To understand the molecular mechanism regulating meristem development in the monocot rice (Oryza sativa), we describe here the isolation and characterization of three floral organ number4 (fon4) alleles and the cloning of the FON4 gene. The fon4 mutants showed abnormal enlargement of the embryonic and vegetative shoot apical meristems (SAMs) and the inflorescence and floral meristems. Likely due to enlarged SAMs, fon4 mutants produced thick culms (stems) and increased numbers of both primary rachis branches and floral organs. We identified FON4 using a map-based cloning approach and found it encodes a small putatively secreted protein, which is the putative ortholog of the Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) gene. FON4 transcripts mainly accumulated in the small group of cells at the apex of the SAMs, whereas the rice ortholog of CLV1 (FON1) is expressed throughout the SAMs, suggesting that the putative FON4 ligand might be sequestered as a possible mechanism for rice meristem regulation. Exogenous application of the peptides FON4p and CLV3p corresponding to the CLV3/ESR-related (CLE) motifs of FON4 and CLV3, respectively, resulted in termination of SAMs in rice, and treatment with CLV3p caused consumption of both rice and Arabidopsis root meristems, suggesting that the CLV pathway in limiting meristem size is conserved in both rice and Arabidopsis. However, exogenous FON4p did not have an obvious effect on limiting both rice and Arabidopsis root meristems, suggesting that the CLE motifs of Arabidopsis CLV3 and FON4 are potentially functionally divergent.

Unlike many wild grasses, domesticated rice cultivars have uniform culm height and panicle size among tillers and the main shoot, which is an important trait for grain yield. However, the genetic basis of this trait remains unknown. Here, we report that DWARF TILLER1 (DWT1) controls the developmental uniformity of the main shoot and tillers in rice (Oryza sativa). Most dwt1 mutant plants develop main shoots with normal height and larger panicles, but dwarf tillers bearing smaller panicles compared with those of the wild type. In addition, dwt1 tillers have shorter internodes with fewer and un-elongated cells compared with the wild type, indicating that DWT1 affects cell division and cell elongation. Map-based cloning revealed that DWT1 encodes a WUSCHEL-related homeobox (WOX) transcription factor homologous to the Arabidopsis WOX8 and WOX9. The DWT1 gene is highly expressed in young panicles, but undetectable in the internodes, suggesting that DWT1 expression is spatially or temporally separated from its effect on the internode growth. Transcriptomic analysis revealed altered expression of genes involved in cell division and cell elongation, cytokinin/gibberellin homeostasis and signaling in dwt1 shorter internodes. Moreover, the non-elongating internodes of dwt1 are insensitive to exogenous gibberellin (GA) treatment, and some of the slender rice1 (slr1) dwt1 double mutant exhibits defective internodes similar to the dwt1 single mutant, suggesting that the DWT1 activity in the internode elongation is directly or indirectly associated with GA signaling. This study reveals a genetic pathway synchronizing the development of tillers and the main shoot, and a new function of WOX genes in balancing branch growth in rice. Plant architecture is important for crop yield. In most plants, branches grow smaller than the main shoot, largely due to the ‘apical dominance’. However, in several cereal crops, including rice, wheat, and barley, the branches (tillers) have a height and size indistinguishable from the main shoot. The genetic basis of uniform tiller growth has remained elusive. We identified DWARF TILLER1, a WUSCHEL-related homeobox (WOX) transcription factor, as a positive regulator of tiller growth. Most dwt1 mutant plants show normal main shoot but dwarf tillers and reduced panicle size. Tiller growth in dwt1 appears to be inhibited by the main shoot, as removal of the main shoot releases the first tiller. The non-elongating internodes in dwt1 show reduced cell number and cell size, while DWT1 was mainly expressed in the panicles but not internodes, suggesting that DWT1 plays a long distance regulatory role in promoting internode elongation. Genome-wide expression analysis revealed that the expression of genes related to cell division and elongation, as well as to homeostasis and signaling of cytokinin and gibberellin were affected in dwt1 un-elongated internodes. This study reveals that a WOX transcription factor controls the growth uniformity of tillers and the main shoot in rice.

A decade of studies on middle cortex (MC) formation in the root endodermis of Arabidopsis (Arabidopsis thaliana) have revealed a complex regulatory network that is orchestrated by several GRAS family transcription factors, including SHORT-ROOT (SHR), SCARECROW (SCR), and SCARECROW-LIKE3 (SCL3). However, how their functions are regulated remains obscure. Here we show that mutations in the SEUSS (SEU) gene led to a higher frequency of MC formation. seu mutants had strongly reduced expression of SHR, SCR, and SCL3, suggesting that SEU positively regulates these genes. Our results further indicate that SEU physically associates with upstream regulatory sequences of SHR, SCR, and SCL3; and that SEU has distinct genetic interactions with these genes in the control of MC formation, with SCL3 being epistatic to SEU. Similar to SCL3, SEU was repressed by the phytohormone GA and induced by the GA biosynthesis inhibitor paclobutrazol, suggesting that SEU acts downstream of GA signaling to regulate MC formation. Consistently, we found that SEU mediates the regulation of SCL3 by GA signaling. Together, our study identifies SEU as a new critical player that integrates GA signaling with transcriptional inputs from the SHR-SCR-SCL3 module to regulate MC formation in the Arabidopsis root.

Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb), a mild gibberellin (GA) deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner. Little is known concerning the identity of regulatory components and signaling pathways that control the growth of meristem cells in plants. Here, we report that rice plants deficient in the AP2/ERF family gene SHOEBOX (SHB) exhibited a severe reduction in the root meristem size. These plants had shorter cells in the root meristems following germination and fewer cells from approximately 5 days after sowing, suggesting that SHB regulates root meristem cell size and number in a developmental stage-specific manner and that cell size participates in the control of root meristem size in rice. SHB is positively regulated by GA signaling and encodes a direct transcriptional activator of the GA biosynthesis gene KS1, indicating that GA modulates its own biosynthesis and consequently the elongation of meristem cells in rice roots via positive feedback regulation on the transcription of SHB and KS1. Consistently, application of exogenous GA restored the size of root meristem cells to normal in shb and paclobutrazol-treated wild-type plants.

Heading date and grain weight are two determining agronomic traits of crop yield. To date, molecular factors controlling both heading date and grain weight have not been identified. Here we report the isolation of a hemizygous mutation, heading and grain weight (hgw), which delays heading and reduces grain weight in rice. Analysis of hgw mutant phenotypes indicate that the hemizygous hgw mutation decreases latitudinal cell number in the lemma and palea, both composing the spikelet hull that is known to determine the size and shape of brown grain. Molecular cloning and characterization of the HGW gene showed that it encodes a novel plant-specific ubiquitin-associated (UBA) domain protein localized in the cytoplasm and nucleus, and functions as a key upstream regulator to promote expressions of heading date- and grain weight-related genes. Moreover, co-expression analysis in rice and Arabidopsis indicated that HGW and its Arabidopsis homolog are co-expressed with genes encoding various components of ubiquitination machinery, implying a fundamental role for the ubiquitination pathway in heading date and grain weight control.

CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins belong to a small peptide family conserved in plants. Recent studies in Arabidopsis and rice have revealed a key role for CLEs in mediating cell–cell communication and stem cell maintenance during plant development, but how CLE signalling controls root development in the rice remains largely unknown. Here it is shown that exogenous application of a synthetic dodeca-amino acid peptide corresponding to the CLE motif of the rice FON2-LIKE CLE PROTEIN2 (FCP2p) protein or overexpression of FCP2 terminates root apical meristem (RAM) activity and impairs late metaxylem formation. FCP2p treatment suppresses the expression of the rice QUIESCENT-CENTER-SPECIFIC HOMEOBOX (QHB) gene, a putative orthologue of Arabidopsis WUSCHEL (WUS)-RELATED HOMEOBOX 5 (WOX5) gene, in both quiescent centre and late metaxylem cells; whereas inducible overexpression of QHB reduces the sensitivity of rice to FCP2p treatment. These results together suggest that in rice RAM maintenance and late metaxylem development are probably controlled by the mutual regulation between FCP2 and QHB. Moreover, a cross-species peptide treatment experiment in Arabidopsis implies that FCP2 has both evolutionarily conserved and species-specific roles in root development.