Regulatory Role of Phytohormones in Maintaining Stem Cells and Boundaries of Stem Cell Niches.

Plants are multicellular organism composed of different types of cells. These all kinds of cells are formed from pluripotent stem cells present at different positions in plant called stem cell niches. All these stem cell niches and their boundaries are maintained by complex regulatory mechanism at molecular level involving different genes, cofactors, and phytohormones. In this chapter, we discussed the regulatory mechanism and models of stem cell maintenance, specifying their boundaries at different stem cell niches.

Coordination of tissue cell polarity by auxin transport and signaling.

Plants coordinate the polarity of hundreds of cells during vein formation, but how they do so is unclear. The prevailing hypothesis proposes that GNOM, a regulator of membrane trafficking, positions PIN-FORMED auxin transporters to the correct side of the plasma membrane; the resulting cell-to-cell, polar transport of auxin would coordinate tissue cell polarity and induce vein formation. Contrary to predictions of the hypothesis, we find that vein formation occurs in the absence of PIN-FORMED or any other intercellular auxin-transporter; that the residual auxin-transport-independent vein-patterning activity relies on auxin signaling; and that a GNOM-dependent signal acts upstream of both auxin transport and signaling to coordinate tissue cell polarity and induce vein formation. Our results reveal synergism between auxin transport and signaling, and their unsuspected control by GNOM in the coordination of tissue cell polarity during vein patterning, one of the most informative expressions of tissue cell polarization in plants.

How plants grow up.

A plant's lateral structures, such as leaves, branches and flowers, literally hinge on the shoot axis, making its integrity and growth fundamental to plant form. In all plants, subapical proliferation within the shoot tip displaces cells downward to extrude the cylindrical stem. Following the transition to flowering, many plants show extensive axial elongation associated with increased subapical proliferation and expansion. However, the cereal grasses also elongate their stems, called culms, due to activity within detached intercalary meristems which displaces cells upward, elevating the grain-bearing inflorescence. Variation in culm length within species is especially relevant to cereal crops, as demonstrated by the high-yielding semi-dwarfed cereals of the Green Revolution. Although previously understudied, recent renewed interest the regulation of subapical and intercalary growth suggests that control of cell division planes, boundary formation and temporal dynamics of differentiation, are likely critical mechanisms coordinating axial growth and development in plants.

Bundle sheath extensions affect leaf structural and physiological plasticity in response to irradiance.

Coordination between structural and physiological traits is key to plants' responses to environmental fluctuations. In heterobaric leaves, bundle sheath extensions (BSEs) increase photosynthetic performance (light-saturated rates of photosynthesis, Amax ) and water transport capacity (leaf hydraulic conductance, Kleaf ). However, it is not clear how BSEs affect these and other leaf developmental and physiological parameters in response to environmental conditions. The obscuravenosa (obv) mutation, found in many commercial tomato varieties, leads to absence of BSEs. We examined structural and physiological traits of tomato heterobaric and homobaric (obv) near-isogenic lines grown at two different irradiance levels. Kleaf , minor vein density, and stomatal pore area index decreased with shading in heterobaric but not in homobaric leaves, which show similarly lower values in both conditions. Homobaric plants, on the other hand, showed increased Amax , leaf intercellular air spaces, and mesophyll surface area exposed to intercellular airspace (Smes ) in comparison with heterobaric plants when both were grown in the shade. BSEs further affected carbon isotope discrimination, a proxy for long-term water-use efficiency. BSEs confer plasticity in traits related to leaf structure and function in response to irradiance levels and might act as a hub integrating leaf structure, photosynthetic function, and water supply and demand.

Endocytosis acts as transport pathway in wood.

In trees, dead and living cells of secondary xylem (wood) function collectively, rendering cell-to-cell communication challenging. Water and solutes are transported over long distances from the roots to the above-ground organs via vessels, the main component of wood, and then radially over short distances to the neighboring cells. This enables proper functioning of trees and integrates whole-plant activity. In this study, tracer loading, immunolocalization experiments and inhibitor assays were used to decipher the mechanisms enabling transport in wood of Acer pseudoplatanus (maple), Fraxinus excelsior (ash) and Populus tremula × tremuloides (poplar) trees. We show that tracer uptake from dead water-conducting vessels, elements of the apoplasm, to living vessel-associated cells (VACs) of the xylem parenchyma of the symplasm system proceeds via the endocytic pathway, including clathrin-mediated and clathrin-independent processes. These findings enhance our understanding of the transport pathways in complex wood tissue, providing experimental evidence of the involvement of VACs and endocytosis in radial uptake from vessels.

Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity.

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (gbs ) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on gbs depending on the technique used to calculate gbs Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of gbs with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of gbs It is intriguing to speculate that PD development is responsive to changes in C4 photosynthetic flux.

Creating Leaf Cell Suspensions for Characterization of Mesophyll and Bundle Sheath Cellular Features.

Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we provide a method for the preparation of leaf cell suspensions that improves upon a previous method by optimizing cellular preservation and cell separation. This technique is accessible to all researchers and amenable for use with all plant species. The leaf suspensions can be used for imaging chloroplast features within a cell that are important for photosynthesis such as size, number, and distribution. However, we also provide examples to illustrate how the cells in the suspensions can be easily stained to image other features, for example pit fields where plasmodesmata are located and organelles such as mitochondria, to improve our understanding of traits that are important for photosynthetic physiology.

Gain-of-function mutation of AtDICE1, encoding a putative endoplasmic reticulum-localized membrane protein, causes defects in anisotropic cell elongation by disturbing cell wall integrity in Arabidopsis.

Background and Aims: Anisotropic cell elongation depends on cell wall relaxation and cellulose microfibril arrangement. The aim of this study was to characterize the molecular function of AtDICE1 encoding a novel transmembrane protein involved in anisotropic cell elongation in Arabidopsis. Methods: Phenotypic characterizations of transgenic Arabidopsis plants mis-regulating AtDICE1 expression with different pharmacological treatments were made, and biochemical, cell biological and transcriptome analyses were performed. Key Results: Upregulation of AtDICE1 in Arabidopsis (35S::AtDICE1) resulted in severe dwarfism, probably caused by defects in anisotropic cell elongation. Epidermal cell swelling was evident in all tissues, and abnormal secondary wall thickenings were observed in pith cells of stems. These phenotypes were reproduced not only by inducible expression of AtDICE1 but also by overexpression of its poplar homologue in Arabidopsis. RNA interference suppression lines of AtDICE1 resulted in no observable phenotypic changes. Interestingly, wild-type plants treated with isoxaben, a cellulose biosynthesis inhibitor, phenocopied the 35S::AtDICE1 plants, suggesting that cellulose biosynthesis was compromised in the 35S::AtDICE1 plants. Indeed, disturbed cortical microtubule arrangements in 35S::AtDICE1/GFP-TuA6 plants were observed, and the cellulose content was significantly reduced in 35S::AtDICE1 plants. A promoter::GUS analysis showed that AtDICE1 is mainly expressed in vascular tissue, and transient expression of GFP:AtDICE1 in tobacco suggests that AtDICE1 is probably localized in the endoplasmic reticulum (ER). In addition, the external N-terminal conserved domain of AtDICE1 was found to be necessary for AtDICE1 function. Whole transcriptome analyses of 35S::AtDICE1 revealed that many genes involved in cell wall modification and stress/defence responses were mis-regulated. Conclusions: AtDICE1, a novel ER-localized transmembrane protein, may contribute to anisotropic cell elongation in the formation of vascular tissue by affecting cellulose biosynthesis.

Organization of Vascular Cells in the Haustorium of the Parasitic Flowering Plant Cuscuta japonica.

The stem parasite dodder, Cuscuta japonica, has evolved a specialized root-like organ, the haustorium, which is differentiated from the stem. In order to take up water and nutrients, C. japonica reprograms haustorial cells to vascular cells, connecting the host's vascular system to its own. However, little is known about vascular differentiation in haustoria. In this study, we first confirmed the temporal and spatial expression profiles of vascular cell type-specific genes, CjAPL, CjSEOR1, CjWOX4 and CjTED7, to examine whether phloem companion cells, developing sieve elements, procambial cells and differentiating xylem cells, respectively, are present in the haustoria. CjAPL and CjSEOR1 decreased, and CjWOX4 showed a transient increase before the onset of xylem vessel formation, and then decreased. CjTED7 increased coincidentally with xylem vessel formation. In situ hybridization demonstrated that CjWOX4-expressing cells and phloem-conducting cells are in close proximity, and occupied a domain distinguishable from xylem vessels, suggesting differentiation of a phloem/procambial domain and a xylem domain in the haustorium. Secondly, expression of regulatory genes that are involved in determination of the fate of procambial cells was investigated. Expression patterns of CjCLE41, CjGSK3 and CjBES1suggested that TDIF-TDR-GSK3-mediated signaling is activated in haustoria. The natural antisense transcript of CjCLE41 was detected in haustoria, implying the sense regulation of CjCLE41. Expression profiles of the regulatory genes, combined with those of cell type-specific marker genes, suggest that reprogramming of haustorial cells to vascular cells is regulated in a way that allows the immediate formation of xylem vessels by alleviating inhibition of xylem differentiation.

OCTOPUS-LIKE 2, a novel player in Arabidopsis root and vascular development, reveals a key role for OCTOPUS family genes in root metaphloem sieve tube differentiation.

Protophloem and metaphloem sieve tubes are essential for transporting carbohydrates and signalling molecules towards sink tissues. OCTOPUS (OPS) was previously identified as an important regulator of protophloem differentiation in Arabidopsis roots. Here, we investigated the role of OCTOPUS-LIKE 2 (OPL2), a gene homologous to OPS. OPL2 expression patterns were analysed, and functional equivalence of OPS and OPL2 was tested. Mutant and double mutant phenotypes were investigated. OPS and OPL2 displayed overlapping expression patterns and a high degree of functional overlap. A mutation in OPL2 revealed redundant functions of OPS and OPL2 in developmental processes in which OPS was known to play a role, notably cotyledon vascular patterning and protophloem development. Moreover, we also uncovered redundant roles for OPS and OPL2 in leaf vascular patterning and, most interestingly, metaphloem sieve tube differentiation. Our results reveal a novel OPS-like protein that, together with OPS, is an important regulator of vascular patterning, root growth and phloem development. OPS and OPL2 are the first genes identified that play a role in metaphloem sieve tube differentiation.

Perturbing phosphoinositide homeostasis oppositely affects vascular differentiation in Arabidopsis thaliana roots.

The plant vascular network consists of specialized phloem and xylem elements that undergo two distinct morphogenetic developmental programs to become transport-functional units. Whereas vacuolar rupture is a determinant step in protoxylem differentiation, protophloem elements never form a big central vacuole. Here, we show that a genetic disturbance of phosphatidylinositol 4,5-bis-phosphate [PtdIns(4,5)P2] homeostasis rewires cell trafficking towards the vacuole in Arabidopsis thaliana roots. Consequently, an enhanced phosphoinositide-mediated vacuolar biogenesis correlates with premature programmed cell death (PCD) and secondary cell wall elaboration in xylem cells. By contrast, vacuolar fusion events in protophloem cells trigger the abnormal formation of big vacuoles, preventing cell clearance and tissue functionality. Removal of the inositol 5' phosphatase COTYLEDON VASCULAR PATTERN 2 from the plasma membrane (PM) by brefeldin A (BFA) treatment increases PtdIns(4,5)P2 content at the PM and disrupts protophloem continuity. Conversely, BFA application abolishes vacuolar fusion events in xylem tissue without preventing PCD, suggesting the existence of additional PtdIns(4,5)P2-dependent cell death mechanisms. Overall, our data indicate that tight PM phosphoinositide homeostasis is required to modulate intracellular trafficking contributing to oppositely regulate vascular differentiation.

Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato.

This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6-8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

Morphological analysis of vessel elements for systematic study of three Zingiberaceae tribes.

Zingiberaceae containing over 1,000 species that are divided into four subfamilies and six tribes. In recent decades, there has been an increase in the number of studies about vessel elements in families of monocotyledon. However, there are still few studies of Zingiberaceae tribes. This study aims to establish systematic significance of studying vessel elements in two subfamilies and three tribes of Zingiberaceae. The vegetative organs of 33 species processed were analysed by light and scanning electron microscopy and Principal Component Analysis was used to elucidate genera boundaries. Characteristics of vessel elements, such as the type of perforation plate, the number of bars and type of parietal thickening, are proved to be important for establishing the relationship among taxa. Scalariform perforation plate and the scalariform parietal thickening are frequent in Zingiberaceae and may be a plesiomorphic condition for this taxon. In the Principal Component Analysis, the most significant characters of the vessel elements were: simple perforation plates and partially pitted parietal thickening, found only in Alpinieae tribe, and 40 or more bars composing the plate in Elettariopsis curtisii, Renealmia chrysotricha, Zingiber spectabile, Z. officinale, Curcuma and Globba species. Vessel elements characters of 18 species of Alpinieae, Zingibereae and Globbeae were first described in this work.

Experimental and Theoretical Methods to Approach the Study of Vascular Patterning in the Plant Shoot.

The plant vascular system provides transport and mechanical support functions that are essential for suitable plant growth and development. In Arabidopsis thaliana (Arabidopsis), the vascular tissues at the shoot inflorescence stems are disposed in organized vascular bundles. The vascular patterning emergence and development within the shoot inflorescence stems is under the control of plant growth regulators (De Rybel et al., Nat Rev Mol Cell Biol 17:30-40, 2016; Caño-Delgado et al., Annu Rev Cell Dev Biol 26:605-637, 2010). By using a combined approach of experimental methods for vascular tissues visualization and quantification together with theoretical methods through mathematical and computational modeling, we have reported that auxin transport and brassinosteroid signaling play complementary roles in the formation of the periodic vascular patterning in the shoot (Ibañes et al., Proc Natl Acad Sci U S A 106:13630-13635, 2009; Fàbregas et al., Plant Signal Behav 5:903-906, 2010; Fàbregas et al., PLoS Genet 11:e1005183, 2015). Here, we report the methodology for the interdisciplinary analysis of the shoot vascular patterning in the plant model Arabidopsis into a handle procedure for visualization, quantification, data analysis, and modeling implementation.

Vascular Morphodynamics During Secondary Growth.

Quantification of vascular morphodynamics during secondary growth has been hampered by the scale of the process. Even in the tiny model plant Arabidopsis thaliana, the xylem can include more than 2000 cells in a single cross section, rendering manual counting impractical. Moreover, due to its deep location, xylem is an inaccessible tissue, limiting live imaging. A novel method to visualize and measure secondary growth progression has been proposed: "the Quantitative Histology" approach. This method is based on a detailed anatomical atlas, and image segmentation coupled with machine learning to automatically extract cell shapes and identify cell type. Here we present a new version of this approach, with a user-friendly interface implemented in the open source software LithoGraphX.

Using CellProfiler to Analyze and Quantify Vascular Morphology.

Computational programs can be used in place of time-consuming, error-prone manual data collection. CellProfiler is a free, open source program that allows researchers to automate image analysis and collect large amounts of phenotypic data relatively easily. Here, we describe how to adapt CellProfiler to analyze cross sections of xylem tissue and use it to gather a variety of information on traits such as cell size, shape, and number. We provide step-by-step instructions to create a typical CellProfiler analysis pipeline, alongside explanations of important modules, options and parameters available to the user.

Exploring key cellular processes and candidate genes regulating the primary thickening growth of Moso underground shoots.

The primary thickening growth of Moso (Phyllostachys edulis) underground shoots largely determines the culm circumference. However, its developmental mechanisms remain largely unknown. Using an integrated anatomy, mathematics and genomics approach, we systematically studied cellular and molecular mechanisms underlying the growth of Moso underground shoots. We discovered that the growth displayed a spiral pattern and pith played an important role in promoting the primary thickening process of Moso underground shoots and driving the evolution of culms with different sizes among different bamboo species. Different with model plants, the shoot apical meristem (SAM) of Moso is composed of six layers of cells. Comparative transcriptome analysis identified a large number of genes related to the vascular tissue formation that were significantly upregulated in a thick wall variant with narrow pith cavity, mildly spiral growth, and flat and enlarged SAM, including those related to plant hormones and those involved in cell wall development. These results provide a systematic perspective on the primary thickening growth of Moso underground shoots, and support a plausible mechanism resulting in the narrow pith cavity, weak spiral growth but increased vascular bundle of the thick wall Moso.

The phylogenetic significance of leaf anatomical traits of southern African Oxalis.

BACKGROUND: The southern African Oxalis radiation is extremely morphologically variable. Despite recent progress in the phylogenetics of the genus, there are few morphological synapomorphies supporting DNA-based clades. Leaflet anatomy can provide an understudied and potentially valuable source of information on the evolutionary history and systematics of this lineage. Fifty-nine leaflet anatomical traits of 109 southern African Oxalis species were assessed in search of phylogenetically significant characters that delineate clades. RESULTS: A combination of 6 leaflet anatomical traits (stomatal position, adaxial epidermal cells, abaxial epidermal cells, mesophyll, sheath around vascular tissue, degree of leaflet conduplication) clearly support various clades defined by previous DNA-based phylogenetic work. Other, mostly continuous leaflet anatomical traits were highly variable and showed less phylogenetic pattern. CONCLUSIONS: Major and unexpected findings include the transition from ancestral hypostomatic leaflets to adaxially-located stomata in the vast majority of southern African Oxalis, the loss of semi-swollen AB epidermal cells and the gain of swollen adaxial and abaxial epidermal cells in selected clades, and multiple changes from ancestral bifacial mesophyll to isobilateral or homogenous mesophyll types. The information gathered in this study will aid in the taxonomic revision of this speciose member of the Greater Cape Floristic Region and provide a basis for future hypotheses regarding its radiation.

Modelling the vasculature of the stem of Cyperus involucratus Rottb.: evidence for three patterns of vascular bundles.

MAIN CONCLUSION: Three independent patterns of vein formation in Cyperus involucratus Rottb. were identified based on rare spontaneous interruptions of scape vein development. A number of developmental anomalies of vascular bundles in Cyperus involucratus Rottb. were identified and they include "turnabout", "absent", "twins", "doublet", amphivasal and various stages of "arrested". These were used to develop a computer program to explain the three vasculature patterns of the scape of (a) ordered deployment of vascular bundles, (b) arrangement of tissues within vascular bundles and (c) orientation of vascular bundles with respect to stem edge. The computer model is a cell-by-cell determination of cell types and facet states.