Transcriptome analyses uncover reliance of endosperm gene expression on Arabidopsis embryonic development.

Endosperm-embryo development in flowering plants is regulated coordinately by signal exchange during seed development. However, such a reciprocal control mechanism has not been clearly identified. In this study, we identified an endosperm-specific gene, LBD35, expressed in an embryonic development-dependent manner, by a comparative transcriptome and cytological analyses of double-fertilized and single-fertilized seeds prepared by using the kokopelli mutant, which frequently induces single fertilization events. Transcriptome analysis using LBD35 as a marker of the central cell fertilization event identified that 141 genes, including 31 genes for small cysteine-rich peptides, are expressed in a double fertilization-dependent manner. Our results reveal possible embryonic signals that regulate endosperm gene expression and provide a practicable method to identify genes involved in the communication during endosperm-embryo development.

Regulatory functions of ROS dynamics via glutathione metabolism and glutathione peroxidase activity in developing rice zygote.

Reactive oxygen species (ROS) play essential roles in plant development and environmental stress responses. In this study, ROS dynamics, the glutathione redox status, the expression and subcellular localization of glutathione peroxidases (GPXs), and the effects of inhibitors of ROS-mediated metabolism were investigated along with fertilization and early zygotic embryogenesis in rice (Oryza sativa). Zygotes and early embryos exhibited developmental arrest upon inhibition of ROS production. Egg cells accumulated high ROS levels, and, after fertilization, intracellular ROS levels progressively declined in zygotes in which de novo expression of GPX1 and 3 was observed through upregulation of the genes. In addition to inhibition of GPX activity, depletion of glutathione impeded early embryonic development and led to failure of the zygote to appropriately decrease H2 O2 levels. Moreover, through monitoring of the glutathione redox status, the developing zygotes exhibited a progressive glutathione oxidation, which became extremely delayed under inhibited GPX activity. Our results provide insights into the importance of ROS dynamics, GPX antioxidant activity, and glutathione redox metabolism during zygotic/embryonic development.

Temperature-dependent fasciation mutants provide a link between mitochondrial RNA processing and lateral root morphogenesis.

Although mechanisms that activate organogenesis in plants are well established, much less is known about the subsequent fine-tuning of cell proliferation, which is crucial for creating properly structured and sized organs. Here we show, through analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis, root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4), that mitochondrial RNA processing is required for limiting cell division during early lateral root (LR) organogenesis. These mutants formed abnormally broadened (i.e. fasciated) LRs under high-temperature conditions due to extra cell division. All TDF proteins localized to mitochondria, where they were found to participate in RNA processing: RRD1 in mRNA deadenylation, and RRD2 and RID4 in mRNA editing. Further analysis suggested that LR fasciation in the TDF mutants is triggered by reactive oxygen species generation caused by defective mitochondrial respiration. Our findings provide novel clues for the physiological significance of mitochondrial activities in plant organogenesis.

Regulation of shoot meristem shape by photoperiodic signaling and phytohormones during floral induction of Arabidopsis.

Floral transition, the onset of plant reproduction, involves changes in shape and identity of the shoot apical meristem (SAM). The change in shape, termed doming, occurs early during floral transition when it is induced by environmental cues such as changes in day-length, but how it is regulated at the cellular level is unknown. We defined the morphological and cellular features of the SAM during floral transition of Arabidopsis thaliana. Both cell number and size increased during doming, and these changes were partially controlled by the gene regulatory network (GRN) that triggers flowering. Furthermore, dynamic modulation of expression of gibberellin (GA) biosynthesis and catabolism enzymes at the SAM contributed to doming. Expression of these enzymes was regulated by two MADS-domain transcription factors implicated in flowering. We provide a temporal and spatial framework for integrating the flowering GRN with cellular changes at the SAM and highlight the role of local regulation of GA.

Genetic and molecular basis of floral induction in Arabidopsis thaliana.

Many plants synchronize their life cycles in response to changing seasons and initiate flowering under favourable environmental conditions to ensure reproductive success. To confer a robust seasonal response, plants use diverse genetic programmes that integrate environmental and endogenous cues and converge on central floral regulatory hubs. Technological advances have allowed us to understand these complex processes more completely. Here, we review recent progress in our understanding of genetic and molecular mechanisms that control flowering in Arabidopsis thaliana.

The sugar transporter SWEET10 acts downstream of FLOWERING LOCUS T during floral transition of Arabidopsis thaliana.

BACKGROUND: Floral transition initiates reproductive development of plants and occurs in response to environmental and endogenous signals. In Arabidopsis thaliana, this process is accelerated by several environmental cues, including exposure to long days. The photoperiod-dependent promotion of flowering involves the transcriptional induction of FLOWERING LOCUS T (FT) in the phloem of the leaf. FT encodes a mobile protein that is transported from the leaves to the shoot apical meristem, where it forms part of a regulatory complex that induces flowering. Whether FT also has biological functions in leaves of wild-type plants remains unclear. RESULTS: In order to address this issue, we first studied the leaf transcriptomic changes associated with FT overexpression in the companion cells of the phloem. We found that FT induces the transcription of SWEET10, which encodes a bidirectional sucrose transporter, specifically in the leaf veins. Moreover, SWEET10 is transcriptionally activated by long photoperiods, and this activation depends on FT and one of its earliest target genes SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1). The ectopic expression of SWEET10 causes early flowering and leads to higher levels of transcription of flowering-time related genes in the shoot apex. CONCLUSIONS: Collectively, our results suggest that the FT-signaling pathway activates the transcription of a sucrose uptake/efflux carrier during floral transition, indicating that it alters the metabolism of flowering plants as well as reprogramming the transcription of floral regulators in the shoot meristem.

Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications.

Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.

BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2 constitute a signaling pathway and modulate CLE peptide-triggered growth inhibition in Arabidopsis root.

Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems.

Mystery in genetics: PUB4 gives a clue to the complex mechanism of CLV signaling pathway in the shoot apical meristem.

Postembryonic growth and development in higher plants are ultimately reliant on the activity of meristems, where the cells divide frequently to provide source cells for new organs and tissues while in part maintain their pluripotent nature as stem cells. The shoot apical meristem (SAM) is maintained throughout the life of plants and responsible for the development of all areal tissues. In Arabidopsis thaliana, the size of SAM is controlled by a peptide ligand, CLAVATA3 (CLV3). Previously, genetic studies have identified several genes that function downstream of CLV3, many of which, intriguingly, encode receptors. Recently we identified an E3 ubiquitin ligase, PLANT U-BOX 4 (PUB4), as a key regulatory component of root meristem maintenance that functions downstream of an exogenous synthetic CLV3 peptide. Here, we report an additional function of PUB4 in the SAM.

A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the Clavata3 (CLV3)/embryo surrounding region-related (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.

Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis.

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (CLE)-like effector proteins. These proteins act as ligand mimics of plant CLE peptides and are required for successful nematode infection. Previously, we showed that the CLV2/CORYNE (CRN) heterodimer receptor complex is required for nematode CLE signaling. However, there was only a partial reduction in nematode infection when this signaling was disrupted, indicating that there might be additional nematode CLE receptors. In this study, we demonstrate that CLV1 and RECEPTOR-LIKE PROTEIN KINASE 2/TOADSTOOL2 (RPK2), two additional receptors that can transmit the CLV3 signal independent of CLV2/CRN for shoot apical meristem maintenance, also play a role in nematode CLE perception. Localization studies showed that both receptors are expressed in nematode-induced syncytia. Infection assays with clv1 and rpk2 single mutants revealed a decrease in both nematode infection and syncytium size. Significantly, further reduction in nematode infection was observed when rpk2 was combined with clv1 and clv2 mutants. Taken together, our results indicate that parallel signaling pathways involving CLV1, CLV2, and RPK2 are important for nematode parasitism.

Mitogen-activated protein kinase regulated by the CLAVATA receptors contributes to shoot apical meristem homeostasis.

In Arabidopsis, the CLAVATA (CLV) pathway operates in the regulation of the size of the stem cell population in the shoot apical meristem (SAM). CLV3 functions as a small peptide ligand to negatively regulate the expression of the WUSCHEL (WUS) transcription factor through three major receptor kinase complexes of CLV1, CLV2-SUPPRESSOR OF LLP1-2 (SOL2)/CORYNE (CRN) and recently identified RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2). Aiming to understand the precise molecular details of CLV3 signaling, we investigated the contribution of phospho-signaling, potentially regulated by these kinase complexes, to the CLV pathway. We detected CLV3-triggered CLV1 phosphorylation, which is also conditioned by the rest of the CLV receptors, presumably by their direct association. Our comprehensive analysis of the activities of the respective CLV receptors on mitogen-activated protein kinases (MAPKs) suggested that the precise balanced regulation of MAPK activity by the CLV receptors is likely to be key for SAM homeostasis.

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis.

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.

Plant meristems: CLAVATA3/ESR-related signaling in the shoot apical meristem and the root apical meristem.

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS-CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species.

The receptor-like kinase SOL2 mediates CLE signaling in Arabidopsis.

Arabidopsis sol2 mutants showed CLV3 peptide resistance. Twenty-six synthetic CLE peptides were examined in the clv1, clv2 and sol2 mutants. sol2 showed different levels of resistance to the various peptides, and the spectrum of peptide resistance was quite similar to that of clv2. SOL2 encoded a receptor-like kinase protein which is identical to CORYNE (CRN). GeneChip analysis revealed that the expression of several genes was altered in the sol2 root tip. Here, we suggest that SOL2, together with CLV2, plays an important role in the regulation of root meristem development through the CLE signaling pathway.

A large family of genes that share homology with CLE domain in Arabidopsis and rice.

Thirty-one CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins are encoded in the Arabidopsis genome, and they are supposed to function as dodecapeptides with two hydroxyproline residues. Twenty-six synthetic CLE peptides, corresponding to the predicted products of the 31 CLE genes, were examined in Arabidopsis and rice. Nineteen CLE peptides induced root meristem consumption, resulting in the short root phenotype in Arabidopsis and rice, whereas no CLE peptides affected the shoot apical meristem in rice. Database searches revealed 47 putative CLE genes in the rice genome. Three of the rice CLE genes, OsCLE502, OsCLE504 and OsCLE506, encode CLE proteins with multiple CLE domains, which are not found in the Arabidopsis genome, and polyproline region was found between these CLE domains. These results indicate conserved and/or diverse CLE functions in each plant species.

Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-related (CLE) peptides in Arabidopsis thaliana and Oryza sativa.

Using 26 chemically synthetic CLAVATA3/ESR (CLE) peptides, which correspond to the predicted products of the 31 Arabidopsis CLE genes, we investigated the CLE peptide function in Arabidopsis and rice. Treatment with some CLE peptides inhibited root elongation in rice as well as in Arabidopsis. It also reduced the size of the shoot apical meristem in Arabidopsis but not in rice. Database searches revealed 47 putative CLE genes in the rice genome and multiple CLE domains in some CLE genes, indicating diverse CLE function in these plants.

A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis.

The Arabidopsis CLAVATA3 (CLV3) gene encodes a stem cell-specific protein presumed to be a precursor of a secreted peptide hormone. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) applied to in situ Arabidopsis tissues determined the structure of a modified 12-amino acid peptide (MCLV3), which was derived from a conserved motif in the CLV3 sequence. Synthetic MCLV3 induced shoot and root meristem consumption as cells differentiated into other organs, displaying the typical phenotype of transgenic plants overexpressing CLV3. These results suggest that the functional peptide of CLV3 is MCLV3.

CLV3/ESR-related (CLE) peptides as intercellular signaling molecules in plants.

For many years, the plant hormones auxin, cytokinin, ethylene, gibberellin, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid have been extensively studied as key regulators of plant growth and development. However, recent biochemical and genetic analyses have revealed that secretory peptides are also responsible for intercellular signaling in plants and regulate various events including wound response, cell division control, and pollen self-incompatibility. We discovered two natural CLAVATA3 (CLV3)/ESR-related (CLE) peptides: tracheary elements differentiation inhibitory factor (TDIF) and CLV3, which are dodecapeptides with two hydroxyproline residues that regulate vascular development and meristem formation, respectively. This discovery enabled us to predict the chemical form of CLE gene products. In the Arabidopsis genome, there are 31 CLE genes that correspond to 26 CLE peptides. The application of all 26 chemically synthesized peptides to plants revealed the existence of distinctive functional groups. From these results, we discuss the functions of CLE peptides in plant development and plant-parasite interactions.