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Shoot apical meristems (SAMs) continuously initiate organ formation and maintain pluripotency through dynamic genetic regulations and cell-to-cell communications. The activity of meristems directly affects the plant's structure by determining the number and arrangement of organs and tissues. We have taken a forward genetic approach to dissect the genetic pathway that controls cell differentiation around the SAM. The rice mutants, adaxial-abaxial bipolar leaf 1 and 2 (abl1 and abl2), produce an ectopic leaf that is fused back-to-back with the fourth leaf, the first leaf produced after embryogenesis. The abaxial-abaxial fusion is associated with the formation of an ectopic shoot meristem at the adaxial base of the fourth leaf primordium. We cloned the ABL1 and ABL2 genes of rice by mapping their chromosomal positions. ABL1 encodes OsHK6, a histidine kinase, and ABL2 encodes a transcription factor, OSHB3 (Class III homeodomain leucine zipper). Expression analyses of these mutant genes as well as OSH1, a rice ortholog of the Arabidopsis STM gene, unveiled a regulatory circuit that controls the formation of an ectopic meristem near the SAM at germination., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Fertilization controls various aspects of cereal growth such as tiller number, leaf size, and panicle size. However, despite such benefits, global chemical fertilizer use must be reduced to achieve sustainable agriculture. Here, based on field transcriptome data from leaf samples collected during rice cultivation, we identify fertilizer responsive genes and focus on Os1900, a gene orthologous to Arabidopsis thaliana MAX1, which is involved in strigolactone biosynthesis. Elaborate genetic and biochemical analyses using CRISPR/Cas9 mutants reveal that Os1900 together with another MAX1-like gene, Os5100, play a critical role in controlling the conversion of carlactone into carlactonoic acid during strigolactone biosynthesis and tillering in rice. Detailed analyses of a series of Os1900 promoter deletion mutations suggest that fertilization controls tiller number in rice through transcriptional regulation of Os1900, and that a few promoter mutations alone can increase tiller numbers and grain yields even under minor-fertilizer conditions, whereas a single defective os1900 mutation does not increase tillers under normal fertilizer condition. Such Os1900 promoter mutations have potential uses in breeding programs for sustainable rice production., (© 2023. The Author(s).)

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Rice leaves consist of three distinct regions along a proximal-distal axis, namely the leaf blade, sheath, and blade-sheath boundary region. Each region has a unique morphology and function, but the genetic programs underlying the development of each region are poorly understood. To fully elucidate rice leaf development and discover genes with unique functions in rice and grasses, it is crucial to explore genome-wide transcriptional profiles during the development of the three regions., Results: In this study, we performed microarray analysis to profile the spatial and temporal patterns of gene expression in the rice leaf using dissected parts of leaves sampled in broad developmental stages. The dynamics in each region revealed that the transcriptomes changed dramatically throughout the progress of tissue differentiation, and those of the leaf blade and sheath differed greatly at the mature stage. Cluster analysis of expression patterns among leaf parts revealed groups of genes that may be involved in specific biological processes related to rice leaf development. Moreover, we found novel genes potentially involved in rice leaf development using a combination of transcriptome data and in situ hybridization, and analyzed their spatial expression patterns at high resolution. We successfully identified multiple genes that exhibit localized expression in tissues characteristic of rice or grass leaves., Conclusions: Although the genetic mechanisms of leaf development have been elucidated in several eudicots, direct application of that information to rice and grasses is not appropriate due to the morphological and developmental differences between them. Our analysis provides not only insights into the development of rice leaves but also expression profiles that serve as a valuable resource for gene discovery. The genes and gene clusters identified in this study may facilitate future research on the unique developmental mechanisms of rice leaves.

Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 ( gle4 ). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Axis formation is a fundamental issue in developmental biology. Axis formation and patterning in plant leaves is crucial for morphology and crop productivity. Here, we reveal the basis of proximal-distal patterning in rice leaves, which consist of a proximal sheath, a distal blade, and boundary organs formed between these two regions. Analysis of the three rice homologs of the Arabidopsis BLADE-ON-PETIOLE1 (BOP1) gene indicates that OsBOPs activate proximal sheath differentiation and suppress distal blade differentiation. Temporal expression changes of OsBOPs are responsible for the developmental changes in the sheath:blade ratio. We further identify that the change in the sheath:blade ratio during the juvenile phase is controlled by the miR156/SPL pathway, which modifies the level and pattern of expression of OsBOPs. OsBOPs are also essential for differentiation of the boundary organs. We propose that OsBOPs, the main regulators of proximal-distal patterning, control temporal changes in the sheath:blade ratio of rice leaves.

Floods impede gas (O 2 and CO 2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Regulation of cell proliferation is crucial for establishing the shape of plant leaves. We have identified MAKIBA3 ( MKB3 ), a loss-of-function mutant of which exhibits a narrowed- and rolled-leaf phenotype in rice. MKB3 was found to be an ortholog of Arabidopsis ANGUSTIFOLIA3 ( AN3 ), which positively regulates cell proliferation. The reduced leaf size of mkb3 plants with enlarged cells and the increased size of MKB3- overexpressing leaves with normal-sized cells indicate that MKB3 is a positive regulator of leaf proliferation and that mkb3 mutation triggers a compensation syndrome, as does Arabidopsis an3 Expression analysis revealed that MKB3 is predominantly expressed on the epidermis of leaf primordia, which is different from the location of AN3 A protein movement assay demonstrated that MKB3 moves from an MKB3- expressing domain to a non-expressing domain, which is required for normal leaf development. Our results suggest that rice MKB3 and Arabidopsis AN3 have conserved functions and effects on leaf development. However, the expression pattern of MKB3 and direction of protein movement are different between rice and Arabidopsis , which might reflect differences in leaf primordia development in these two species., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

In several eudicot species, one copy of each member of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, WOX1 and WOX3, is redundantly or differentially involved in lateral leaf outgrowth, whereas only the WOX3 gene regulating the lateral domain of leaf development has been reported in grass. In this study, we show that a WOX3 gene, LEAF LATERAL SYMMETRY1 (LSY1), regulates lateral leaf development in a different manner ftom that of other duplicated paralogs of WOX3, NARROW LEAF2 (NAL2)/NAL3, in rice. A loss-of-function mutant of LSY1 exhibited an asymmetrical defect from early leaf development, which is different from a symmetric defect in a double loss-of-function mutant of NAL2/3, whereas the expression of both genes was observed in a similar domain in the margins of leaf primordia. Unlike NAL2/3, overexpression of LSY1 produced malformed leaves whose margins were curled adaxially. Expression domains and the level of adaxial/abaxial marker genes were affected in the LSY1-overexpressing plants, indicating that LSY1 is involved in regulation of adaxial-abaxial patterning at the margins of the leaf primordia. Additive phenotypes in some leaf traits of lsy1 nal2/3 triple mutants and the unchanged level of NAL2/3 expression in the lsy1 background suggested that LSY1 regulates lateral leaf development independently of NAL2/3. Our results indicated that all of the rice WOX3 genes are involved in leaf lateral outgrowth, but the functions of LSY1 and NAL2/3 have diverged. We propose that the function of WOX3 and the regulatory mode of leaf development in rice are comparable with those of WOX1/WOX3 in eudicot species.

Juvenile-to-adult phase transition is an important shift for the acquisition of adult vegetative characteristics and subsequent reproductive competence. We identified a recessive precocious (pre) mutant exhibiting a long leaf phenotype in rice. The long leaf phenotype is conspicuous in the second to the fourth leaves, which are juvenile and juvenile-to-adult transition leaves. We found that morphological and physiological traits, such as midrib formation, shoot meristem size, photosynthetic rate and plastochron, in juvenile and juvenile-to-adult transition stages of the pre mutant have precociously acquired adult characteristics. In agreement with these results, expression patterns of miR156 and miR172, which are microRNAs regulating phase change, support the accelerated juvenile-to-adult phase change in the pre mutant. The mutated gene encodes an allene oxide synthase (OsAOS1), which is a key enzyme for the biosynthesis of jasmonic acid (JA). The pre mutant showed a low level of JA and enhanced sensitivity to gibberellic acid, which promotes the phase change in some plant species. We also show that prolonged plastochron in the pre mutant is caused by accelerated PLASTOCHRON1 (PLA1) function. The present study reveals a substantial role of JA as a negative regulator of vegetative phase change., (© 2016. Published by The Company of Biologists Ltd.)

Embryogenesis in rice is different from that of most dicotolydonous plants in that it shows a non-stereotypic cell division pattern, formation of dorsal-ventral polarity, and endogenous initiation of the radicle. To reveal the transcriptional features associated with developmental events during rice early embryogenesis, we used microarray analysis coupled with laser microdissection to obtain both spatial and temporal transcription profiles. Our results allowed us to determine spatial expression foci for each expressed gene in the globular embryo, which revealed the importance of phytohormone-related genes and a suite of transcription factors to early embryogenesis. Our analysis showed the polarized expression of a small number of genes along the apical-basal and dorsal-ventral axes in the globular embryo, which tended to fluctuate in later developmental stages. We also analyzed gene expression patterns in the early globular embryo and how this relates to expression in embryonic organs at later stages. We confirmed the accuracy of the expression patterns found by microarray analysis of embryo subdomains using in situ hybridization. Our study identified homologous genes from Arabidopsis thaliana with known functions in embryogenesis in addition to unique and uncharacterized genes that show polarized expression patterns during embryogenesis. The results of this study are presented in a database to provide a framework for spatiotemporal gene expression during rice embryogenesis, to serve as a resource for future functional analysis of genes, and as a basis for comparative studies of plant embryogenesis., (© 2016. Published by The Company of Biologists Ltd.)

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

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.

Similarity of gene expression across a wide range of biological conditions can be efficiently used in characterization of gene function. We have constructed a rice gene coexpression database, RiceFREND (http://ricefrend.dna.affrc.go.jp/), to identify gene modules with similar expression profiles and provide a platform for more accurate prediction of gene functions. Coexpression analysis of 27 201 genes was performed against 815 microarray data derived from expression profiling of various organs and tissues at different developmental stages, mature organs throughout the growth from transplanting until harvesting in the field and plant hormone treatment conditions, using a single microarray platform. The database is provided with two search options, namely, 'single guide gene search' and 'multiple guide gene search' to efficiently retrieve information on coexpressed genes. A user-friendly web interface facilitates visualization and interpretation of gene coexpression networks in HyperTree, Cytoscape Web and Graphviz formats. In addition, analysis tools for identification of enriched Gene Ontology terms and cis-elements provide clue for better prediction of biological functions associated with the coexpressed genes. These features allow users to clarify gene functions and gene regulatory networks that could lead to a more thorough understanding of many complex agronomic traits.

Phyllotaxy is defined as the spatial arrangement of leaves on the stem. The mechanism responsible for this extremely regular pattern is one of the most fascinating enigmas in plant biology. In this study, we identified a gene regulating the phyllotactic pattern in rice. Loss-of-function mutants of the DECUSSATE (DEC) gene displayed a phyllotactic conversion from normal distichous pattern to decussate. The dec mutants had an enlarged shoot apical meristem with enhanced cell division activity. In contrast to the shoot apical meristem, the size of the root apical meristem in the dec mutants was reduced, and cell division activity was suppressed. These phenotypes indicate that DEC has opposite functions in the shoot apical meristem and root apical meristem. Map-based cloning revealed that DEC encodes a plant-specific protein containing a glutamine-rich region and a conserved motif. Although its molecular function is unclear, the conserved domain is shared with fungi and animals. Expression analysis showed that several type A response regulator genes that act in the cytokinin signaling pathway were down-regulated in the dec mutant. In addition, dec seedlings showed a reduced responsiveness to exogenous cytokinin. Our results suggest that DEC controls the phyllotactic pattern by affecting cytokinin signaling in rice.

RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE), which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs) and are processed into small RNAs (small interfering RNAs, siRNAs) that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA) to suppress host silencing. Members of the microRNA820 (miR820) gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements., Competing Interests: The authors have declared that no competing interests exist.

Rice PLASTOCHRON 1 (PLA1) and PLA2 genes regulate leaf maturation and plastochron, and their loss-of-function mutants exhibit small organs and rapid leaf emergence. They encode a cytochrome P450 protein CYP78A11 and an RNA-binding protein, respectively. Their homologs in Arabidopsis and maize are also associated with plant development/organ size. Despite the importance of PLA genes in plant development, their molecular functions remain unknown. Here, we investigated how PLA1 and PLA2 genes are related to phytohormones. We found that gibberellin (GA) is the major phytohormone that promotes PLA1 and PLA2 expression. GA induced PLA1 and PLA2 expression, and conversely the GA-inhibitor uniconazole suppressed PLA1 and PLA2 expression. In pla1-4 and pla2-1 seedlings, expression levels of GA biosynthesis genes and the signal transduction gene were similar to those in wild-type seedlings. GA treatment slightly down-regulated the GA biosynthesis gene GA20ox2 and up-regulated the GA-catabolizing gene GA2ox4, whereas the GA biosynthesis inhibitor uniconazole up-regulated GA20ox2 and down-regulated GA2ox4 both in wild-type and pla mutants, suggesting that the GA feedback mechanism is not impaired in pla1 and pla2. To reveal how GA signal transduction affects the expression of PLA1 and PLA2, PLA expression in GA-signaling mutants was examined. In GA-insensitive mutant, gid1 and less-sensitive mutant, Slr1-d1, PLA1 and PLA2 expression was down-regulated. On the other hand, the expression levels of PLA1 and PLA2 were highly enhanced in a GA-constitutive-active mutant, slr1-1, causing ectopic overexpression. These results indicate that both PLA1 and PLA2 act downstream of the GA signal transduction pathway to regulate leaf development.

The temporal and spatial control of meristem identity is a key element in plant development. To better understand the molecular mechanisms that regulate inflorescence and flower architecture, we characterized the rice aberrant panicle organization 2 (apo2) mutant which exhibits small panicles with reduced number of primary branches due to the precocious formation of spikelet meristems. The apo2 mutants also display a shortened plastochron in the vegetative phase, late flowering, aberrant floral organ identities and loss of floral meristem determinacy. Map-based cloning revealed that APO2 is identical to previously reported RFL gene, the rice ortholog of the Arabidopsis LEAFY (LFY) gene. Further analysis indicated that APO2/RFL and APO1, the rice ortholog of Arabidopsis UNUSUAL FLORAL ORGANS, act cooperatively to control inflorescence and flower development. The present study revealed functional differences between APO2/RFL and LFY. In particular, APO2/RFL and LFY act oppositely on inflorescence development. Therefore, the genetic mechanisms for controlling inflorescence architecture have evolutionarily diverged between rice (monocots) and Arabidopsis (eudicots)., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The rice peter pan syndrome-1 (pps-1) mutant shows a prolonged juvenile phase and early flowering. Although the early vegetative phase and flowering time of pps-1 have been closely examined, the phenotypes in the late vegetative and reproductive phases are not yet well understood. In the ninth leaf blade of pps-1, the relative length of the midrib was comparable to the sixth leaf blade of wild-type. Moreover, pps-1 had a small inflorescence meristem and small panicles. These phenotypes indicate that in pps-1 the juvenile phase coexists with the late vegetative phase, resulting in small panicles. Gibberellin is known to promote the juvenile-adult phase transition. d18-k is dwarf and has a prolonged juvenile phase. Double mutant (d18-k pps-1) showed the same phenotype as the pps-1, indicating that PPS is upstream of GA biosynthetic genes.

Because plant reproductive development occurs only in adult plants, the juvenile-to-adult phase change is an indispensable part of the plant life cycle. We identified two allelic mutants, peter pan syndrome-1 (pps-1) and pps-2, that prolong the juvenile phase in rice (Oryza sativa) and showed that rice PPS is an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC1. The pps-1 mutant exhibits delayed expression of miR156 and miR172 and the suppression of GA biosynthetic genes, reducing the GA(3) content in this mutant. In spite of its prolonged juvenile phase, the pps-1 mutant flowers early, and this is associated with derepression of RAP1B expression in pps-1 plants independently of the Hd1-Hd3a/RFT1 photoperiodic pathway. PPS is strongly expressed in the fourth and fifth leaves, suggesting that it regulates the onset of the adult phase downstream of MORI1 and upstream of miR156 and miR172. Its ability to regulate the vegetative phase change and the time of flowering suggests that rice PPS acquired novel functions during the evolution of rice/monocots.

In rice (Oryza sativa), trans-acting small interfering RNA (ta-siRNA) is essential for shoot development, including shoot apical meristem (SAM) formation and leaf morphogenesis. The rice wavy leaf1 (waf1) mutant has been identified as an embryonic mutant resembling shoot organization1 (sho1) and sho2, homologs of a loss-of-function mutant of DICER-LIKE4 and a hypomorphic mutant of ARGONAUTE7, respectively, which both act in the ta-siRNA production pathway. About half of the waf1 mutants showed seedling lethality due to defects in SAM maintenance, but the rest survived to the reproductive phase and exhibited pleiotropic phenotypes in leaf morphology and floral development. Map-based cloning of WAF1 revealed that it encodes an RNA methyltransferase, a homolog of Arabidopsis (Arabidopsis thaliana) HUA ENHANCER1. The reduced accumulation of small RNAs in waf1 indicated that the stability of the small RNA was decreased. Despite the greatly reduced level of microRNAs and ta-siRNA, microarray and reverse transcription-polymerase chain reaction experiments revealed that the expression levels of their target genes were not always enhanced. A double mutant between sho and waf1 showed an enhanced SAM defect, suggesting that the amount and/or quality of ta-siRNA is crucial for SAM maintenance. Our results indicate that stabilization of small RNAs by WAF1 is indispensable for rice development, especially for SAM maintenance and leaf morphogenesis governed by the ta-siRNA pathway. In addition, the inconsistent relationship between the amount of small RNAs and the level of the target mRNA in waf1 suggest that there is a complex regulatory mechanism that modifies the effects of microRNA/ta-siRNA on the expression of the target gene.

Leaf veins have a complex network pattern. Formation of this vein pattern has been widely studied as a model of tissue pattern formation in plants. To understand the molecular mechanism governing the vascular patterning process, we isolated the rice mutant, commissural vein excessive1 (coe1). The coe1 mutants had short commissural vein (CV) intervals and produced clustered CVs. Application of 1-N-naphthylphthalamic acid and brefeldin A decreased CV intervals, and application of 1-naphthaleneacetic acid increased CV intervals in wild-type rice; however, coe1 mutants were insensitive to these chemicals. COE1 encodes a leucine-rich repeat receptor-like kinase, whose amino acid sequence is similar to that of brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and which is localized at the plasma membrane. Because of the sequence similarity of COE1 to BAK1, we also examined the involvement of brassinosteroids in CV formation. Brassinolide, an active brassinosteroid, decreased the CV intervals of wild-type rice, and brassinazole, an inhibitor of brassinosteroid biosynthesis, increased the CV intervals of wild-type rice, but coe1 mutants showed insensitivity to these chemicals. These results suggest that auxin and brassinosteroids regulate CV intervals in opposite directions, and COE1 may regulate CV intervals downstream of auxin and brassinosteroid signals., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development.

Numerous genes are involved in the regulation of plant development, including those that regulate floral homeotic genes, We identified two recessive allelic rice mutants, open beak-1 (opb-1) and opb-2, which exhibited pleiotropic defects in leaf morphogenesis, inflorescence architecture, and floral organ identity. Abnormal cell proliferation was observed in the leaves and spikelets, and ectopic or overexpression of several class 1 knox genes was detected; thus, the abnormal cell proliferation in opb mutants is probably caused by ectopic class 1 knox gene expression. The opb mutants also had defects in floral organ identity, resulting in the development of mosaic organs, including gluminous lodicules, staminoid lodicules, and pistiloid stamens. These results, together with the reduced expression of a class B gene, indicate that OPB positively regulates the expression of class B genes. Map-based cloning revealed that OPB encodes a transcription factor that is orthologous to the Arabidopsis JAGGED gene and is expressed in leaf primordia, inflorescence meristem, rachis branch meristems, floral meristem, and floral organ primordia. Taken together, our data suggest that the OPB gene affects cellular proliferation and floral organ identity through the regulation of class 1 knox genes and floral homeotic genes.

Most aerial parts of the plant body are products of the continuous activity of the shoot apical meristem (SAM). Leaves are the major component of the aerial plant body, and their temporal and spatial distribution mainly determines shoot architecture. Here we report the identification of the rice gene PLASTOCHRON3 (PLA3)/GOLIATH (GO) that regulates various developmental processes including the rate of leaf initiation (the plastochron). PLA3/GO encodes a glutamate carboxypeptidase, which is thought to catabolize small acidic peptides and produce small signaling molecules. pla3 exhibits similar phenotypes to pla1 and pla2- a shortened plastochron, precocious leaf maturation and rachis branch-to-shoot conversion in the reproductive phase. However, in contrast to pla1 and pla2, pla3 showed pleiotropic phenotypes including enlarged embryo, seed vivipary, defects in SAM maintenance and aberrant leaf morphology. Consistent with these pleiotropic phenotypes, PLA3 is expressed in the whole plant body, and is involved in plant hormone homeostasis. Double mutant analysis revealed that PLA1, PLA2 and PLA3 are regulated independently but function redundantly. Our results suggest that PLA3 modulates various signaling pathways associated with a number of developmental processes.

We describe how rice leaves are regionalized and regulated along the central-marginal axis. The shoot organization2 (sho2) mutant, a weak allele of SHOOTLESS4 that is a ZIPPY/ARGONAUTE7 homolog in rice, shows a variety of leaf abnormalities; filamentous leaves, bi- or trifurcated leaves, separation of the filamentous structure from the leaf blade or deletion of the margin. All of these phenotypes can be interpreted as combinatorial defects in the growth of the central, lateral and marginal domains along the central-marginal axis, on the condition that the growth of the central domain is predominant. The leaf founder cells for the lateral and marginal domains are recruited normally in sho2, indicating that sho2 is defective in the growth of leaf domains after the founder cells are recruited. The expression pattern of SHO2 in the outer layer of the shoot apical meristem and the adaxial surface of the leaf, as well as the altered expression of HD-ZIP III and ETTIN homologs in the central domain of sho2 leaves, suggest that normal development of the central domain is a prerequisite for the synchronous growth of the three domains. This synchrony is thought to be mediated by a small interfering RNA-dependent process.

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner.

We isolated flattened shoot meristem (fsm) mutants in rice that showed defective seedling growth and died in the vegetative phase. Since most fsm plants had flat and small shoot apical meristems (SAMs), we suggest that FSM is required for proper SAM maintenance. FSM encodes a putative ortholog of Arabidopsis FASCIATA1 (FAS1) that corresponds to the p150 subunit of chromatin assembly factor-1 (CAF-1). FSM is expressed patchily in tissues with actively dividing cells, suggesting a tight association of FSM with specific cell-cycle phases. Double-target in situ hybridization counterstained with cell-cycle marker genes revealed that FSM is expressed mainly in the G(1) phase. In fsm, expressions of the two marker genes representing S- and G(2)- to M-phases were enhanced in SAM, despite a reduced number of cells in SAM, suggesting that S- and G(2)-phases are prolonged in fsm. In addition, developmental events in fsm leaves took place at the proper time, indicating that the temporal regulation of development occurs independently of the cell-cycle period. In contrast to the fasciated phenotype of Arabidopsis fas1, fsm showed size reduction of SAM. The opposite phenotypes between fsm and fas1 indicate that the SAM maintenance is regulated differently between rice and Arabidopsis.

Cleistogamy is an efficient strategy for preventing gene flow from genetically modified (GM) crops. We identified a cleistogamous mutant of rice harbouring a missense mutation (the 45th residue isoleucine to threonine; I45T) in the class-B MADS-box gene SUPERWOMAN1 (SPW1), which specifies the identities of lodicules (equivalent to petals) and stamens. In the mutant, spw1-cls, the stamens are normal, but the lodicules are transformed homeotically to lodicule-glume mosaic organs, thereby engendering cleistogamy. Since this mutation does not affect other agronomic traits, it can be used in crosses to produce transgenic lines that do not cause environmental perturbation. Molecular analysis revealed that the reduced heterodimerization ability of SPW1(I45T) with its counterpart class-B proteins OsMADS2 and OsMADS4 caused altered lodicule identity. spw1-cls is the first useful mutant for practical gene containment in GM rice. Cleistogamy is possible in many cereals by engineering class-B floral homeotic genes and thereby inducing lodicule identity changes.

The shoot apical meristem (SAM) is a group of stem cells that are responsible for plant development. Mutations in rice SHOOTLESS2 (SHL2), SHL4/SHOOT ORGANIZATION2 (SHO2), and SHO1 cause complete deletion or abnormal formation of the SAM. In this study we showed that defects in SAM formation in shl mutants are associated with the loss of expression of the homeodomain-leucine zipper (HD-ZIPIII) family genes. Rice SHL2, SHL4/SHO2, and SHO1 encoded orthologues of Arabidopsis RNA-dependent RNA polymerase 6, ARGONAUTE (AGO) 7, and DICER-like 4, respectively, whose mutations affect leaf development through the trans-acting siRNA (ta-siRNA) pathway. This suggested that the ta-siRNA pathway regulates the critical step of SAM formation during rice embryogenesis. The gain-of-function experiment by the ectopic expression of SHL4 resulted in reduced accumulation of an microRNA, miR166, and partial adaxialization of leaves, supporting a role for the ta-siRNA pathway in the maintenance of leaf polarity as previously reported in maize. Analysis of the spatiotemporal expression patterns of HD-ZIPIII and miR166 in wild-type and shl mutant embryos suggested that the loss of HD-ZIPIII expression in the SAM region of the developing embryo is the result of ectopic expression of miR166. Our analysis of shl mutants demonstrated that HD-ZIPIII expression regulated by miR166 is sensitive to the ta-siRNA pathway during SAM formation in rice embryogenesis.

In order to facilitate the functional analysis of rice genes, we produced about 50,000 insertion lines with the endogenous retrotransposon Tos17. Phenotypes of these lines in the M2 generation were observed in the field and characterized based on 53 phenotype descriptors. Nearly half of the lines showed more than one mutant phenotype. The most frequently observed phenotype was low fertility, followed by dwarfism. Phenotype data with photographs of each line are stored in the Tos17 mutant panel web-based database with a dataset of sequences flanking Tos17 insertion points in the rice genome (http://tos.nias.affrc.go.jp/). This combination of phenotypic and flanking sequence data will stimulate the functional analysis of rice genes.

In higher plants, the process of embryogenesis establishes the plant body plan (body axes). On the basis of positional information specified by the body axes, the shoot apical meristem (SAM) and root apical meristem (RAM) differentiate at fixed positions early in embryogenesis. After germination, SAM and RAM are responsible for the development of the above-ground and below-ground parts, respectively, of the plant. Because of the importance of SAM function in plant development, the mechanisms of SAM formation during embryogenesis and of SAM maintenance and function in post-embryonic development are priority questions in plant developmental biology. Recent advances in molecular and genetic analysis of morphogenetic mutations in Arabidopsis have revealed several components required for SAM formation, maintenance and function. Although these processes are fundamental to the life cycle of every plant, conservation of the components does not explain the diversity of plant morphologies. Rice is used as a model plant of the grass family and of monocots because of the progress in research infrastructure, especially the collection of unique mutations and genome information. In comparison with the dicot Arabidopsis, rice has many unique organs or processes of development. This review summarizes what is known of the processes of SAM formation, maintenance and function in rice.

In higher plants, leaves initiate in constant spatial and temporal patterns. Although the pattern of leaf initiation is a key element of plant shoot architecture, little is known about how the time interval between initiation events, termed plastochron, is regulated. Here, we present a detailed analysis of plastochron2 (pla2), a rice (Oryza sativa) mutant that exhibits shortened plastochron and precocious maturation of leaves during the vegetative phase and ectopic shoot formation during the reproductive phase. The corresponding PLA2 gene is revealed to be an orthologue of terminal ear1, a maize (Zea mays) gene that encodes a MEI2-like RNA binding protein. PLA2 is expressed predominantly in young leaf primordia. We show that PLA2 normally acts to retard the rate of leaf maturation but does so independently of PLA1, which encodes a member of the P450 family. Based on these analyses, we propose a model in which plastochron is determined by signals from immature leaves that act non-cell-autonomously in the shoot apical meristem to inhibit the initiation of new leaves.

Rice is becoming a model plant in monocotyledons and a model cereal crop. For better understanding of the rice plant, it is essential to elucidate the developmental programs of the life cycle. To date, several attempts have been made in rice to categorize the developmental processes of some organs into substages. These studies are based exclusively on the morphological and anatomical viewpoints. Recent advancement in genetics and molecular biology has given us new aspects of developmental processes. In this review, we first describe the phasic development of the rice plant, and then describe in detail the developmental courses of major organs, leaf, root and spikelet, and specific organs/tissues. Also, for the facility of future studies, we propose a staging system for each organ.

During postembryonic development of higher plants, the shoot apical meristem produces lateral organs in a regular spacing (phyllotaxy) and a regular timing (plastochron). Molecular analysis of mutants associated with phyllotaxy and plastochron would greatly increase understanding of the developmental mechanism of plant architecture because phyllotaxy and plastochron are fundamental regulators of plant architecture. pla1 of rice is not only a plastochron mutant showing rapid leaf initiation without affecting phyllotaxy, but also a heterochronic mutant showing ectopic shoot formation in the reproductive phase. Thus, pla1 provides a tool for analyzing the molecular basis of temporal regulation in leaf development. In this work, we isolated the PLA1 gene by map-based cloning. The identified PLA1 gene encodes a cytochrome P450, CYP78A11, which potentially catalyzes substances controlling plant development. PLA1 is expressed in developing leaf primordia, bracts of the panicle, and elongating internodes, but not in the shoot apical meristem. The expression pattern and mutant phenotype suggest that the PLA1 gene acting in developing leaf primordia affects the timing of successive leaf initiation and the termination of vegetative growth.

Asymmetric cell division is one of the most important mechanisms in the diversification of cell function and fate. In Arabidopsis, SCARECROW (SCR) is essential for the asymmetric division of the cortex/endodermis progenitor cell in the root. To learn more about how SCR is involved in asymmetric division, we analyzed the rice SCR (OsSCR) expression. In the root tip, OsSCR expression was observed in the endodermal cell layer and downregulated in the daughter cortex cell after asymmetric division, just as with Arabidopsis SCR. In leaf primordia, expression of OsSCR was observed in stomatal and ligule formation. In stomatal development, OsSCR was specifically expressed in the stomatal cell files before formation of guard mother cells (GMCs), and then, its expression was localized in GMCs, when the first asymmetric division occurred to generate the GMCs. Before the second asymmetric division of subsidiary mother cells (SMCs), localized OsSCR expression was observed in SMCs in the area close to the GMCs. Before these asymmetric divisions, the localization of OsSCR mRNA in GMC-forming cells and SMCs was observed in the area of the daughter GMC and subsidiary cells. OsSCR expression was also observed in the initiation area of ligule formation, and its downregulation occurred in the inner L2 cells generated by asymmetric division. Based on these observations, we proposed that OsSCR is involved not only in the asymmetric division of the cortex/endodermis progenitor cell but also during stomata and ligule formation by establishing the polarization of cytoplasm.

To characterize the SHL2 and SHL1 genes in detail, we analyzed three strains carrying weak alleles of SHL2, shl2-6, shl2-7, and shl2-8, and one weak allele of SHL1, shl1-3. In contrast to strong alleles, which result in lack of shoot meristem, strains bearing these weak alleles formed shoot meristem frequently during embryogenesis. In shl2-6 and shl2-7 mutants, the meristem was lost during seed development. Only the shl2-8 mutant could survive after germination, but it showed abnormal initiation pattern and morphology of leaves. In strains bearing the weak alleles, the shoot meristem was composed of a small number of indeterminate cells and ultimately converted into leaf primordium. The shl1-3 mutant showed phenotypes similar to those of shl2-8. Thus SHL2 and SHL1 are required for both initiation and maintenance of shoot meristem. In shl2 mutants, there was a positive correlation between the size of the expression domain of OSH1 representing the number of indeterminate cells, the frequency of shoot meristem initiation, and the duration of meristem survival. Thus the shoot meristem will not initiate in an "all-or-nothing" fashion, but is formed in various degrees depending on the strength of the alleles. Double-mutant analyses indicate that SHL2 functions upstream of SHO to establish proper organization of the shoot meristem.