Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severly affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.     

Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice

Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.     

Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants

The rice homeobox gene OSH15 (Oryza sativa homeobox) is a member of the knotted1-type homeobox gene family. We report here on the identification and characterization of a loss-of-function mutation in OSH15 from a library of retrotransposon-tagged lines of rice. Based on the phenotype and map position, we have identified three independent deletion alleles of the locus among conventional morphological mutants. All of these recessive mutations, which are considered to be null alleles, exhibit defects in internode elongation. Introduction of a 14 kbp genomic DNA fragment that includes all exons, introns and 5'- and 3'- flanking sequences of OSH15 complemented the defects in internode elongation, confirming that they were caused by the loss-of-function of OSH15. Internodes of the mutants had abnormal-shaped epidermal and hypodermal cells and showed an unusual arrangement of small vascular bundles. These mutations demonstrate a role for OSH15 in the development of rice internodes. This is the first evidence that the knotted1-type homeobox genes have roles other than shoot apical meristem formation and/or maintenance in plant development.     

Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice

The mechanism regulating the pattern of leaf initiation was analyzed by using shoot organization (sho) mutants derived from three loci (SHO1, SHO2, and SHO3). In the early vegetative phase, sho mutants show an increased rate of leaf production with random phyllotaxy. The resulting leaves are malformed, threadlike, or short and narrow. Their shoot apical meristems are relatively low and wide, that is, flat shaped, although their shape and size are highly variable among plants of the same genotype. Statistical analysis reveals that the shape of the shoot meristem rather than its size is closely correlated with the variations of plastochron and phyllotaxy. Rapid and random leaf production in sho mutants is correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a rice homeobox gene, OSH1. These changes in the organization and behavior of the shoot apical meristems suggest that sho mutants have fewer indeterminate cells and more determinate cells than wild type, with many cells acting as leaf founder cells. Thus, the SHO genes have an important role in maintaining the proper organization of the shoot apical meristem, which is essential for the normal initiation pattern of leaf primordia.     

The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model

We have isolated and characterized four tobacco homeobox genes, NTH1, NTH9, NTH20, NTH22 (Nicotiana tabacum homeobox) which belong to the class 1 knotted1-type family of homeobox genes. Comparison of the inferred amino acid sequences of the ELK homeodomains of these genes and previously reported kn1-type class 1 proteins has revealed that the four new tobacco genes belong to distinct subclasses, suggesting that each NTH gene may have distinct functions. Using in situ hybridization and by analysing the distribution of GUS activity in tobacco plants transformed with NTH promoter::GUS constructs, localized expression of the three NTH genes was observed in the shoot apical meristem (SAM). In the vegetative SAM, NTH1 and NTH15 showed overlapping expression in the corpus, NTH20 was expressed in the peripheral zone, and NTH9 was predominantly expressed in the rib zone. The expression patterns of the different NTH genes correspond to regions predicted by the cytohistological zonation model, suggesting that each NTH gene specifies the function of the SAM zone with which it is associated.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems

Members of the class 1 knotted-like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.     

Over-expression of a tobacco homeobox gene, NTH15 , decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase

Ectopic expression of the homeobox gene, NTH15 ( Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA₁. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA₁₉ to GA₂₀ was blocked in transgenic tobacco plants overexpressing NTH15 . To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3β-hydroxylase. RNA gel blot analysis revealed that the expression of one gene ( Ntc12 , encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA₂₀ or GA₁ but not by GA₁₉. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12 , resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.     

Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes

The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented.     

The patterns of gene expression in the tomato shoot apical meristem.

In this paper, we describe the synthesis of a cDNA library from the vegetative shoot apical meristem and the analysis of clones selected from it. Using in situ hybridization, we characterized the patterns of expression of these genes in the tomato shoot apical meristem, as well as the patterns obtained from other sources. The results from the analysis of 15 cDNAs indicated the following six main patterns of gene expression in the shoot apical meristem: overall expression, zero expression, expression limited to the epidermis, expression excluded from the epidermis, punctate expression, and expression elevated in the flanks of the meristem. The patterns observed and the nature and number of the genes showing these patterns necessitate a reinterpretation of the models of meristem structure and function. In particular, the data suggest a compartmentation within the shoot apical meristem based on the spatial expression of particular subsets of genes. This paper also reports on the specific and precise criteria essential for the correct identification of meristem-specific gene expression. The data give new insight into the molecular organization of the shoot apical meristem and provide the framework for a detailed dissection of the factors controlling this organization.     

Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes.

The regulatory mechanism of shoot apical meristem (SAM) initiation is an important subject in developmental plant biology. We characterized nine recessive mutations derived from four independent loci (SHL1-SHL4) causing the deletion of the SAM. Radicles were produced in these mutant embryos. Concomitant with the loss of SAM, two embryo-specific organs, coleoptile and epiblast, were lost, but the scutellum was formed normally. Therefore, differentiation of radicle and scutellum is regulated independently of SAM, but that of coleoptile and epiblast may depend on SAM. Regeneration experiments using adventitious shoots from the scutellum-derived calli showed that no adventitious shoots were regenerated in any shl mutant. However, small adventitious leaves were observed in both mutant and wild-type calli, but they soon became necrotic and showed no extensive growth. Thus, leaf primordia can initiate in the absence of SAM, but their extensive growth requires the SAM. An in situ hybridization experiment using a rice homeobox gene, OSH1, as a probe revealed that shl1 and shl2 modified the expression domain of OSH1, but normal expression of OSH1 was observed in shl3 and shl4 embryos. Accordingly, SHL1 and SHL2 function upstream of OSH1, and SHL3 and SHL4 downstream or independently of OSH1. These shl mutants are useful for elucidating the genetic program driving SAM initiation and for unraveling the interrelationships among various organs in grass embryos.     

Over-expression of tobacco knotted1-type class1 homeobox genes alters various leaf morphology

We compared the phenotypes of transgenic tobacco plants over-expressing various knotted1-type class1 homeobox genes. All transformants showed abnormal leaf morphology, with the degree of abnormality depending upon the Nicotiana tabacum homeobox (NTH) gene that was over-expressed. Tobacco plants over-expressing NTH1 or NTH9 showed a relatively weak phenotype, while NTH15 and NTH20 over-expressing plants exhibited severe alterations, with occasional ectopic shoot formation on the leaves. Plants over-expressing NTH22 had a relatively severe phenotype, but did not form any ectopic shoots. These results indicate that all of the NTH genes can influence leaf development from the shoot apical meristem, but that the effect varies with the gene. Based on phylogenetic analysis of the NTH genes and comparison of the phenotypes of plants over-expressing them, we suggest that the kn1-type class1 family can be divided into two subgroups, and that the differences in their ability to induce the abnormal phenotype corresponds to the structures of their conserved domains.     

Regional expression of the rice KN1-type homeobox gene family during embryo, shoot, and flower development.

We report the isolation, sequence, and pattern of gene expression of members of the KNOTTED1 (KN1)-type class 1 homeobox gene family from rice. Phylogenetic analysis and mapping of the rice genome revealed that all of the rice homeobox genes that we have isolated have one or two direct homologs in maize. Of the homeobox genes that we tested, all exhibited expression in a restricted region of the embryo that defines the position at which the shoot apical meristem (SAM) would eventually develop, prior to visible organ formation. Several distinct spatial and temporal expression patterns were observed for the different genes in this region. After shoot formation, the expression patterns of these homeobox genes were variable in the region of the SAM. These results suggest that the rice KN1-type class 1 homeobox genes function cooperatively to establish the SAM before shoot formation and that after shoot formation, their functions differ.     

KNOX homeobox genes are sufficient in maintaining cultured cells in an undifferentiated state in rice

We produced transgenic rice calli, which constitutively express each of four KNOX family class 1 homeobox genes of rice, OSH1, OSH16, OSH15, and OSH71, and found that constitutive and ectopic expression of such genes inhibits normal regeneration from transformed calli, which showed continuous growth around their shoot-regenerating stages. Transgenic calli transferred onto regeneration medium began to display green spots, a sign of regeneration, but most of the transformants continued to propagate green spots at given stages. In the normal shoot-regeneration process of calli, expression of endogenous OSH1 was restricted in presumptive shoot-regenerating regions of calli and not observed in other areas. This restricted expression pattern should be required for further differentiation of the regenerating shoots. Thus our present results support the proposed function that KNOX family class 1 homeobox genes play a role in the formation and maintenance of the undetermined meristematic state of cells.     

KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis.

Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.     

Expression of CENTRORADIALIS (CEN) and CEN-like genes in tobacco reveals a conserved mechanism controlling phase change in diverse species.

Plant species exhibit two primary forms of flowering architecture, namely, indeterminate and determinate. Antirrhinum is an indeterminate species in which shoots grow indefinitely and only generate flowers from their periphery. Tobacco is a determinate species in which shoot meristems terminate by converting to a flower. We show that tobacco is responsive to the CENTRORADIALIS (CEN) gene, which is required for indeterminate growth of the shoot meristem in Antirrhinum. Tobacco plants overexpressing CEN have an extended vegetative phase, delaying the switch to flowering. Therefore, CEN defines a conserved system controlling shoot meristem identity and plant architecture in diverse species. To understand the underlying basis for differences between determinate and indeterminate architectures, we isolated CEN-like genes from tobacco (CET genes). In tobacco, the CET genes most similar to CEN are not expressed in the main shoot meristem; their expression is restricted to vegetative axillary meristems. As vegetative meristems develop into flowering shoots, CET genes are downregulated as floral meristem identity genes are upregulated. Our results suggest a general model for tobacco, Antirrhinum, and Arabidopsis, whereby the complementary expression patterns of CEN-like genes and floral meristem identity genes underlie different plant architectures.