Dynamic analysis of epidermal cell divisions identifies specific roles for COP10 in Arabidopsis stomatal lineage development

Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.     

MicroRNA-mediated regulation of stomatal development in Arabidopsis

The proper number and distribution of stomata are essential for the efficient exchange of gases between the atmosphere and the aerial parts of plants. We show that the density and development of stomatal complexes on the epidermis of Arabidopsis thaliana leaves depend, in part, on the microRNA-mediated regulation of Agamous-like16 (AGL16), which is a member of the MADS box protein family. AGL16 mRNA is targeted for sequence-specific degradation by miR824, a recently evolved microRNA conserved in the Brassicaceae and encoded at a single genetic locus. Primary stomatal complexes can give rise to higher-order complexes derived from satellite meristemoids. Expression of a miR824-resistant AGL16 mRNA, but not the wild-type AGL16 mRNA, in transgenic plants increased the incidence of stomata in higher-order complexes. By contrast, reduced expression of AGL16 mRNA in the agl16-1 deficiency mutant and in transgenic lines overexpressing miR824 decreased the incidence of stomata in higher-order complexes. These findings and the nonoverlapping patterns of AGL16 mRNA and miR824 localization led us to propose that the miR824/AGL16 pathway functions in the satellite meristemoid lineage of stomatal development.     

BASL and EPF2 act independently to regulate asymmetric divisions during stomatal development

The initiation of stomatal development in the developing Arabidopsis epidermis is characterized by an asymmetric ‘entry’ division in which a small cell, known as a meristemoid, and a larger daughter cell is formed. The meristemoid may undergo further asymmetric divisions, regenerating a meristemoid each time, before differentiating into a guard mother cell which divides symmetrically to form a pair of guard cells surrounding a stomatal pore. Recently EPF2 and BASL have emerged as regulators of these asymmetric divisions and here we present results indicating that these two factors operate independently to control stomatal developmentKey words: stomata, development, meristemoids, asymmetric cell division, leaf epidermis, cell polarity, peptide signal     

AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades have been implicated in regulating various aspects of plant development, including somatic cytokinesis. The evolution of expanded plant MAPK gene families has enabled the diversification of potential MAPK cascades, but functionally overlapping components are also well documented. Here we report that Arabidopsis MPK4, an MAPK that was previously described as a regulator of disease resistance, can interact with and be phosphorylated by the cytokinesis-related MAP kinase kinase, AtMKK6. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to form a normal intersporal callose wall after male meiosis, and thus cannot complete meiotic cytokinesis. Nevertheless, the multinucleate mpk4 microspores subsequently proceed through mitotic cytokinesis, resulting in enlarged mature pollen grains that possess increased sets of the tricellular structure. This pollen development phenotype is reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants, and protein-protein interaction analysis defines a putative signalling module linking AtNACK2/TES/STUD, AtANP3, AtMKK6 and AtMPK4 together as a cascade that facilitates male-specific meiotic cytokinesis in Arabidopsis.     

FRL1 is required for petal and sepal development in Arabidopsis

A novel flower mutant, frl1 (frill 1) was isolated in Arabidopsis thaliana. The frl1 mutant has serrated petals and sepals but the other floral and vegetative organs appear to be normal. To analyse the role of the FRL1 gene, morphological, cytological and double mutant analyses were carried out. The frl1 flower had broader petals and sepals as compared with the wild-type. The distal region of frl1 petals contained fewer epidermal cells but their size was variable and generally larger than that in the wild-type. However, no significant difference was found in the basal region. Observations of the early petal development revealed that the morphology of the developing frl1 petal was normal until the middle of stage 9, but the frl1 phenotype became apparent in stages later than 10. Furthermore, larger nuclei with varied sizes were observed in the distal region of frl1 petals, but not in this region in wild-type petals. This strongly suggests that abnormal endo-reduplication had occurred. These observations indicate that the frl1 mutation affects the number of cell divisions and the subsequent cell expansion during the late stage of petal lamina formation, and that FRL1 might be maintaining the mitotic state or suppressing the transition to the endo-reduplication cycle. Double mutants with the homeotic mutants apetala3-1 and agamous showed additive phenotypes. Ectopic petals in the third whorl of fr11 ag flowers were serrated, indicating that the FRL1 gene acts in petal and sepal development in an organ-specific manner.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

SOS4, a pyridoxal kinase gene,is required for root hair development in Arabidopsis

Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.     

A lectin receptor-like kinase is required for pollen development in Arabidopsis

Lectin receptor-like kinases (Lectin RLKs) are a large family of receptor-like kinases with an extracellular legume lectin-like domain. There are approximately 45 such receptor kinases in Arabidopsis thaliana. Surprisingly, although receptor-like kinases in general are well investigated in Arabidopsis, relatively little is known about the functions of members of the Lectin RLK family. A number of studies implicated members of this family in various functions, such as disease resistance, stress responses, hormone signaling, and legume-rhizobium symbiosis. Our current work demonstrated that mutation in one Lectin RLK gene led to male sterility in Arabidopsis. The sterility was due to defects in pollen development. Pollen development proceeded normally in the mutant until anther stage 8. After that, all pollen grains deformed and collapsed. Mature pollen grains were much smaller than wild-type pollen grains, glued together, and totally collapsed. Therefore, the mutant was named sgc, standing for small, glued-together, and collapsed pollen mutant. The mutant phenotype appeared to be caused by an unidentified sporophytic defect due to the mutation. As revealed by analysis of the promoter-GUS transgenic plants and the gene expression analysis using RT-PCR, the gene showed an interesting temporal and spatial expression pattern: it had no or a low expression in young flowers (roughly before anther stage 6), reached a maximum level around stages 6-7, and then declined gradually to a very low level in young siliques. No expression was detected in microspores or pollen. Together, our data demonstrated that SGC Lectin RLK plays a critical role in pollen development.     

Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis

Arabidopsis NSN1 encodes a nucleolar GTP-binding protein and is required for flower development. Defective flowers were formed in heterozygous nsn1/+?plants. Homozygous nsn1 plants were dwarf and exhibited severe defects in reproduction. Arrests in embryo development in nsn1 could occur at any stage of embryogenesis. Cotyledon initiation and development during embryogenesis were distorted in nsn1 plants. At the seedling stage, cotyledons and leaves of nsn1 formed upward curls. The curled leaves developed meristem-like outgrowths or hyperplasia tissues in the adaxial epidermis. Long and enlarged pavement cells, characteristic of the abaxial epidermis of wild type plants, were found in the adaxial epidermis in nsn1 leaves, suggesting a disoriented leaf polarity in the mutant. The important role of NSN1 in embryo development and leaf differentiation was consistent with the high level expression of the NSN1 gene in the developing embryos and the primordia of cotyledons and leaves. The CLAVATA 3 (CLV3) gene, a stem cell marker in the Arabidopsis shoot apical meristem (SAM), was expressed in expanded regions surrounding the SAM of nsn1 plants, and induced ectopically in the meristem-like outgrowths in cotyledons and leaves. The nsn1 mutation up-regulated the expression levels of several genes implicated in the meristem identity and the abaxial cell fate, and repressed the expression of other genes related to the specification of cotyledon boundary and abaxial identity. These results demonstrate that NSN1 represents a novel GTPase required for embryogenesis, leaf development and leaf polarity establishment in Arabidopsis.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

Light-harvesting chlorophyll a/b-binding proteins are required for stomatal response to abscisic acid in Arabidopsis

The light-harvesting chlorophyll a/b binding proteins (LHCB) are perhaps the most abundant membrane proteins in nature. It is reported here that the down-regulation or disruption of any member of the LHCB family, LHCB1, LHCB2, LHCB3, LHCB4, LHCB5, or LHCB6, reduces responsiveness of stomatal movement to ABA, and therefore results in a decrease in plant tolerance to drought stress in Arabidopsis thaliana. By contrast, over-expression of a LHCB member, LHCB6, enhances stomatal sensitivity to ABA. In addition, the reactive oxygen species (ROS) homeostasis and a set of ABA-responsive genes are altered in the lhcb mutants. These data demonstrate that LHCBs play a positive role in guard cell signalling in response to ABA and suggest that they may be involved in ABA signalling partly by modulating ROS homeostasis.     

Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development

WD40 repeat proteins similar to yeast MSI1 are conserved in animals and plants, in which they participate in complexes involved in chromatin metabolism. Although MSI1-like proteins are well characterised biochemically, their function in the development of multicellular eukaryotes is not well understood. We constructed Arabidopsis plants in which the AtMSI1 protein level was altered. Strong ectopic expression of AtMSI1 produced no visible altered phenotype, but reduction of AtMSI1 dramatically affected development. The primary shoot apical meristem was unable to develop organs after the transition to flowering. Flowers that developed on floral shoots from axillary meristems experienced a progressive loss of floral morphology, including a reduction in size of the petals and stamens and the development of carpel-like sepals. Ovule development was disrupted in all flowers, resulting in complete female sterility. Molecular analysis of the mutant plants revealed that AtMSI1 is required to maintain the correct temporal and organ-specific expression of homeotic genes, including AGAMOUS and APETALA2. In contrast, FAS1 and FAS2, which together with AtMSI1 form the chromatin assembly complex CAF-1, are not required for repression of these genes. Therefore, AtMSI1 has specific functions in addition to CAF-1-mediated chromatin assembly. Efficient formation of heterochromatin, but not methylation of centromeric DNA repeats, depends on AtMSI1 presence demonstrating a key role of AtMSI1 in maintenance of chromatin structure.     

H2S is involved in ABA-mediated stomatal movement through MPK4 to alleviate drought stress in Arabidopsis thaliana

Plant and Soil - Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in multiple physiological processes in both animals and plants. Mitogen-activated protein kinase (MAPK) is...