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Patterning of the polar axis during the early leaf developmental stage is established by cell-to-cell communication between the shoot apical meristem (SAM) and the leaf primordia. In a previous study, we showed that the DRL1 gene, which encodes a homolog of the Elongator-associated protein KTI12 of yeast, acts as a positive regulator of adaxial leaf patterning and shoot meristem activity. To determine the evolutionally conserved functions of DRL1, we performed a comparison of the deduced amino acid sequence of DRL1 and its yeast homolog, KTI12, and found that while overall homology was low, well-conserved domains were presented. DRL1 contained two conserved plant-specific domains. Expression of the DRL1 gene in a yeast KTI12-deficient yeast mutant suppressed the growth retardation phenotype, but did not rescue the caffeine sensitivity, indicating that the role of Arabidopsis Elongator-associated protein is partially conserved with yeast KTI12, but may have changed between yeast and plants in response to caffeine during the course of evolution. In addition, elevated expression of DRL1 gene triggered zymocin sensitivity, while overexpression of KTI12 maintained zymocin resistance, indicating that the function of Arabidopsis DRL1 may not overlap with yeast KTI12 with regards to toxin sensitivity. In this study, expression analysis showed that class-I KNOX genes were downregulated in the shoot apex, and that YAB and KAN were upregulated in leaves of the Arabidopsis drl1-101 mutant. Our results provide insight into the communication network between the SAM and leaf primordia required for the establishment of leaf polarity by mediating histone acetylation or through other mechanisms.

In plants, heteroblasty reflects the morphological adaptation during leaf development according to the external environmental condition and affects the final shape and size of organ. Among parameters displaying heteroblasty, leaf index is an important and typical one to represent the shape and size of simple leaves. Leaf index factor is eventually determined by cell proliferation and cell expansion in leaf blades. Although several regulators and their mechanisms controlling the cell division and cell expansion in leaf development have been studied, it does not fully provide a blueprint of organ formation and morphogenesis during environmental changes. To investigate genes and their mechanisms controlling leaf index during leaf development, we carried out molecular-genetic and physiological experiments using an Arabidopsis mutant. In this study, we identified macrophylla (mac) which had enlarged leaves. In detail, the mac mutant showed alteration in leaf index and cell expansion in direction of width and length, resulting in not only modification of leaf shape but also disruption of heteroblasty. Molecular-genetic studies indicated that mac mutant had point mutation in ROTUDJFOLIA3 (ROT3) gene involved in brassinosteroid biosynthesis and was an allele of rod-I mutant. We named it rnac/rot3-5 mutant. The expression of ROT3 gene was controlled by negative feedback inhibition by the treatment of brassinosteroid hormone, suggesting that ROT3 gene was involved in brassinosteroid biosynthesis. in dark condition, in addition, the expression of ROT3 gene was up-regulated and mac/rot3-5 mutant showed lower response, compare to wild type in petiole elongation. This study suggests that ROT3 gene has an important role in control of leaf index during leaf expansion process for proper environmental adaptation, such as shade avoidance syndrome, via the control of brassinosteroid biosynthesis.

SUMMARYCtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations,and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana,although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN wouldfunction in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, preciseverification has been lacking. In this paper, we isolated a homologous gene (MAN)ofAN from liverwort,Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completelycomplemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that existsinANproteinsisolatedfromother plantspecies.WeconstructedseveralplasmidsforexpressingmodifiedANswith amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutationsin the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examinedlocalization of AN using several techniques. Our results demonstrated that AN localizes on punctuatestructures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted anunexpected link between leaf development and membrane trafficking. We should reconsider the roles andevolutionary traits of AN based on these findings.Keywords: ANGUSTIFOLIA, CtBP/BARS, Golgi, Marchantia polymorpha, TGN.INTRODUCTIONLeaf development is divided into two processes, cell prolif-eration and cell differentiation. In the cell differentiationprocess, genetic pathways controlling leaf cell shape havebeen revealed by the characterization of two mutants ofArabidopsis thaliana (hereafter Arabidopsis): angustifolia(an) and rotundifolia3 (rot3). The an mutant was originallyisolated by Re´dei (1962) as a mutant having a narrow leafand twisted fruit. Later studies reported that an has tric-homes with two branches (Hu¨lskampet al., 1994) and its leafcells are narrower and thicker than wild-type cells (Tsukayaet al., 1994; Tsuge et al., 1996). In contrast, rot3 has short,rounded leaves resulting from reduced cell expansion alongthe leaf length direction (Tsuge et al., 1996). These axis-specific defects in cell expansion demonstrate two inde-pendent polarity-dependent cell expansion systems (Tsugeet al., 1996; Tsukaya, 2002).

ANGUSTIFOLIA (AN), the first gene encoding a putative C-terminal binding protein (CtBP) identified in plants, controls leaf cell width in a polarity-dependent manner by regulating the arrangement of cortical microtubules. However, several differences in the sequence of AN have been detected in its conserved CtBP domains, which are transcriptional co-repressors in animals; thus, it is unclear whether AN is a transcriptional co-repressor in plants and how it may function. In this study, we found that AN possesses incomplete D2-HDH and GxGxxG(17x)D motifs, which confer CtBP dehydrogenase activity and NAD-binding for interaction with PxDLS motifs, respectively. In addition, full-length AN was unable to couple the reduction of pyruvate to lactic acid with the oxidation of NADH to NAD+, suggesting that it might not have dehydrogenase activity. Moreover, we found that AN has an additional short open reading frame (ORF), which was identified as an upstream ORF (uORF), in its 5′-untranslated region that overlaps with the start codon of the AN gene. Transcriptional analysis revealed that the uORF and AN ORF are transcribed as a single molecule, indicating that the uORF might influence AN transcription during leaf development.

The processes for leaf development in dicotyledonous plants are surprisingly complex, while the mechanism of controlling and coordinating them is poorly understood. To characterize the fundamental features of the leaf development of Arabidopsis, we first attempted to isolate mutants that alter leaf morphology. Here, leaf morphological mutant of Arabidopsis, lanceolata1 (lan1) which has small and narrow leaves have isolated and characterized. To clarify the function of LAN1 in organ development, we characterized lan1-7 mutant using an anatomical and genetic approach. The lan1-7 mutant had reduced size of foliage leaves and reduced dimensions of stems. A reduction both in cell size and in cell number was evident at the cellular level in the lan1 mutant, revealing that LAN1 gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. From the analysis of heterogeneous plant with lan1 mutation and 35S-AG transgenic plant, AG gene is revealed to regulate leaf morphology under the control of 35S promoter. Thus, MADS-box gene was revealed to have some relationship to that of LAN1 gene at certain stage in leaf development processes.

CtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations, and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana, although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN would function in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, precise verification has been lacking. In this paper, we isolated a homologous gene (MAN) of AN from liverwort, Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completely complemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that exists in AN proteins isolated from other plant species. We constructed several plasmids for expressing modified ANs with amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutations in the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examined localization of AN using several techniques. Our results demonstrated that AN localizes on punctuate structures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted an unexpected link between leaf development and membrane trafficking. We should reconsider the roles and evolutionary traits of AN based on these findings.

ANGUSTIFOLIA (AN) controls leaf morphology in the plant Arabidopsis thaliana. Previous studies on sequence similarity demonstrated that the closest proteins to AN are members of animal C-terminal-binding proteins (CtBPs) found in nematodes, arthropods, and vertebrates. Drosophila CtBP (dCtBP) functions as a transcriptional corepressor for deoxyribonucleic acid (DNA)-binding repressors containing the short amino acid motif, PXDLS, to regulate tissue specification and segmentation during early embryogenesis. It has previously been shown that AN was thought to repress transcription similar to the function of CtBPs; however, AN lacks some of the structural features that are conserved in animal CtBPs. In this paper, we examined whether AN is functionally related to dCtBP. Firstly, we re-examined sequence similarity among AN and various CtBPs from several representative species in the plant and animal kingdoms. Secondly, yeast two-hybrid assays demonstrated that AN failed to interact with an authentic CtBP-interacting factor, adenovirus E1A oncoprotein bearing the PXDLS motif. Thirdly, AN tethered to DNA was unable to repress the expression of reporter genes in transgenic Drosophila embryos. Fourthly, overexpression assays suggested that dCtBP and AN function differently in Drosophila tissues. Finally, AN failed to rescue the zygotic lethality caused by dCtBP loss-of-function. These data, taken together, suggest that AN is functionally distinct from dCtBP. Likely, ancestral CtBPs acquired corepressor function (capability of both repression and binding to repressors containing the PXDLS motif) after the animal–plant divergence but before the protostome–deuterostome split. We therefore propose to categorize AN as a subfamily member within the CtBP/BARS/RIBEYE/AN superfamily.

ANGUSTIFOLIA (AN) is the first C-terminal binding protein (CtBP) gene from plants and controls leaf width and pattern of trichome branching in Arabidopsis thaliana (L.) Heynh. We characterized an ortholog of AN from Ipomoea nil (L.) Roth (Japanese morning glory) and designated it Ipomoea nil's AN (IAN). IAN is a single-copy gene in the genome and is expressed ubiquitously in various organs of I. nil. IAN contains not only a D2-HDH motif, which is highly conserved within the CtBP family, but also LXCXE, NLS and PEST motifs, which are specific to the AN subfamily. The expression of IAN cDNA driven by the cauliflower mosaic virus 35S promoter restored a defect in leaf expansion in the leaf width direction in the angustifolia-1 (an-1) mutant of Arabidopsis, suggesting that IAN retains a common function with AN. In contrast, the complementation by IAN of a defect in the trichome branching pattern on the leaf surface of the an-1 mutant was less effective than that observed for leaf shape. These results suggest that the mechanisms by which AN regulates leaf width and trichome branching are separable.

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.