Kiyotaka Okada

Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation.

The pin-formed mutant pin 1-1, one of the Arabidopsis flower mutants, has several structural abnormalities in inflorescence axes, flowers, and leaves. In some cases, pin1-1 forms a flower with abnormal structure (wide petals, no stamens, pistil-like structure with no ovules in the ovary) at the top of inflorescence axes. In other cases, no floral buds are formed on the axes. An independently isolated allelic mutant (pin1-2) shows similar phenotypes. These mutant phenotypes are exactly the same in wild-type plants cultured in the presence of chemical compounds known as auxin polar transport inhibitors: 9-hydroxyfluorene-9-carboxylic acid or N-(1-naphthyl)phthalamic acid. We tested the polar transport activity of indole-3-acetic acid and the endogenous amount of free indole-3-acetic acid in the tissue of inflorescence axes of the pin1 mutants and wild type. The polar transport activity in the pin 1-1 mutant and in the pin1-2 mutant was decreased to 14% and 7% of wild type, respectively. These observations strongly suggest that the normal level of polar transport activity in the inflorescence axes is required in early developmental stages of floral bud formation in Arabidopsis and that the primary function of the pin1 gene is auxin polar transport in the inflorescence axis.

Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains

SummaryThe efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.

Genetic analyses of signalling in flower development using Arabidopsis

Flower development can be divided into four major steps: phase transition from vegetative to reproductive growth, formation of inflorescence meristem, formation and identity determination of floral organs, and growth and maturation of floral organs. Intercellular and intracellular signalling mechanisms must have important roles in each step of flower development, because it requires cell division, cell growth, and cell differentiation in a concerted fashion. Molecular genetic analysis of the process has started by isolation of a series of mutants with unusual flowering time, with aberrant structure in inflorescence and in flowers, and with no self-fertilization. At present more than 60 genes are identified from Arabidopsis thaliana and some of them have cloned. Although the information is still limited, several types of signalling systems are revealed. In this review, we summarize the present genetic aspects of the signalling network underlying the processes of flower development.

A Comprehensive Expression Analysis of the Arabidopsis MICRORNA165/6 Gene Family during Embryogenesis Reveals a Conserved Role in Meristem Specification and a Non-cell-autonomous Function

One of the most fundamental events in plant ontogeny is the specification of the shoot and root apical meristem (SAM and RAM) in embryogenesis. In Arabidopsis, the restricted expression of class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs) at the central-apical domain of early embryos is required for the correct specification of the SAM and RAM. Because the expression of HD-ZIP III TFs is suppressed by microRNA165/166 (miR165/6), elucidation of the sites of miR165/6 production and their activity range is a key to understanding the molecular basis of SAM and RAM specification in embryogenesis. Here, we present a comprehensive reporter analysis of all nine Arabidopsis MICRORNA165/166 (MIR165/6) genes during embryogenesis. We show that five MIR165/6 genes are transcribed in a largely conserved pattern in embryos, with their expression being preferentially focused at the basal-peripheral region of embryos. Our analysis also indicated that MIR165/6 transcription does not depend on SCARECROW (SCR) function in early embryos, in contrast to its requirement in post-embryonic roots. Furthermore, by observing the expression pattern of the miR-resistant PHBmu-GFP (green fluorescent protein) reporter, in either the presence or absence of the MIR165Amu transgene, which targets PHBmu-GFP, we obtained data that indicate a non-cell-autonomous function for miR165 in early embryos. These results suggest that miR165, and possibly miR166 as well, has the capacity to act as a positional cue from the basal-peripheral region of early embryos, and remotely controls SAM and RAM specification with their non-cell-autonomous function.

Succinic Semialdehyde Dehydrogenase is Involved in the Robust Patterning of Arabidopsis Leaves along the Adaxial–Abaxial Axis

Polarity along the adaxial-abaxial axis of the leaf is essential for leaf development and morphogenesis. One of the genes that encodes a putative transcription factor regulating adaxial-abaxial polarity, FILAMENTOUS FLOWER (FIL), is expressed in the abaxial region of the leaf primordia. However, the molecular mechanisms controlling the polarized expression of FIL remain unclear. Here, we analyzed an enlarged fil expression domain1 (enf1) mutant of Arabidopsis, which forms both abaxialized leaves and adaxialized leaves. The ENF1 gene encodes SUCCINIC SEMIALDEHYDE DEHYDROGENASE (SSADH), which catalyzes the conversion of succinic semialdehyde (SSA) to succinate. The enf1 phenotype was suppressed by an additional mutation in GAMMA-AMINOBUTYRIC ACID AMINOTRANSFERASE1 (GABAT1), which encodes an SSA-producing enzyme, suggesting that SSA or its derivatives is the metabolite responsible for the defect in the adaxial-abaxial axis-dependent gene expression of enf1. In the shoot apical meristem, GABAT1 was expressed in the outermost layer but SSADH was not. Exogenous application of SSA induced adaxial characters on the abaxial side of the newly developed leaves. We suggest that a GABA shunt metabolite, SSA or its close derivatives, is involved in the robust leaf patterning and structure along the adaxial-abaxial axis.

Aspects of recent developments in mutational studies of plant signaling pathways

Kiyotaka Okada’ and Yoshiro Shimura’t *Division 1 of Gene Expression and Regulation National Institute for Basic Biology Okazaki 444 Japan tDepartment of Biophysics Faculty of Science Kyoto University Kyoto 606 Japan As multicellular organisms, vascular plants have devel- oped unique signaling pathways that function in growth and development. Several kinds of stimuli initiate morpho- logical changes in different phases of the life cycle of vas- cular plants. Direction of growth of shoots and roots is altered by physical stimuli such as gravity, light, or touch- ing. Light also triggers developmental processes, includ- ing seed germination, leaf formation, chloroplast develop- ment, flowering, and fruit ripening. In addition to these physical stimuli, chemical com- pounds are effecters of plant growth and development. When plants are treated with sufficient amounts of plant hormones, drastic changes are induced in growth rate and organ morphology. Some polyamines and polysaccha- rides modulate growth patterns. Wounding and infection with parasitic bacteria, fungi, animals, and plants also cause abnormal cell growth and differentiation. The signal- ing pathways working in these stimulus-response interac- tions are considered to be composed of the following steps: stimulus perception, signal transport, signal trans- duction, modulation of gene expression, and cell growth and differentiation. Distinct from animals, responses in plants are usually slower and continue for longer times, because plant be-

NO VEIN Mediates Auxin-Dependent Specification and Patterning in the Arabidopsis Embryo, Shoot, and Root

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type–specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type–specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.

Reaction-diffusion pattern in shoot apical meristem of plants.

A fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain this issue. Experimental evidence of reaction-diffusion patterns in living organisms was first provided by the pigmentation pattern on the skin of fishes in 1995. However, whether or not this mechanism plays an essential role in developmental events of living organisms remains elusive. Here we show that a reaction-diffusion model can successfully explain the shoot apical meristem (SAM) development of plants. SAM of plants resides in the top of each shoot and consists of a central zone (CZ) and a surrounding peripheral zone (PZ). SAM contains stem cells and continuously produces new organs throughout the lifespan. Molecular genetic studies using Arabidopsis thaliana revealed that the formation and maintenance of the SAM are essentially regulated by the feedback interaction between WUSHCEL (WUS) and CLAVATA (CLV). We developed a mathematical model of the SAM based on a reaction-diffusion dynamics of the WUS-CLV interaction, incorporating cell division and the spatial restriction of the dynamics. Our model explains the various SAM patterns observed in plants, for example, homeostatic control of SAM size in the wild type, enlarged or fasciated SAM in clv mutants, and initiation of ectopic secondary meristems from an initial flattened SAM in wus mutant. In addition, the model is supported by comparing its prediction with the expression pattern of WUS in the wus mutant. Furthermore, the model can account for many experimental results including reorganization processes caused by the CZ ablation and by incision through the meristem center. We thus conclude that the reaction-diffusion dynamics is probably indispensable for the SAM development of plants.

Pattern dynamics in adaxial-abaxial specific gene expression are modulated by a plastid retrograde signal during Arabidopsis thaliana leaf development.

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.