Flower meristem maintenance by TILLERS ABSENT 1 is essential for ovule development in rice.

Plant development depends on the activity of pluripotent stem cells in meristems, such as the shoot apical meristem and the flower meristem. In Arabidopsis thaliana, WUSCHEL (WUS) is essential for stem cell homeostasis in meristems and integument differentiation in ovule development. In rice (Oryza sativa), the WUS ortholog TILLERS ABSENT 1 (TAB1) promotes stem cell fate in axillary meristem development, but its function is unrelated to shoot apical meristem maintenance in vegetative development. In this study, we examined the role of TAB1 in flower development. The ovule, which originates directly from the flower meristem, failed to differentiate in tab1 mutants, suggesting that TAB1 is required for ovule formation. Expression of a stem cell marker was completely absent in the flower meristem at the ovule initiation stage, indicating that TAB1 is essential for stem cell maintenance in the 'final' flower meristem. The ovule defect in tab1 was partially rescued by floral organ number 2 mutation, which causes overproliferation of stem cells. Collectively, it is likely that TAB1 promotes ovule formation by maintaining stem cells at a later stage of flower development.

Genetic basis controlling rice plant architecture and its modification for breeding.

The shoot and root system architectures are fundamental for crop productivity. During the history of artificial selection of domestication and post-domestication breeding, the architecture of rice has significantly changed from its wild ancestor to fulfil requirements in agriculture. We review the recent studies on developmental biology in rice by focusing on components determining rice plant architecture; shoot meristems, leaves, tillers, stems, inflorescences and roots. We also highlight natural variations that affected these structures and were utilized in cultivars. Importantly, many core regulators identified from developmental mutants have been utilized in breeding as weak alleles moderately affecting these architectures. Given a surge of functional genomics and genome editing, the genetic mechanisms underlying the rice plant architecture discussed here will provide a theoretical basis to push breeding further forward not only in rice but also in other crops and their wild relatives.

CURLED LATER1 encoding the largest subunit of the Elongator complex has a unique role in leaf development and meristem function in rice.

The Elongator complex, which is conserved in eukaryotes, has multiple roles in diverse organisms. In Arabidopsis thaliana, Elongator is shown to be involved in development, hormone action and environmental responses. However, except for Arabidopsis, our knowledge of its function is poor in plants. In this study, we initially carried out a genetic analysis to characterize a rice mutant with narrow and curled leaves, termed curled later1 (cur1). The cur1 mutant displayed a heteroblastic change, whereby the mutant leaf phenotype appeared specifically at a later adult phase of vegetative development. The shoot apical meristem (SAM) was small and the leaf initiation rate was low, suggesting that the activity of the SAM seemed to be partially reduced in cur1. We then revealed that CUR1 encodes a yeast ELP1-like protein, the largest subunit of Elongator. Furthermore, disruption of OsELP3 encoding the catalytic subunit of Elongator resulted in phenotypes similar to those of cur1, including the timing of the appearance of mutant phenotypes. Thus, Elongator activity seems to be specifically required for leaf development at the late vegetative phase. Transcriptome analysis showed that genes involved in protein quality control were highly upregulated in the cur1 shoot apex at the later vegetative phase, suggesting the restoration of impaired proteins probably produced by partial defects in translational control due to the loss of function of Elongator. The differences in the mutant phenotype and gene expression profile between CUR1 and its Arabidopsis ortholog suggest that Elongator has evolved to play a unique role in rice development.

Function of the TRY C-terminal region artificially fused with its homologous transcription factors inducing root hair differentiation in Arabidopsis.

TRIPTYCHON (TRY) is one of the R3-MYB transcription factors. Its extended C-terminal 19 amino-acid region (CTRY) is considered to affect the ability of root hair differentiation in Arabidopsis. Here, to further understand the function of CTRY, it, together with GFP, was artificially fused with TRY homologs, CPC and ETC1, which do not contain such extended regions and induce root hair differentiation. Arabidopsis transgenic plants carrying the fusion proteins, CPC-CTRY-GFP and ETC1-CTRY-GFP, induced root hair differentiation as observed in those carrying the original proteins without CTRY. The expression levels of the fusion proteins in the transgenic plants were essentially the same as those of the original proteins, although their subcellular localization to nuclei of root epidermal cells was slightly changed by CTRY. Therefore, CTRY does not affect the ability of CPC and ETC1 to induce root hair differentiation when artificially fused, and its function may be restricted in TRY.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

DWARF WITH SLENDER LEAF1 Encoding a Histone Deacetylase Plays Diverse Roles in Rice Development.

In plants, reversible histone acetylation and deacetylation play a crucial role in various biological activities, including development and the response to environmental stress. Histone deacetylation, which is generally associated with gene silencing, is catalyzed by multiple histone deacetylases (HDACs). Our understanding of HDAC function in plant development has accumulated from molecular genetic studies in Arabidopsis thaliana. By contrast, how HDACs contribute to the development of rice (Oryza sativa) is poorly understood and no rice mutants of HDAC have been reported. Here we have characterized a new rice mutant showing semi-dwarfism, which we named dwarf with slender leaf1 (dsl1). The mutant showed pleiotropic defects in both vegetative and reproductive developments; e.g. dsl1 produced short and narrow leaves, accompanied by a reduction in the number and size of vascular bundles. The semi-dwarf phenotype was due to suppression of the elongation of some culm (stem) internodes. Interestingly, despite this suppression of the upper internodes, the elongation and generation of lower internodes were slightly enhanced. Inflorescence and spikelet development were also affected by the dsl1 mutation. Some of the observed morphological defects were related to a reduction in cell numbers, in addition to reduced cell division in leaf primordia revealed by in situ hybridization analysis, suggesting the possibility that DSL1 is involved in cell division control. Gene cloning revealed that DSL1 encodes an HDAC belonging to the reduced potassium dependence3/histone deacetylase1 family. Collectively, our study shows that the HDAC DSL1 plays diverse and important roles in development in rice.

Antagonistic action of TILLERS ABSENT1 and FLORAL ORGAN NUMBER2 regulates stem cell maintenance during axillary meristem development in rice.

Shoot branches are formed from the axillary meristem and their formation is a key process in plant development. Although our understanding of the mechanisms underlying stem cell maintenance in the shoot apical meristem (SAM) is progressing, our knowledge of these mechanisms during the process of axillary meristem development is insufficient. To elucidate the genetic mechanisms underlying axillary meristem development in rice (Oryza sativa), we undertook a molecular genetic analysis focusing on TILLERS ABSENT1 (TAB1) and FLORAL ORGAN NUMBER2 (FON2), respective orthologs of the WUSCHEL and CLAVATA3 genes involved in SAM maintenance in Arabidopsis (Arabidopsis thaliana). We revealed that stem cells were established at an early stage of axillary meristem development in the wild-type, but were not maintained in tab1. By contrast, the stem cell region and TAB1 expression domain were expanded in fon2, and FON2 overexpression inhibited axillary meristem formation. These results indicate that TAB1 is required to maintain stem cells during axillary meristem development, whereas FON2 negatively regulates stem cell fate by restricting TAB1 expression. Thus, the genetic pathway regulating SAM maintenance in Arabidopsis seems to have been recruited to play a specific role within a narrow developmental window - namely, axillary meristem establishment - in rice.

Transcriptional Corepressor ASP1 and CLV-Like Signaling Regulate Meristem Maintenance in Rice.

Stem cell homeostasis is maintained by the WUSCHEL-CLAVATA (WUS-CLV) negative feedback loop in Arabidopsis (Arabidopsis thaliana). In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2) functions in the negative regulation of stem cell proliferation, similar to Arabidopsis CLV3 In this study, through genetic enhancer analysis, we found that loss of function of ABERRANT SPIKELET AND PANICLE1 (ASP1), encoding an Arabidopsis TOPLESS (TPL)-like transcriptional corepressor, enhances the fon2 flower phenotype, which displayed an increase in floral organ number. In the fon2 asp1 double mutant, the inflorescence was severely affected, resulting in bifurcation of the main axis (rachis), a phenotype that has not previously been reported. The stem cells showed marked overproliferation in fon2 asp1, resulting in extreme enlargement and splitting of the inflorescence meristem. These results suggest that ASP1 and FON2 synergistically regulate stem cell maintenance in rice. Unexpectedly, genetic analysis indicated that TILLERS ABSENT1, the rice ortholog of WUS, is not involved in promoting stem cell proliferation in this meristem. Transcriptome analysis suggested that ASP1 and FON signaling negatively regulate a set of genes with similar functions, and they act on these genes in concert. Taken together, our results suggest that TPL-like corepressor activity plays a crucial role in meristem maintenance, and that stem cell proliferation is properly maintained via the cooperation of ASP1 and FON2.

Rice Flower Development Revisited: Regulation of Carpel Specification and Flower Meristem Determinacy.

The ABC model in flower development represents a milestone of plant developmental studies and is essentially conserved across a wide range of angiosperm species. Despite this overall conservation, individual genes in the ABC model are not necessarily conserved and sometimes play a species-specific role, depending on the plant. We previously reported that carpels are specified by the YABBY gene DROOPING LEAF (DL) in rice (Oryza sativa), which bears flowers that are distinct from those of eudicots. In contrast, another group reported that carpels are specified by two class C genes, OsMADS3 and OsMADS58. Here, we have addressed this controversial issue by phenotypic characterization of floral homeotic gene mutants. Analysis of a complete loss-of-function mutant of OsMADS3 and OsMADS58 revealed that carpel-like organs expressing DL were formed in the absence of the two class C genes. Furthermore, no known flower organs including carpels were specified in a double mutant of DL and SUPERWOMAN1 (a class B gene), which expresses only class C genes in whorls 3 and 4. These results suggest that, in contrast to Arabidopsis, class C genes are not a key regulator for carpel specification in rice. Instead, they seem to be involved in the elaboration of carpel morphology rather than its specification. Our phenotypic analysis also revealed that, similar to its Arabidopsis ortholog CRABS CLAW, DL plays an important function in regulating flower meristem determinacy in addition to carpel specification.

Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1.

Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem.

Genetic Enhancer Analysis Reveals that FLORAL ORGAN NUMBER2 and OsMADS3 Co-operatively Regulate Maintenance and Determinacy of the Flower Meristem in Rice.

Meristems such as the shoot apical meristem and flower meristem (FM) act as a reservoir of stem cells, which reproduce themselves and supply daughter cells for the differentiation of lateral organs. In Oryza sativa (rice), the FLORAL ORGAN NUMBER2 (FON2) gene, which is similar to Arabidopsis CLAVATA3, is involved in meristem maintenance. In fon2 mutants, the numbers of floral organs are increased due to an enlargement of the FM. To identify new factors regulating meristem maintenance in rice, we performed a genetic screening of mutants that enhanced the fon2 mutation, and found a mutant line (2B-424) in which pistil number was dramatically increased. By using a map-based approach and next-generation sequencing, we found that the line 2B-424 had a complete loss-of-function mutation (a large deletion) in OsMADS3, a class C MADS-box gene that is known to be involved in stamen specification. Disruption of OsMADS3 in the fon2 mutant by CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology caused a flower phenotype similar to that of 2B-424, confirming that the gene responsible for enhancement of fon2 was OsMADS3. Morphological analysis showed that the fon2 and osmads3 mutations synergistically affected pistil development and FM determinacy. We also found that whorl 3 was duplicated in mature flowers and the FM was enlarged at an early developmental stage in severe osmads3 single mutants. These findings suggest that OsMADS3 is involved not only in FM determinacy in late flower development but also in FM activity in early flower development.

Three TOB1-related YABBY genes are required to maintain proper function of the spikelet and branch meristems in rice.

YABBY genes play important roles in the development of lateral organs such as leaves and floral organs in Angiosperms. However, the function of YABBY genes is poorly understood in monocots. We focused on three rice (Oryza sativa) YABBY genes, TONGARI-BOUSHI (TOB1, TOB2, TOB3), which are closely related to Arabidopsis (Arabidopsis thaliana) FILAMENTOUS FLOWER (FIL). To elucidate the function of these YABBY genes, we employed a reverse genetic approach. TOB genes were expressed in bract and lateral organ primordia, but not in meristems. RNAi knockdown of TOB2 or TOB3 in the tob1 mutant caused abnormal spikelet development. Furthermore, simultaneous knockdown of both TOB2 and TOB3 in tob1 affected not only spikelet, but also inflorescence development. In severe cases, the inflorescences comprised naked branches without spikelets. Analysis of inflorescence development at an early stage showed that the observed phenotypic defects were closely associated with a failure to initiate and maintain reproductive meristems. These results indicate that the TOB genes regulate the maintenance and fate of all reproductive meristems. It is likely that the function of FIL/TOB clade YABBY genes has been conserved between Arabidopsis and rice to maintain the proper function of meristems, even though these genes are expressed in lateral organ primordia.

WUSCHEL-RELATED HOMEOBOX4 is involved in meristem maintenance and is negatively regulated by the CLE gene FCP1 in rice.

The shoot apical meristem is the ultimate source of the cells that constitute the entire aboveground portion of the plant body. In Arabidopsis thaliana, meristem maintenance is regulated by the negative feedback loop of WUSCHEL-CLAVATA (WUS-CLV). Although CLV-like genes, such as FLORAL ORGAN NUMBER1 (FON1) and FON2, have been shown to be involved in maintenance of the reproductive meristems in rice (Oryza sativa), current understanding of meristem maintenance remains insufficient. In this article, we demonstrate that the FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2 genes encoding proteins with similar CLE domains are involved in negative regulation of meristem maintenance in the vegetative phase. In addition, we found that WUSCHEL-RELATED HOMEOBOX4 (WOX4) promotes the undifferentiated state of the meristem in rice and that WOX4 function is associated with cytokinin action. Consistent with similarities in the shoot apical meristem phenotypes caused by overexpression of FCP1 and downregulation of WOX4, expression of WOX4 was negatively regulated by FCP1 (FCP2). Thus, FCP1/2 and WOX4 are likely to be involved in maintenance of the vegetative meristem in rice.

The YABBY gene TONGARI-BOUSHI1 is involved in lateral organ development and maintenance of meristem organization in the rice spikelet.

The meristem initiates lateral organs in a regular manner, and proper communication between the meristem and the lateral organs ensures the normal development of plants. Here, we show that mutation of the rice (Oryza sativa) gene TONGARI-BOUSHI1 (TOB1) results in pleiotropic phenotypes in spikelets, such as the formation of a cone-shaped organ instead of the lemma or palea, the development of two florets in a spikelet, or premature termination of the floret meristem, in addition to reduced growth of the lemma or palea and elongation of the awn. These phenotypes seem to result from not only failure in growth of the lateral organs, but also defects in maintenance and organization of the meristem. For example, the cone-shaped organ develops as a ring-like primordium from an initial stage, suggesting that regulation of organ initiation in the meristem may be compromised. TOB1 encodes a YABBY protein, which is closely related to FILAMENTOUS FLOWER in Arabidopsis thaliana, and is expressed in the lateral organ primordia without any patterns of polarization. No TOB1 expression is detected in the meristem, so TOB1 may act non-cell autonomously to maintain proper meristem organization and is therefore likely to play an important role in rice spikelet development.

YABBY and diverged KNOX1 genes shape nodes and internodes in the stem.

Plant stems comprise nodes and internodes that specialize in solute exchange and elongation. However, their boundaries are not well defined, and how these basic units arise remains elusive. In rice with clear nodes and internodes, we found that one subclade of class I knotted1-like homeobox (KNOX1) genes for shoot meristem indeterminacy restricts node differentiation and allows internode formation by repressing YABBY genes for leaf development and genes from another node-specific KNOX1 subclade. YABBYs promote nodal vascular differentiation and limit stem elongation. YABBY and node-specific KNOX1 genes specify the pulvinus, which further elaborates the nodal structure for gravitropism. Notably, this KNOX1 subclade organization is specific to seed plants. We propose that nodes and internodes are distinct domains specified by YABBY-KNOX1 cross-regulation that diverged in early seed plants.

Grass meristems I: shoot apical meristem maintenance, axillary meristem determinacy and the floral transition.

The vegetative and reproductive shoot architectures displayed by members of the grass family are critical to reproductive success, and thus agronomic yield. Variation in shoot architecture is explained by the maintenance, activity and determinacy of meristems, pools of pluripotent stem cells responsible for post-embryonic plant growth. This review summarizes recent progress in understanding the major properties of grass shoot meristems, focusing on vegetative phase meristems and the floral transition, primarily in rice and maize. Major areas of interest include: the control of meristem homeostasis by the CLAVATA-WUSCHEL pathway and by hormones such as cytokinin; the initiation of axillary meristems and the control of axillary meristem dormancy; and the environmental and endogenous cues that regulate flowering time. In an accompanying paper, Tanaka et al. review subsequent stages of shoot development, including current knowledge of reproductive meristem determinacy and the fate transitions associated with these meristems.

Grass meristems II: inflorescence architecture, flower development and meristem fate.

Plant development depends on the activity of various types of meristems that generate organs such as leaves and floral organs throughout the life cycle. Grass species produce complex inflorescences and unique flowers. The grass inflorescence is composed of different types of branches, including a specialized branch called a spikelet. The spikelet is a special unit of the inflorescence and forms one to several florets, depending on the species. In the floret, floral organs such as perianth organs, carpels and stamens are formed. In Arabidopsis, because the inflorescence meristem (IM) forms the floral meristems (FMs) directly on its flanks, the change of meristem fate is relatively simple. In contrast, in grasses, different types of meristem, such as the IM, the branch meristem (BM), the spikelet pair meristem (SPM) in some grasses, the spikelet meristem (SM) and the FM, are responsible for the elaboration of their complex inflorescences and flowers. Therefore, sequential changes of meristem fate are required, and a number of genes involved in the specification of the fate of each meristem have been identified. In this review, we focus on the following issues concerning the fate of the reproductive meristems in two grass species, maize (Zea mays) and rice (Oryza sativa): (i) meristem regulation during inflorescence development; (ii) specification and fate change of the BM and the SM; (iii) determinacy of the FM; and (iv) communication between the meristem and lateral organs.

Complementation and protein localization analyses of R3 MYBs in an Arabidopsis caprice mutant.

Root hairs play vital roles in plant growth since they enable the efficient absorption of water and nutrients from the soil. Recent advances in Arabidopsis research have provided a deeper understanding of the molecular genetic mechanisms underlying root hair differentiation. CAPRICE (CPC) and its four homologs, which belong to the CPC gene family and encode R3 MYB transcription factors, play central roles in root hair differentiation. In this study, to better understand the functional specificity and contribution of these five CPC family genes, we conducted phenotypic and expression analyses of the CPC family proteins in a cpc mutant background. As a result, ENHANCER OF TRY AND CPC1 (ETC1) and ETC3 were found to complement the hairless root phenotype of the cpc mutant, as did CPC, whereas TRIPTYCHON (TRY) and ETC2 did not rescue the cpc phenotype. Protein expression analysis revealed that GFP fluorescence was nearly undetectable in pCPC::TRY:GFP/cpc and pCPC::ETC2:GFP/cpc plants, supporting the incapability of root hair formation in these plants. Interestingly, the fluorescence intensity of the CPC:GFP fusion protein was weaker than that of ETC1:GFP and ETC3:GFP fusion proteins. These results were inconsistent with the result of the phenotypic analysis, in which the three genes promoted root hair formation to almost the same degree in the cpc mutant background. We further discuss the discrepancy between the root hair phenotypes and the expression levels of CPC family proteins.

The homeotic gene long sterile lemma (G1) specifies sterile lemma identity in the rice spikelet.

The mechanism of floral organ specification is principally conserved in angiosperms, as demonstrated by the ABC model. By contrast, mechanisms that regulate the development of organs or structures specific to a group of species remain unclear. Grasses have unique inflorescence units, comprising spikelets and florets. In the genus Oryza (rice), the single spikelet consists of a fertile floret subtended by a lemma and a palea, two sterile lemmas, and rudimentary glumes. Each sterile lemma is a tiny glume-like organ with a smooth surface. Here, we have examined a long sterile lemma1 (g1) mutant, in which the sterile lemma is enlarged like the lemma. Detailed phenotypic analysis reveals that the large sterile lemma in the g1 mutant appears to be caused by homeotic transformation of the sterile lemma into a lemma, suggesting that G1 is involved in the repression of lemma identity to specify the sterile lemma. Gene isolation reveals that G1 is a member of a plant-specific gene family that encodes proteins with a previously uncharacterized domain, named here ALOG (Arabidopsis LSH1 and Oryza G1). G1 mRNA is expressed in sterile lemma primordia throughout their development, and G1 protein is localized in the nucleus. A trans-activation assay using the yeast GAL4 system suggests that G1 is involved in transcriptional regulation. Repression of lemma identity by G1 is consistent with a hypothesis proposed to explain the morphological evolution of rice spikelets. We also show that a wild rice species, Oryza grandiglumis, that forms large sterile lemmas has serious mutations in the G1 gene.