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Endoplasmic reticulum (ER)–derived organelles, ER bodies, participate in the defense against herbivores in Brassicaceae plants. ER bodies accumulate β-glucosidases, which hydrolyze specialized thioglucosides known as glucosinolates to generate bioactive substances. In Arabidopsis thaliana , the leaf ER (LER) bodies are formed in large pavement cells, which are found in the petioles, margins and blades of rosette leaves. However, the regulatory mechanisms involved in establishing large pavement cells are unknown. Here, we show that the ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) transcription factor regulates the formation of LER bodies in large pavement cells of rosette leaves. Overexpression of ATML1 enhanced the expression of LER body–related genes and the number of LER body–containing large pavement cells, whereas its knock-out resulted in opposite effects. ATML1 enhances endoreduplication and cell size through LOSS OF GIANT CELLS FROM ORGANS (LGO). Although the overexpression and knock-out of LGO affected the appearance of large pavement cells in Arabidopsis , the effect on LER body–related gene expression and LER body formation was weak. LER body–containing large pavement cells were also found in Eutrema salsugineum , another Brassicaceae species. Our results demonstrate that ATML1 establishes large pavement cells to induce LER body formation in Brassicaceae plants and thereby possibly contribute to the defense against herbivores. [ABSTRACT FROM AUTHOR]

The epidermis of shoot organs in plants develops from the outermost layer (L1) of the shoot apical meristem. In Arabidopsis, a pair of homeobox genes, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2), play a role in regulating the expression of L1-specific genes. atml1-1 pdf2-1 double mutants show striking defects in the differentiation of shoot epidermal cells. However, because atml1-1 and pdf2-1 have a T-DNA inserted downstream of the respective homeobox sequences, these alleles may not represent null mutations. Here we characterized additional mutant alleles that have a T-DNA insertion at different positions of each gene. Double mutants of a strong atml1-3 allele with each pdf2 allele were found to cause embryonic arrest at the globular stage. Although with low frequency, all double mutant combinations of a weak atml1-1 allele with each pdf2 allele germinated and showed phenotypes defective in shoot epidermal cell differentiation. We further confirmed that transgenic induction of PDF2 fused to the Drosophila Engrailed repressor domain temporarily interferes with epidermal cell differentiation in the wild-type background. These results indicate that ATML1 and PDF2 act redundantly as a positive regulator of shoot epidermal cell differentiation and at least one copy of these genes is essential for embryo development. [ABSTRACT FROM AUTHOR]

Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layerspecific DEK1 down-regulation, we show that L1- and 35Sinduced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells. [ABSTRACT FROM AUTHOR]

Cotton fibres are a subset of single epidermal cells that elongate from the seed coat to produce the long cellulose strands or lint used for spinning into yarn. To identify genes that might regulate lint fibre initiation, expression profiles of 0 days post-anthesis (dpa) whole ovules from six reduced fibre or fibreless mutants were compared with wild-type linted cotton using cDNA microarrays. Numerous clones were differentially expressed, but when only those genes that are normally expressed in the ovule outer integument (where fibres develop) were considered, just 13 different cDNA clones were down-regulated in some or all of the mutants. These included: a Myb transcription factor (GhMyb25) similar to the Antirrhinum Myb AmMIXTA, a putative homeodomain protein (related to Arabidopsis ATML1), a cyclin D gene, some previously identified fibre-expressed structural and metabolic genes, such as lipid transfer protein, α-expansin and sucrose synthase, as well as some unknown genes. Laser capture microdissection and reverse transcription–PCR were used to show that both the GhMyb25 and the homeodomain gene were predominantly ovule specific and were up-regulated on the day of anthesis in fibre initials relative to adjacent non-fibre ovule epidermal cells. Their spatial and temporal expression pattern therefore coincided with the time and location of fibre initiation. Constitutive overexpression of GhMyb25 in transgenic tobacco resulted in an increase in branched long-stalked leaf trichomes. The involvement of cell cycle genes prompted DNA content measurements that indicated that fibre initials, like leaf trichomes, undergo DNA endoreduplication. Cotton fibre initiation therefore has some parallels with leaf trichome development, although the detailed molecular mechanisms are clearly different. [ABSTRACT FROM AUTHOR]

Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis , three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE , meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase. [ABSTRACT FROM AUTHOR]

In many eukaryotic organisms, homeobox genes are important regulators that specify the cell fate and body plan in early embryogenesis. In this study, a gene designated OSTF1 (Oryza sativa transcription factor 1) encoding a homeodomain protein in rice was isolated and characterized. The encoded OSTF1, although sharing only approximately 51% sequence identity with other HD-GL2 members, contains four characteristic motifs (an N-terminal acidic region, a homeodomain, a truncated leucine zipper, and a START domain). OSTF1 was detected as a single copy gene in rice. The transcripts were absent in young panicle or mature spikelet before anthesis, but appeared very early in the pollinated grain with a transient profile. In vegetative tissues examined, expression was only detectable in root. In situ hybridization analysis on developing grains revealed that OSTF1 was strongly and uniformly expressed in the embryo at the globular stage and preferentially localized to the protoderm at 3–6 d after pollination. Expression was also detectable in the integument and throughout the endosperm. Although OSTF1 is not closely related to the remaining HD-GL2 members in sequences, this gene exhibits an analogous epidermis-preferential expression pattern. [ABSTRACT FROM PUBLISHER]

Female gametophyte (FG) is crucial for reproduction in flowering plants. Arabidopsis thaliana produces Polygonum -type FGs, which consist of an egg cell, two synergid cells, three antipodal cells and a central cell. Egg cell and central cell are the two female gametes that give rise to the embryo and surrounding endosperm, respectively, after fertilization. During the development of a FG, a single megaspore produced by meiosis undergoes three rounds of mitosis to produce an eight-nucleate cell. A seven-celled FG is formed after cellularization. The central cell initially contains two polar nuclei that fuse during female gametogenesis to form the secondary nucleus. In this study, we developed a gene induction system for analyzing the functions of various genes in developing Arabidopsis FGs. This system allows transgene expression in developing FGs using the heat-inducible Cre- loxP recombination system and FG-specific embryo sac 2 (ES2) promoter. Efficient gene induction was achieved in FGs by incubating flower buds and isolated pistils at 35°C for short periods of time (1–5 min). Gene induction was also induced in developing FGs by heat treatment of isolated ovules using the infrared laser-evoked gene operator (IR-LEGO) system. Expression of a dominant-negative mutant of Sad1/UNC84 (SUN) proteins in developing FGs using the gene induction system developed in this study caused defects in polar nuclear fusion, indicating the roles of SUN proteins in this process. This strategy represents a new tool for analyzing the functions of genes in FG development and FG functions. [ABSTRACT FROM AUTHOR]

The endosperm and embryo originate from the fertilized central cell and egg cell through a programmed series of cell division and differentiation events. Characterization of more vital genes involved in endosperm and embryo development can help us to understand the regulatory mechanism in more depth. In this study, we found that loss of NAA10 and NAA15, the catalytic and auxiliary subunits of Arabidopsis thaliana N-terminal acetyltransferase A (AtNatA), respectively, led to severely delayed and incomplete endosperm cellularization, accompanied by disordered cell division in the early embryo. Studies on the marker genes/lines of the endosperm (AGL62-GFP, pDD19::GFP, pDD22::NLS-GFP and N9185) and embryo (STM, FIL, SCR and WOX5) in naa10/naa15 mutants showed that expression patterns of these markers were significantly affected, which were tightly associated with the defective feature of endosperm cellularization and embryo cell differentiation. Subsequently, embryonic complementation rescued the abortive embryos, but failed to initiate endosperm cellularization properly, further confirming the essential role of AtNatA in both endosperm and embryo development. Moreover, repression of AGL62 in naa10 and naa15 restored the endosperm cellularization, suggesting that NAA10/NAA15 functions in initiation of endosperm cellularization by inhibiting the expression of AGL62 in Arabidopsis. Therefore, NAA10 and NAA15 could be considered as crucial factors involved in promoting endosperm cellularization in Arabidopsis. [ABSTRACT FROM AUTHOR]

Growth and development are tightly co-ordinated events in the lifetime of living organisms. In temperate bamboo plants, spring is the season when environmental conditions are suitable for the emergence of new shoots. Previous studies demonstrated that bamboo plants undergo an energyconsuming 'fast stem growth' phase. However, the events during the initiation of stem elongation in bamboo are poorly understood. To understand the onset of bamboo stem growth, we performed hormone and transcriptome profiling of tissue regions in newly elongating shoots of the Moso bamboo Phyllostachys edulis. The growth hormones auxins, cytokinins and gibberellins accumulated in the shoot apex, while the stress hormones ABA, salicylic acid (SA) and jasmonic acid (JA) are predominantly found in the lower part of the stem. The mature basal part of the stem showed enrichment of transcripts associated with cell wall metabolism and biosynthesis of phenylpropanoid metabolites, such as lignin. In the young upper stem region, expression of cell formation- and DNA synthesis-related genes was enriched. Moreover, the apical region showed enhanced expression of genes involved in meristem maintenance, leaf differentiation and development, abaxial/adaxial polarity and flowering. Our findings integrate the spatial regulation of hormones and transcriptome programs during the initiation of bamboo stem growth. [ABSTRACT FROM AUTHOR]

Maintenance of organ separation is one of the essential phenomena for normal plant development. We have identified and analyzed ONION3 (ONI3), which is required for avoiding organ fusions in rice. Loss-of-function mutations of ONI3, which were identified as mutants with ectopic expression of KNOX genes in leaves and morphologically resembling KNOX overexpressors, showed abnormal organ fusions in developing shoots. The mutant seedlings showed fusions between neighboring organs and also within an organ; they stopped growing soon after germination and subsequently died. ONI3 was shown to encode an enzyme that is most similar to Arabidopsis HOTHEAD and is involved in biosynthesis of long-chain fatty acids. Expression analyses showed that ONI3 was specifically expressed in the outermost cell layer in the shoot apex throughout life cycle, and the oni3 mutants had an aberrant outermost cell layer. Our results together with previous studies suggest that long-chain fatty acids are required for avoiding organ fusions and promoting normal shoot development in rice. [ABSTRACT FROM PUBLISHER]

Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the β subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels. [ABSTRACT FROM PUBLISHER]

A plant’s surface is covered with epicuticular wax, which protects plants from inappropriate environmental conditions such as drought and pathogen attack. Very-long-chain fatty acids (VLCFAs) are the main component of epicuticular wax on the surface of above-ground organs. Here we show that a fatty acid elongase catalyzing an elongation reaction of VLCFAs is required for shoot development in rice. onion2 (oni2) mutants produced very small shoots in which leaves were fused to each other, and ceased growing after germination. The midrib of oni2 leaf blades was not developed correctly. Molecular cloning showed that ONI2 encodes a fatty acid elongase, which catalyzes the first step of elongation reactions of a carbon chain of VLCFAs, and oni2 had a reduced amount of VLCFAs. Expression analysis showed that ONI2 is specifically expressed in the outermost cell layer of young lateral organs. These results suggest that ONI2 is a layer 1-specific gene required for development of the entire shoot and that VLCFAs play an essential role in normal shoot development in rice. [ABSTRACT FROM AUTHOR]

The molecular basis underlying the development, maintenance and function of companion cells in plants is largely unknown. The identification of several genes expressed specifically in companion cells implies the contribution of specific transcriptional elements to the identity of companion cells. However, less is known about the companion cell-specific transcriptional regulation of promoters. We established a novel assay method using gene-gun delivery of partially deleted promoters to string-containing vascular bundles excised from the petiole of Brassica napus for the rapid identification of cis-elements. To test this system, we analyzed the Arabidopsis METALLOTHIONEIN 2B (MT2B) gene, which is expressed in companion cells. The assay revealed a 49-bp region possessing two predicted cis-regulatory elements: a G-box and an evening element-related sequence (EEr), and EEr showed higher activity. We confirmed the reliability of the result with stable transformants harboring a deleted MT2B promoter:GUS transgene. The lack of EEr completely eliminated the MT2B-like expression, but the lack of G-box did not eliminate it. We conclude that EEr is a major cis-regulatory element of the MT2B promoter. Our method will help to explain the transcriptional background of companion cells through the rapid identification of cis-regulatory regions. [ABSTRACT FROM PUBLISHER]

In higher plants, the main elements of the fundamental body plan, the apical-basal and radial patterns, are established during embryogenesis. We have isolated several globular embryo (gle) mutants of rice that fail to develop any embryonic organs. We expected that these gle mutants might include mutants defective in their radial pattern formation ability. We developed two markers specifically staining the L2 and L3 layers (OsSCR and OsPNH1, respectively) and characterized the gle mutants by using these markers in addition to the already developed markers Roc1 (marker for the L1 layer), Ramy1A (marker of the L1 layer of the epithelium), and OSH1 (marker of the apical region). One of the gle mutants, gle4, expressed Roc1 and Ramy1A at the normal positions, but other markers exhibited an abnormal expression pattern; that is, both OsPNH1 and OsSCR were expressed in the central region of the embryo and OSH1 expression was not observed. Calli from the gle4 epithelium regenerated plants with abnormal morphologies. These results indicate that the GLE4 gene is involved in radial pattern formation during rice embryogenesis to differentiate the L2 and L3 layers, but is not involved in the establishment of the L1 layer or in the formation of embryonic organs. [ABSTRACT FROM AUTHOR]

The surfaces of higher plants are characterized by epidermis, which usually consists of a single layer of cells. The epidermis is derived from the outer cell layer of the embryo or protoderm, which arises as a result of periclinal cell division. After seed germination, most of the epidermal cells of the aerial parts of plants are derived from the outer cell layer of the shoot apical meristem (the L1 layer). Thus, knowledge of how the protoderm and/or L1 layer is established is fundamental to understanding the morphogenesis of higher plants. Here, we report the isolation of a gene encoding an Arabidopsis homologue (ACR4) of the maize putative receptor kinase CRINKLY4 (CR4), which is involved in epidermal differentiation. The domain organization of the predicted amino acid sequence of ACR4 is essentially identical to that of CR4. ACR4-GFP fusion protein localized to the cell surface when expressed in tobacco cell (BY-2) culture. ACR4 transcripts were detected in all the organs of the Arabidopsis plant. In developing embryos and shoot apices, ACR4 transcripts accumulated in protoderm and epidermis at relatively higher levels than in the inner tissues. Over-expression of antisense ACR4 in Arabidopsis plants resulted in malformation of embryos to varying degrees. These results suggest that ACR4 is, at a minimum, involved in the normal morphogenesis of embryos, most likely through properly differentiating protoderm cells. [ABSTRACT FROM PUBLISHER]

We examined spatial and temporal expression patterns of four carrot HD-Zip I homeobox genes in somatic embryos. The mRNAs for CHB3, CHB4 and CHB5 were accumulated preferentially in the innermost cortical cell layers of the embryo axis in the torpedo-shaped embryo. In contrast, the accumulation of CHB6 mRNA was restricted to procambial cells of the heart- and torpedo-shaped embryos. In the embryonic cotyledons and the hypocotyl of the seedlings, all of the mRNAs for the four genes were located in the vascular tissues. These findings indicate that different HD-Zip I homeobox genes may be involved in the differentiation of specific tissues during somatic embryogenesis. [ABSTRACT FROM AUTHOR]

The shoot apical meristem (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largely unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower 1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, PDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed. [ABSTRACT FROM AUTHOR]