Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

Developmental programmes in floral organ formation

In contrast to animals, organogenesis in plants is continuous, allowing development in response to intrinsic and extrinsic signals. Organs arise from primordia formed on the flanks of meristems. The apical meristem produces primordia that acquire leaf identity, while floral meristems form primordia which develop into four organ types: sepals, petals, stamens and carpels. The production of mature organs involves two distinct processes, the initiation of organ primordia and the establishment of meristem, primordia and cell identities. Here we concentrate on floral organogenesis in Arabidopsis and examine the extent to which these processes utilize similar control mechanisms and regulatory molecules.     

The early phase change gene in maize

Recessive mutations of the early phase change (epc) gene in maize affect several aspects of plant development. These mutations were identified initially because of their striking effect on vegetative phase change. In certain genetic backgrounds, epc mutations reduce the duration of the juvenile vegetative phase of development and cause early flowering, but they have little or no effect on the number of adult leaves. Except for a transient delay in leaf production during germination, mutant plants initiate leaves at a normal rate both during and after embryogenesis. Thus, the early flowering phenotype of epc mutations is explained completely by their effect on the expression of the juvenile phase. The observation that epc mutations block the rejuvenation of leaf primordia in excised shoot apices supports the conclusion that epc is required for the expression of juvenile traits. This phenotype suggests that epc functions normally to promote the expression of the juvenile phase of shoot development and to suppress the expression of the adult phase and that floral induction is initiated by the transition to the adult phase. epc mutations are epistatic to the gibberellin-deficient mutation dwarf1 and interact additively with the dominant gain-of-function mutations Teopod1, Teopod2, and Teopod3. Genetic backgrounds that enhance the mutant phenotype of epc demonstrate that, in addition to its role in phase change, epc is required for the maintenance of the shoot apical meristem, leaf initiation, and root initiation.     

PLASTOCHRON2 regulates leaf initiation and maturation 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.     

The arabidopsis serrate gene encodes a zinc-finger protein required for normal shoot development

Organogenesis in plants depends upon the proper regulation of many genes, but how such necessary changes in gene expression are coordinated is largely unknown. The serrate (se) mutant of Arabidopsis displays defects in the initiation and elaboration of cotyledons and post-embryonic lateral organs. Cloning the SE gene revealed that it encodes a protein with a single, C(2)H(2)-type, zinc finger related to genes in other eukaryotes. Consistent with a role in organogenesis, the SE gene is transcribed in shoot meristems and in emerging organ primordia throughout development. Expression of the SE cDNA under the control of a heterologous promoter caused both accelerated and arrested plant growth, and these phenotypes were due to overexpression and co-suppression of the SE gene, respectively. Our analysis of the se mutant and the SE gene suggests a role for the SE gene product in regulating changes in gene expression via chromatin modification. Consistent with this proposed function, a synergistic double mutant phenotype was seen for plants mutant at both the SE locus and the locus encoding the largest subunit of chromatin assembly factor I.     

LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems

Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.     

Lateral root organogenesis - from cell to organ

Unlike locomotive organisms capable of actively approaching essential resources, sessile plants must efficiently exploit their habitat for water and nutrients. This involves root-mediated underground interactions allowing plants to adapt to soils of diverse qualities. The root system of plants is a dynamic structure that modulates primary root growth and root branching by continuous integration of environmental inputs, such as nutrition availability, soil aeration, humidity, or salinity. Root branching is an extremely flexible means to rapidly adjust the overall surface of the root system and plants have evolved efficient control mechanisms, including, firstly initiation, when and where to start lateral root formation; secondly lateral root primordia organogenesis, during which the development of primordia can be arrested for a certain time; and thirdly lateral root emergence. Our review will focus on the most recent advances in understanding the molecular mechanisms involved in the regulation of lateral root initiation and organogenesis with the main focus on root system of the model plant Arabidopsis thaliana.     

Developmental role and auxin responsiveness of Class III homeodomain leucine zipper gene family members in rice

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.     

Arabidopsis mutants with increased organ regeneration in tissue culture are more competent to respond to hormonal signals

Shoots and roots can be regenerated through organogenesis in tissue culture by subjecting plant explants to the appropriate regime of hormone treatments. In an effort to understand the control of shoot organogenesis, we screened for mutants in Arabidopsis thaliana (L.) Heynh. Columbia ecotype for enhanced shoot development at sub-optimal concentrations of cytokinin. Mutants in four different complementation groups were identified, one of which represents a new locus named increased organ regeneration1 (ire1) and another that is allelic to the previously identified pom1/erh2 mutant. Although the mutants were selected for their response to cytokinin, they were neither hypersensitive to, nor were they over-producers of cytokinins. The mutations identified in this study not only promote more robust shoot production in tissue culture, but also enhance green-callus and root formation. We interpret this to mean that, in tissue culture, IRE genes act before organ specification during the time when root explants acquire the competency to respond to organ formation signals. In normal plant development, IRE genes may down-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root organogenesis.     

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.     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.     

Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death

The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B(1)-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.     

The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis

Ubiquitin-mediated protein modification plays a key role in many cellular signal transduction pathways. The Arabidopsis gene XBAT32 encodes a protein containing an ankyrin repeat domain at the N-terminal half and a RING finger motif. The XBAT32 protein is capable of ubiquitinating itself. Mutation in XBAT32 causes a number of phenotypes including severe defects in lateral root production and in the expression of the cell division marker CYCB1;1::GUS . The XBAT32 gene is expressed abundantly in the vascular system of the primary root, but not in newly formed lateral root primordia. Treatment with auxin increases the expression of XBAT32 in the primary root and partially rescues the lateral root defect in xbat32 - 1 mutant plants. Thus, XBAT32 is a novel ubiquitin ligase required for lateral root initiation.     

Winter bud content according to position in 3-year-old branching systems of 'Granny Smith' apple

An investigation was made of the number of preformed organs in winter buds of 3-year-old reiterated complexes of the 'Granny Smith' cultivar. Winter bud content was studied with respect to bud position: terminal buds were compared on both long shoots and spurs according to branching order and shoot age, while axillary buds were compared between three zones (distal, median and proximal) along 1-year-old annual shoots in order 1. The percentage of winter buds that differentiated into inflorescences was determined and the flowers in each bud were counted for each bud category. The other organ categories considered were scales and leaf primordia. The results confirmed that a certain number of organs must be initiated before floral differentiation occurred. The minimum limit was estimated at about 15 organs on average, including scales. Total number of lateral organs formed was shown to vary with both bud position and meristem age, increasing from newly formed meristems to 1- and 2-year-old meristems on different shoot types. These differences in bud organogenesis depending on bud position, were consistent with the morphogenetic gradients observed in apple tree architecture. Axillary buds did not contain more than 15 organs on average and this low organogenetic activity of the meristems was related to a low number of flowers per bud. In contrast, the other bud categories contained more than 15 differentiated organs on average and a trade-off was observed between leaf and flower primordia. The ratio between the number of leaf and flower primordia per bud varied with shoot type. When the terminal buds on long shoots and spurs were compared, those on long shoots showed more flowers and a higher ratio of leaf to flower primordia.     

Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.     

From organelle to organ: ZRIZI MATE-Type transporter is an organelle transporter that enhances organ initiation

Plant architecture is a predictable but flexible trait. The timing and position of organ initiation from the shoot apical meristem (SAM) contribute to the final plant form. While much progress has been made recently in understanding how the site of leaf initiation is determined, the mechanism underlying the temporal interval between leaf primordia is still largely unknown. The Arabidopsis ZRIZI (ZRZ) gene belongs to a large gene family encoding multidrug and toxic compound extrusion (MATE) transporters. Unique among plant MATE transporters identified so far, ZRZ is localized to the membrane of a small organelle, possibly the mitochondria. Plants overexpressing ZRZ in initiating leaves are short, produce leaves much faster than wild-type plants and show enhanced growth of axillary buds. These results suggest that ZRZ is involved in communicating a leaf-borne signal that determines the rate of organ initiation.     

Characterization of the Lotus japonicus symbiotic mutant lot1 that shows a reduced nodule number and distorted trichomes

We isolated a recessive symbiotic mutant of Lotus japonicus that defines a genetic locus, LOT1 (for low nodulation and trichome distortion). The nodule number per plant of the mutant was about one-fifth of that of the wild type. The lot1 mutant showed a moderate dwarf phenotype and distorted trichomes, but its root hairs showed no apparent differences to those of the wild type. Infection thread formation after inoculation of Mesorhizobium loti was repressed in lot1 compared to that in the wild type. The nodule primordia of lot1 did not result in any aborted nodule-like structure, all nodules becoming mature and exhibiting high nitrogen fixation activity. The mutant was normally colonized by mycorrhizal fungi. lot1 also showed higher sensitivity to nitrate than the wild type. The grown-up seedlings of lot1 were insensitive to any ethylene treatments with regard to nodulation, although the mutant showed normal triple response on germination. It is conceivable that a nodulation-specific ethylene signaling pathway is constitutively activated in the mutant. Grafting experiments with lot1 and wild-type seedlings suggested that the root genotype mainly determines the low nodulation phenotype of the mutant, while the trichome distortion is regulated by the shoot genotype. Grafting of har1-4 shoots to lot1 roots resulted in an intermediate nodule number, i.e. more than that of lot1 and less than that of har1-4. Putative double mutants of lot1 and har1 also showed intermediate nodulation. Thus, it was indicated that LOT1 is involved in a distinct signal transduction pathway independent of HAR1.