Division Of Guard Mother Cells Research Articles

An R2R3 MYB transcription factor PsFLP regulates the symmetric division of guard mother cells during stomatal development in Pisum sativum.

MYB transcriptional regulators belong to one of the most significant transcription factors families in plants, among which R2R3-MYB transcription factors are involved in plant growth and development, hormone signal transduction, and stress response. Two R2R3-MYB transcription factors, FLP and its paralogous AtMYB88, redundantly regulate the symmetrical division of guard mother cells (GMCs), and abiotic stress response in Arabidopsis thaliana. Only one orthologue gene of FLP was identified in pea (Pisum sativum FLP; PsFLP). In this study, we explored the gene function of PsFLP by virus-induced gene silencing (VIGS) technology. The phenotypic analysis displayed that the silencing of PsFLP expression led to the abnormal development of stomata and the emergence of multiple guard cells tightly united. In addition, the abnormal stomata of flp could be fully rescued by PsFLP driven by the FLP promoter. In conclusion, the results showed that PsFLP plays a conservative negative role in regulating the symmetric division of GMC during stomatal development. Based on real-time quantitative PCR, the relative expressions of AAO3, NCED3, and SnRK2.3 significantly increased in the flp pFLP::PsFLP plants compared to mutant, indicating that PsFLP might be involved in drought stress response. Thus, PsFLP regulates the genes related to cell cycle division during the stomatal development of peas and participates in response to drought stress. The study provides a basis for further research on its function and application in leguminous crop breeding.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

A Rice R2R3-Type MYB Transcription Factor OsFLP Positively Regulates Drought Stress Response via OsNAC

Abiotic stresses adversely affect plant growth and the yield of crops worldwide. R2R3-MYB transcriptional factors have been found to be vital for plants to confer stress response. In Arabidopsis, FOUR LIPS (FLP, MYB124) and its paralogous MYB88 function redundantly regulated the symmetric division of guard mother cells (GMCs) and abiotic stress response. Here, OsFLP was identified as an R2R3-MYB transcriptional activator and localized in the nucleus. OsFLP was transiently induced by drought, salt stress and abscisic acid (ABA). Overexpression of OsFLP showed enhanced tolerance to drought and salt stresses. The stomatal density in OsFLP-OE plants was not changed, whereas the stomatal closure was sensitive to ABA treatment compared to wild-type plants. In contrast, OsFLP-RNAi plants had abnormal stomata and were sensitive to drought. Moreover, the transcripts of stomatal closure-related genes DST and peroxidase 24 precursor, which are identified as downstream of OsNAC1, were inhibited in OsFLP-RNAi plants. The yeast-one-hybrid assay indicated that OsFLP can specifically bind and positively regulate OsNAC1 and OsNAC6. Meanwhile, stress response genes, such as OsLEA3 and OsDREB2A, were up-regulated in OsFLP-OE plants. These findings suggested that OsFLP positively participates in drought stress, mainly through regulating regulators’ transcripts of OsNAC1 and OsNAC6.

OsBC1L1 and OsBC1L8 function in stomatal development in rice

Stomata that are bordered by pairs of guard cells are specialized for regulating gas exchange and transpiration in plants. The stomatal morphology of grass is unique, characterized by two dumbbell-shaped guard cells flanked by two lateral subsidiary cells. This morphology and developmental pattern enable grass stomata to respond to environmental signals efficiently. In this study, we demonstrated that knockout either OsBC1L1 or OsBC1L8, two close homologs of OsBC1L family causes no discernible defects in rice stomatal development, however, the double knockout mutant osbc1l1 osbc1l8 exhibits excess stomatal production and stomatal clustering. OsBC1L1 overexpression also causes abnormal stomatal patterning in rice. Moreover, osbc1l1 osbc1l8 has many defective stomata complexes with only one subsidiary cell. The expression of OsSPCH2 and OsFAMA, two genes key to stomatal development is both down-regulated in osbc1l1 osbc1l8. In contrast, overexpressing OsBC1L1 suppresses only the expression of OsSPCH2. Both OsBC1L1 and OsBC1L8 could be detected to be localized at the cell plate and plasma membrane during cell division of guard mother cells and subsidiary mother cells. Taken together, these results suggest that OsBC1L1 and OsBC1L8 play essential roles in the development of rice stomatal complex likely through their involvement in cell reproduction.

Arabidopsis thaliana Leaf Epidermal Guard Cells: A Model for Studying Chloroplast Proliferation and Partitioning in Plants.

The existence of numerous chloroplasts in photosynthetic cells is a general feature of plants. Chloroplast biogenesis and inheritance involve two distinct mechanisms: proliferation of chloroplasts by binary fission and partitioning of chloroplasts into daughter cells during cell division. The mechanism of chloroplast number coordination in a given cell type is a fundamental question. Stomatal guard cells (GCs) in the plant shoot epidermis generally contain several to tens of chloroplasts per cell. Thus far, chloroplast number at the stomatal (GC pair) level has generally been used as a convenient marker for identifying hybrid species or estimating the ploidy level of a given plant tissue. Here, we report that Arabidopsis thaliana leaf GCs represent a useful system for investigating the unexploited aspects of chloroplast number control in plant cells. In contrast to a general notion based on analyses of leaf mesophyll chloroplasts, a small difference was detected in the GC chloroplast number among three Arabidopsis ecotypes (Columbia, Landsberg erecta, and Wassilewskija). Fluorescence microscopy often detected dividing GC chloroplasts with the FtsZ1 ring not only at the early stage of leaf expansion but also at the late stage. Compensatory chloroplast expansion, a phenomenon well documented in leaf mesophyll cells of chloroplast division mutants and transgenic plants, could take place between paired GCs in wild-type leaves. Furthermore, modest chloroplast number per GC as well as symmetric division of guard mother cells for GC formation suggests that Arabidopsis GCs would facilitate the analysis of chloroplast partitioning, based on chloroplast counting at the individual cell level.

BZU2/ZmMUTE controls symmetrical division of guard mother cell and specifies neighbor cell fate in maize.

Intercellular communication in adjacent cell layers determines cell fate and polarity, thus orchestrating tissue specification and differentiation. Here we use the maize stomatal apparatus as a model to investigate cell fate determination. Mutations in ZmBZU2 (bizui2, bzu2) confer a complete absence of subsidiary cells (SCs) and normal guard cells (GCs), leading to failure of formation of mature stomatal complexes. Nuclear polarization and actin accumulation at the interface between subsidiary mother cells (SMCs) and guard mother cells (GMCs), an essential pre-requisite for asymmetric cell division, did not occur in Zmbzu2 mutants. ZmBZU2 encodes a basic helix-loop-helix (bHLH) transcription factor, which is an ortholog of AtMUTE in Arabidopsis (BZU2/ZmMUTE). We found that a number of genes implicated in stomatal development are transcriptionally regulated by BZU2/ZmMUTE. In particular, BZU2/ZmMUTE directly binds to the promoters of PAN1 and PAN2, two early regulators of protodermal cell fate and SMC polarization, consistent with the low levels of transcription of these genes observed in bzu2-1 mutants. BZU2/ZmMUTE has the cell-to-cell mobility characteristic similar to that of BdMUTE in Brachypodium distachyon. Unexpectedly, BZU2/ZmMUTE is expressed in GMC from the asymmetric division stage to the GMC division stage, and especially in the SMC establishment stage. Taken together, these data imply that BZU2/ZmMUTE is required for early events in SMC polarization and differentiation as well as for the last symmetrical division of GMCs to produce the two GCs, and is a master determinant of the cell fate of its neighbors through cell-to-cell communication.

Aminocyclopropane-1-carboxylic acid is a key regulator of guard mother cell terminal division in Arabidopsis thaliana.

Stomata have a critical function in the exchange of gases and water vapor between plants and their environment. Stomatal development is under the rigorous control of many regulators. The last step of development is the terminal division of guard mother cells (GMC) into two guard cells (GC). It is still unclear how the symmetric division of GMCs is regulated. Here, we show that the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) is required for the symmetric division of GMCs into GCs in Arabidopsis. Exogenous application of the ACC biosynthesis inhibitor aminoethoxyvinylglycine (AVG) induced the formation of single guard cells (SGCs). Correspondingly, an acs octuple-mutant with extremely low endogenous ACC also developed SGCs, and exogenous ACC dramatically decreased the number of SGCs in this mutant whereas exogenous ethephon (which is gradually converted into ethylene) had no effect. Furthermore, neither blocking of endogenous ethylene synthesis nor disruption of ethylene signaling transduction could induce the production of SGCs. Further investigation indicated that ACC promoted the division of GMCs in fama-1 and flp-1myb88 mutants whereas AVG inhibited it. Moreover, ACC positively regulated the expression of CDKB1;1 and CYCA2;3 in the fama-1 and flp-1myb88 mutants. The SGC number was not affected by ACC or AVG in cdkb1;11;2 and cyca2;234 mutants. Taken together, the results demonstrate that ACC itself, but not ethylene, positively modulates the symmetric division of GMCs in a manner that is dependent on CDKB1s and CYCA2s.

A2-type cyclin is required for the asymmetric entry division in rice stomatal development.

In rice, and other major cereal grass crops, stomata are arranged in linear files parallel to the long growth axis of leaves. Each stomatal unit comprises two dumbbell-shaped guard cells flanked by two subsidiary cells. These morphological and developmental characteristics enable grass stomata to respond to environmental changes more efficiently. Cyclin-dependent kinases (CDKs) and their cyclin partners co-ordinate cell proliferation and differentiation during the development of multicellular organisms. In contrast to animals, plants have many more types and members of cyclins. In Arabidopsis, four A2-type cyclins (CYCA2s) function redundantly in regulating CDKB1 activity to promote the asymmetric division for stomatal initiation and the symmetric division of guard mother cells (GMCs). In this study, we examine the function of the single A2-type cyclin in rice, OsCYCA2;1, as well the single B1-type CDK, OsCDKB1;1. Cross-species complementation tests demonstrated that OsCYCA2;1 and OsCDKB1;1 could complement the defective stomatal phenotypes of Arabidopsis cyca2 and cdkb1 mutants, but also could suppress DNA endoduplication and cell enlargement. The early asymmetric divisions that establish the stomatal lineages are often missing within the stomatal cell files of OsCYCA2;1-RNAi rice transgenic lines, leading to a significantly reduced stomatal production. However, GMC divisions are not disrupted either in OsCYCA2;1-RNAi or in OsCDKB1;1-RNAi rice transgenic lines as expected. Our results demonstrate a conserved but diverged function and behavior of rice A2-type cyclins, which might be associated with the distinct stomatal development pathways between rice and Arabidopsis.

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.

Requirement for A-type cyclin-dependent kinase and cyclins for the terminal division in the stomatal lineage of Arabidopsis

The Arabidopsis stoma is a specialized epidermal valve made up of a pair of guard cells around a pore whose aperture controls gas exchange between the shoot and atmosphere. Guard cells (GCs) are produced by a symmetric division of guard mother cells (GMCs). The R2R3-MYB transcription factor FOUR LIPS (FLP) and its paralogue MYB88 restrict the division of a GMC to one. Previously, the upstream regions of several core cell cycle genes were identified as the direct targets of FLP/MYB88, including the B-type cyclin-dependent kinase CDKB1;1 and A2-type cyclin CYCA2;3. Here we show that CDKA;1 is also an immediate direct target of FLP/MYB88 through the binding to cis-regulatory elements in the CDKA;1 promoter region. CDKA;1 activity is required not only for normal GMC divisions but also for the excessive cell overproliferation in flp myb88 mutant GMCs. The impaired defects of GMC division in cdkb1;1 1;2 mutants could be partially rescued by a stage-specific expression of CDKA;1. Although targeted overexpression of CDKA;1 does not affect stomatal development, ectopic expression of the D3-type cyclin CYCD3;2 induces GC subdivision, resulting in a stoma with 3-4 GCs instead of the normal two. Co-overexpression of CDKA;1 with CYCD3;2, but not with CYCA2;3, confers a synergistic effect with respect to GC subdivision. Thus, in addition to a role in stomatal formative asymmetric divisions at early developmental stages, CDKA;1 is needed in triggering GMC symmetric divisions at the late stage of stomatal development. However, timely down-regulation of CDKA;1-CYCD3 activity is required for restriction of GC proliferation.

Variable timing of developmental progression in the stomatal pathway in Arabidopsis cotyledons.

• Stomatal production depends on the rates of precursor cell formation (e.g. the formation of meristemoids by asymmetric division), and of developmental progression (e.g. the division of guard mother cells). It is not known whether these rates follow steady-state kinetics or are variable. • The timing of development was scored in Arabidopsis cotyledons in fixed and living tissue using the dental resin impression method. • Cotyledons exhibited much less of a longitudinal gradient in stomatal formation than leaves. The timing of the appearance of stomatal and precursor cells during cotyledon development varied between individual plants. Precursor cell formation ceased much earlier in the adaxial than in the abaxial epidermis. Meristemoids are precursors that form guard mother cells. The ratio of these cell types varied greatly in different plants as well as in the same epidermis through time. There was also considerable variability in the duration of the meristemoid stage between individual cell lineages. • Precursor cell production follows non-steady-state kinetics. Early steps in the pathway are not necessarily synchronized, but later steps, such as the conversion of meristemoids to guard mother cells, sometimes appear to be coordinated.

Microtubule organization during development of stomatal complexes inLolium rigidum

Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.

Structure and development of normal and abnormal stomata in the seedlings of some Cruciferae

The structure and ontogeny of normal and abnormal stomata in the seedlings of 16 species of the Cruciferae have been presented. The mature stomata are anomocytic, paracytic, anisocytic, helicocytic and with a single subsidiary cell. The ontogeny of the anisocytic, paracytic, helicocytic and stoma with a single subsidiary cell conforms to the syndetocheilic or mesogenous, while that of anomocytic to haplocheilic or perigenous type. Several types of aberrant stomatal formations such as: single guard cell; contiguoas single-guard cells; contiguous stomatal; degeneration of guard cell/s; persistert stomatal cell; amitotic division of guard mother cell nucleus followed by pore formation; stoma with double pores; binucleate guard cell; division of guard cell; incomplete stoma with unequal guard cells; cytoplasmic connection; ring-shaped division of guard mother cell and uncommon wall thickening have been noticed. Aberrant stomatal developments are naturally occurring and not induced.