogene have been identified in the Arabidopsis genome. With a reverse genetic approach, we have constructed a "phenome-ready unimutant collection" of the GRAS genes in Arabidopsis thaliana. Of this collection, we focused on loss-of-function mutations in 23 novel GRAS members. Under standard conditions, homozygous mutants have no obvious morphological phenotypes compared with those of wild-type plants. Expression analysis of GRAS genes using quantitative real-time RT-PCR (qRT-PCR), microarray data mining, and promoter::GUS reporter fusions revealed their tissue-specific expression patterns. Our analysis of protein-protein interaction and subcellular localization of individual GRAS members indicated their roles as transcription regulators. In our yeast two-hybrid (Y2H) assay, we confirmed the protein-protein interaction between SHORT-ROOT (SHR) and SCARECROW (SCR). Furthermore, we identified a new SHR-interacting protein, SCARECROW-LIKE23 (SCL23), which is the most closely related to SCR. Our large-scale analysis provides a comprehensive evaluation on the Arabidopsis GRAS members, and also our phenome-ready unimutant collection will be a useful resource to better understand individual GRAS proteins that play diverse roles in plant growth and development.