Hirokazu Tsukaya

Stable establishment of cotyledon identity during embryogenesis in Arabidopsis by ANGUSTIFOLIA3 and HANABA TARANU.

In seed plants, the shoot apical and root apical meristems form at the apical and basal poles of the embryonic axis, and leaves form at the flanks of the shoot apical meristem. ANGUSTIFOLIA3/GRF INTERACTING FACTOR1 (AN3/GIF1) encodes a putative transcriptional co-activator involved in various aspects of shoot development, including the maintenance of shoot apical meristems, cell proliferation and expansion in leaf primordia, and adaxial/abaxial patterning of leaves. Here, we report a novel function of AN3 involved in developmental fate establishment. We characterised an an3-like mutant that was found to be an allele of hanaba taranu (han), named han-30, and examined its genetic interactions with an3. an3 han double mutants exhibited severe defects in cotyledon development such that ectopic roots were formed at the apical region of the embryo, as confirmed by pWOX5::GFP expression. Additionally, gif2 enhanced the ectopic root phenotype of an3 han. Although the auxin accumulation pattern of the embryo was correct in an3 han-30, based on DR5rev::GFP expression at the globular stage, expression of the PLETHORA1 (PLT1), a master regulator of root development, expanded from the basal embryonic region to the apical region during the same developmental stage. Furthermore, the plt1 mutation suppressed ectopic root formation in an3 han. These data suggest that establishing cotyledon identity requires both AN3 and HAN to repress ectopic root formation by repressing PLT1 expression.

The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation

We previously showed that the ANGUSTIFOLIA (AN) gene regulates the width of leaves of Arabidopsis thaliana, by controlling the polar elongation of leaf cells. In the present study, we found that the abnormal arrangement of cortical microtubules (MTs) in an leaf cells appeared to account entirely for the abnormal shape of the cells. It suggested that the AN gene might regulate the polarity of cell growth by controlling the arrangement of cortical MTs. We cloned the AN gene using a map‐based strategy and identified it as the first member of the CtBP family to be found in plants. Wild‐type AN cDNA reversed the narrow‐leaved phenotype and the abnormal arrangement of cortical MTs of the an‐1 mutation. In the animal kingdom, CtBPs self‐associate and act as co‐repressors of transcription. The AN protein can also self‐associate in the yeast two‐hybrid system. Furthermore, microarray analysis suggested that the AN gene might regulate the expression of certain genes, e.g. the gene involved in formation of cell walls, MERI5. A discussion of the molecular mechanisms involved in the leaf shape regulation is presented based on our observations.

Organ shape and size: a lesson from studies of leaf morphogenesis.

Control of the shape and size of indeterminate organs, such as roots and stems, is directly related to the control of the shape and size of the cells in these organs, as predicted by orthodox cell theory. For example, the polarity-dependent growth of leaf cells directly affects the polar expansion of leaves. Thus, the control of leaf shape is related to the control of the shape of cells within the leaf, as suggested by cell theory. By contrast, in determinate organs, such as leaves, the number of cells does not necessarily reflect organ shape or size. Genetic evidence shows that a compensatory system(s) is involved in leaf morphogenesis, and that an increase in cell volume can be triggered by a decrease in cell number and vice versa. Studies of chimeric leaves also suggest interaction between leaf cells that coordinates the behaviour of these cells at the organ level. Moreover, leaf size also appears to be coordinated at the whole-plant level. The recently hypothesised neo cell theory describes how leaf shape- and size-control mechanisms control leaf shape at the organ-level via cell-cell interaction.

The evolution and functional significance of leaf shape in the angiosperms

Angiosperm leaves manifest a remarkable diversity of shapes that range from developmental sequences within a shoot and within crown response to microenvironment to variation among species within and between communities and among orders or families. It is generally assumed that because photosynthetic leaves are critical to plant growth and survival, variation in their shape reflects natural selection operating on function. Several non-mutually exclusive theories have been proposed to explain leaf shape diversity. These include: thermoregulation of leaves especially in arid and hot environments, hydraulic constraints, patterns of leaf expansion in deciduous species, biomechanical constraints, adaptations to avoid herbivory, adaptations to optimise light interception and even that leaf shape variation is a response to selection on flower form. However, the relative importance, or likelihood, of each of these factors is unclear. Here we review the evolutionary context of leaf shape diversification, discuss the proximal mechanisms that generate the diversity in extant systems, and consider the evidence for each the above hypotheses in the context of the functional significance of leaf shape. The synthesis of these broad ranging areas helps to identify points of conceptual convergence for ongoing discussion and integrated directions for future research.

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.

The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis.

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.

Analysis of Leaf Development in fugu Mutants of Arabidopsis Reveals Three Compensation Modes That Modulate Cell Expansion in Determinate Organs

In multicellular organisms, the coordination of cell proliferation and expansion is fundamental for proper organogenesis, yet the molecular mechanisms involved in this coordination are largely unexplored. In plant leaves, the existence of this coordination is suggested by compensation, in which a decrease in cell number triggers an increase in mature cell size. To elucidate the mechanisms of compensation, we isolated five new Arabidopsis (Arabidopsis thaliana) mutants (fugu1–fugu5) that exhibit compensation. These mutants were characterized together with angustifolia3 (an3), erecta (er), and a KIP-RELATED PROTEIN2 (KRP2) overexpressor, which were previously reported to exhibit compensation. Time-course analyses of leaf development revealed that enhanced cell expansion in fugu2-1, fugu5-1, an3-4, and er-102 mutants is induced postmitotically, indicating that cell enlargement is not caused by the uncoupling of cell division from cell growth. In each of the mutants, either the rate or duration of cell expansion was selectively enhanced. In contrast, we found that enhanced cell expansion in KRP2 overexpressor occurs during cell proliferation. We further demonstrated that enhanced cell expansion occurs in cotyledons with dynamics similar to that in leaves. In contrast, cell expansion was not enhanced in roots even though they exhibit decreased cell numbers. Thus, compensation was confirmed to occur preferentially in determinate organs. Flow cytometric analyses revealed that increases in ploidy level are not always required to trigger compensation, suggesting that compensation is only partially mediated by ploidy-dependent processes. Our results suggest that compensation reflects an organ-wide coordination of cell proliferation and expansion in determinate organs, and involves at least three different expansion pathways.

The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in the control of heteroblasty.

Regulation of cell number and cell size is essential for controlling the shape and size of leaves. Here, we report a novel class of Arabidopsis thaliana mutants, more and smaller cells 1 (msc1)-msc3, which have increased cell number and decreased cell size in leaves. msc1 has a miR156-resistant mutation in the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15) gene. msc2 and msc3 are new alleles of paused and squint mutants, respectively. All msc mutants showed accelerated heteroblasty, a phenomenon in which several morphological traits of leaves change along with phase change. Consistent with this finding, in the wild type, leaves at higher nodes (adult leaves) also have increased cell number and reduced cell size compared with those at lower nodes (juvenile leaves). These facts indicate that precocious acquisition of adult leaf characteristics in the msc mutants may cause alterations in the number and size of cells, and that heteroblasty plays an important role in the regulation of cell number and size. In agreement with this suggestion, such heteroblasty-associated changes in cell number and size are severely inhibited by the constitutive overexpression of miR156 and are promoted by the elevated expression of miR156-insensitive forms of SPL genes. By contrast, rdr6, sgs3, zip, arf3 and arf4 mutations, which affect progression of heteroblasty, had little or no effect on number or size of cells. These results suggest that cell number and size are mainly regulated by an SPL-dependent pathway rather than by a tasiR-ARF-dependent pathway.

Involvement of auxin and brassinosteroid in the regulation of petiole elongation under the shade.

Plants grown under a canopy recognize changes in light quality and modify their growth patterns; this modification is known as shade avoidance syndrome. In leaves, leaf blade expansion is suppressed, whereas petiole elongation is promoted under the shade. However, the mechanisms that control these responses are largely unclear. Here, we demonstrate that both auxin and brassinosteroid (BR) are required for the normal leaf responses to shade in Arabidopsis (Arabidopsis thaliana). The microarray analysis of leaf blades and petioles treated with end-of-day far-red light (EODFR) revealed that almost half of the genes induced by the treatment in both parts were previously identified as auxin-responsive genes. Likewise, BR-responsive genes were overrepresented in the EODFR-induced genes. Hence, the auxin and BR responses were elevated by EODFR treatment in both leaf blades and petioles, although opposing growth responses were observed in these two parts. The analysis of the auxin-deficient doc1/big mutant and the BR-deficient rot3/cyp90c1 mutant further indicates that auxin and BR were equally required for the normal petiole elongation response to the shade stimulus. In addition, the spotlight irradiation experiment revealed that phytochrome in leaf blades but not that in petioles regulated petiole elongation, which was probably mediated through regulation of the auxin/BR responses in petioles. On the basis of these findings, we conclude that auxin and BR cooperatively promote petiole elongation in response to the shade stimulus under the control of phytochrome in the leaf blade.

Controlling size in multicellular organs: focus on the leaf.

In leaves, cells get larger as cell division decreases or the ploidy increases. This might seem a logical response, but the controls are more complicated.