TOUSLED participates in apical tissue formation during gynoecium development in Arabidopsis.

Mutations at the TOUSLED (TSL) protein kinase locus in Arabidopsis cause reduced differentiation of apical gynoecial tissues and eliminate the fusion of the style and septum. TSL expression becomes confined to the developing style by stage 13, where it may promote expansion of tissues. Double mutant analysis suggests that ETTIN interacts with TSL, possibly by restricting TSL expression to apical regions. TSL, LEUNIG, and PERIANTHIA appear to participate in pathways of redundant function during the development of specific gynoecial tissues. TSL and LEUNIG most likely function in similar pathways during ovule development. TSL acts independently of the function of the organ identity genes AGAMOUS and APETALA2, and it is required for the formation of specific tissues in ectopic carpels. Mutations in TSL, ETTIN, PERIANTHIA, and LEUNIG all affect floral organ number as well as gynoecium morphology. Their respective wild-type loci must therefore play important roles in early floral meristem development during initiation of organ primordia in addition to their functions during regional differentiation within developing gynoecial primordia.     

Specific expression of the AGL1 MADS-box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development

Several members of the MADS-box gene family have been shown to be important regulators of flower development, controlling such well-studied early events as the formation of the floral meristem and the specification of floral organ identity. Other floral-specific MADS-box genes, of as yet unknown function, have been isolated by homology and are proposed to be part of a regulatory hierarchy controlling flower development. Some of these genes might regulate later aspects of flower development, such as development of individual floral organs, which is less well studied at the molecular level. This paper presents a detailed analysis of the expression pattern of one such gene from Arabidopsis , AGL1 , using RNA in situ hybridization. It is found that AGL1 is specifically expressed in particular regions of the gynoecium and ovule, only during and after floral development stage 7. AGL1 expression at the tip of the growing carpel primordia, along the margins of the ovary valves in developing and mature gynoecia and in specific regions of developing and mature ovules provides important insights into the possible roles of AGL1 . It is proposed that AGL1 may have regulatory functions in the structural definition and/or function of the valve margins, in axis maintenance during ovule development, in nutritional supply to the growing ovule and embryo sac, and in pollen tube guidance. In the floral homeotic mutants ag-1 , ap3-3 and ap2-2 , AGL1 mRNA is expressed in an organ-dependent manner, suggesting that AGL1 is a carpel-specific gene and as such ultimately depends upon the carpel identity gene AG for proper gene expression.     

Involvement of CUP-SHAPED COTYLEDON genes in gynoecium and ovule development in Arabidopsis thaliana

When mutations in CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 are combined, severe defects involving fusion of sepals and of stamens occur in Arabidopsis flowers. In addition, septa of gynoecia do not fuse along the length of the ovaries and many ovules have their growth arrested. CUC2 is expressed at the tips of septal primordia during gynoecium development and at the boundary between nucellus and chalaza during ovule development. These expression patterns are partially consistent with the phenotype of the mutant gynoecium. CUC2 mRNA is also shown to be expressed at the boundaries between meristems and organ primordia during both the vegetative and reproductive phases. This expression pattern indicates that CUC2 is generally involved in organ separation in shoot and floral meristems.     

The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation

In higher plants, molecular mechanisms regulating shoot apical meristem (SAM) formation and organ separation are largely unknown. The CUC1 (CUP-SHAPED COTYLEDON1) and CUC2 are functionally redundant genes that are involved in these processes. We cloned the CUC1 gene by a map-based approach, and found that it encodes a NAC-domain protein highly homologous to CUC2. CUC1 mRNA was detected in the presumptive SAM during embryogenesis, and at the boundaries between floral organ primordia. Surprisingly, overexpression of CUC1 was sufficient to induce adventitious shoots on the adaxial surface of cotyledons. Expression analyses in the overexpressor and in loss-of-function mutants suggest that CUC1 acts upstream of the SHOOT MERISTEMLESS gene.     

ROOT HAIR DEFECTIVE 2 vesicular delivery to the apical plasma membrane domain during Arabidopsis root hair development

Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.

Structural sterols might participate in delivering GFP-RHD2 to the apical plasma membrane of developing root hairs.     

The essential gene EMB1611 maintains shoot apical meristem function during Arabidopsis development

The Arabidopsis thaliana genome contains hundreds of genes essential for seed development. Because null mutations in these genes cause embryo lethality, their specific molecular and developmental functions are largely unknown. Here, we identify a role for EMB1611/MEE22 , an essential gene in Arabidopsis, in shoot apical meristem maintenance. EMB1611 encodes a large, novel protein with N-terminal coiled-coil regions and two putative transmembrane domains. We show that the partial loss-of-function emb1611-2 mutation causes a range of pleiotropic developmental phenotypes, most dramatically a progressive loss of shoot apical meristem function that causes premature meristem termination. emb1611-2 plants display disorganization of the shoot meristem cell layers early in development, and an associated stem cell fate change to an organogenic identity. Genetic and molecular analysis indicates that EMB1611 is required for maintenance of the CLV-WUS stem cell regulatory pathway in the shoot meristem, but also has WUS -independent activity. In addition, emb1611-2 plants have reduced shoot and root growth, and their rosette leaves form trichomes with extra branches, a defect we associate with an increase in endoreduplication. Our data indicate that EMB1611 functions to maintain cells, particularly those in the shoot meristem, roots and developing rosette leaves, in a proliferative or uncommitted state.     

Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development

LRR-extensins (LRX) form a family of structural cell wall proteins containing a receptor-like domain. The functional analysis of Arabidopsis LRX1 has shown that it is involved in cell morphogenesis of root hairs. In this work, we have studied LRX2, a paralog of LRX1. LRX2 expression is mainly found in roots and is responsive to factors promoting or repressing root hair formation. The function of LRX1 and LRX2 was tested by the expression of a truncated LRX2 and different LRX1/LRX2 chimaeric proteins. Using complementation of the lrx1 phenotype as the parameter for protein function, our experiments indicate that LRX1 and LRX2 are functionally similar but show differences in their activity. Genetic analysis revealed that single lrx2 mutants do not show any defect in root hair morphogenesis, but synergistically interact with the lrx1 mutation. lrx1/lrx2 double mutants have a significantly enhanced lrx1 phenotype, resulting in frequent rupture of the root hairs soon after their initiation. Analysis of the root hair cell wall ultrastructure by transmission electron microscopy (TEM) revealed the formation of osmophilic aggregates within the wall, as well as local disintegration of the wall structure in the double mutant, but not in wild-type plants. Our results indicate that LRX1 and LRX2 have overlapping functions in root hair formation, and that they likely regulate cell morphogenesis by promoting proper development of the cell wall.     

AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues

Plants employ a specialized transport system composed of separate influx and efflux carriers to mobilize the plant hormone auxin between its site(s) of synthesis and action. Mutations within the permease-like AUX1 protein significantly reduce the rate of carrier-mediated auxin uptake within Arabidopsis roots, conferring an agravitropic phenotype. We are able to bypass the defect within auxin uptake and restore the gravitropic root phenotype of aux1 by growing mutant seedlings in the presence of the membrane-permeable synthetic auxin, 1-naphthaleneacetic acid. We illustrate that AUX1 expression overlaps that previously described for the auxin efflux carrier, AtPIN2, using transgenic lines expressing an AUX1 promoter::uidA (GUS) gene. Finally, we demonstrate that AUX1 regulates gravitropic curvature by acting in unison with the auxin efflux carrier to co-ordinate the localized redistribution of auxin within the Arabidopsis root apex. Our results provide the first example of a developmental role for the auxin influx carrier within higher plants and supply new insight into the molecular basis of gravitropic signalling.     

Arabidopsis (Brassicaceae) flower development and gynoecium patterning in wild type and Ettin mutants

Screening for mutations that alter flower development in Arabidopsis has led to the identification of two general types of genetic loci: those affecting meristem and organ identity, and those affecting growth and development independent of identity. ettin (ett) mutants belong to the latter class and exhibit pleiotropic phenotypes distinct from previously described Arabidopsis mutants. These phenotypes include increases in sepal and petal number, decreases in stamen number and anther locule number, and gross alteration of tissue patterning in the gynoecium. To determine when and how differences in ett floral meristems originate, flower development was compared between the wild type and ett mutants. ett floral meristems exhibit increases in abaxial sepal and petal primordia number without apparent increases in meristem size. Extra sepal and petal primordia develop into normal organs. In contrast, stamen and carpel primordia exhibit alterations in shape and form, subsequent to premature elongation of the terminal floral meristem. Phenotypes are allele-strength dependent. The stigma develops precociously and style differentiation is basally and abaxially misplaced in ett gynoecia. The data are discussed in the context of a model suggesting that two concentric boundaries specify the apical-basal pattern of gynoecium differentiation.     

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.     

DDB2, DDB1A and DET1 exhibit complex interactions during Arabidopsis development

Damaged DNA-binding proteins 1 and 2 (DDB1 and DDB2) are subunits of the damaged DNA-binding protein complex (DDB). DDB1 is also found in the same complex as DE-ETIOLATED 1 (DET1), a negative regulator of light-mediated responses in plants. Arabidopsis has two DDB1 homologs, DDB1A and DDB1B. ddb1a single mutants have no visible phenotype while ddb1b mutants are lethal. We have identified a partial loss-of-function allele of DDB2. To understand the genetic interaction among DDB2, DDB1A, and DET1 during Arabidopsis light signaling, we generated single, double, and triple mutants. det1 ddb2 partially enhances the short hypocotyl and suppresses the high anthocyanin content of dark-grown det1 and suppresses the low chlorophyll content, early flowering time (days), and small rosette diameter of light-grown det1. No significant differences were observed between det1 ddb1a and det1 ddb1a ddb2 in rosette diameter, dark hypocotyl length, and anthocyanin content, suggesting that these are DDB1A-dependent phenotypes. In contrast, det1 ddb1a ddb2 showed higher chlorophyll content and later flowering time than det1 ddb1a, indicating that these are DDB1A-independent phenotypes. We propose that the DDB1A-dependent phenotypes indicate a competition between DDB2- and DET1-containing complexes for available DDB1A, while, for DDB1A-independent phenotypes, DDB1B is able to fulfill this role.