Kiu-Hyung Cho

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.

Structurally related Arabidopsis ANGUSTIFOLIA is functionally distinct from the transcriptional corepressor CtBP

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.

Comparative Analysis of the Conserved Functions of Arabidopsis DRL1 and Yeast KTI12

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.

Comparative Analysis of Local Green Tea in Korea by STS-RFLP

Consumption of green tea has increased along with increasing concern regarding healthier lifestyles, and many brands of green tea are sold with a label indicating the region of Korea in which the tea was produced. However, there is little information on identifying the difference between the green tea cultivars according to the region they were grown. Here, 9 green tea cultivars collected from Hadong region, Bosung region, China and Japan were subjected to the STS-RFLP analysis. Using the coding and noncoding DNA regions of genes related to the phenylpropanoid pathway, such as phe-nylalanine ammonia-lyase, chalcone synthase and dihydroflavonol 4-reductase, we have identified the differences between green tea cultivars according to the region they were grown in. In this study, we showed a STS-RFLP method of green tea analysis which easily distinguished different kinds of tea using the primers as described. In addition, we identified that the green tea cultivars from Hadong and Bosung displayed a different profile when PAL intron was digested with Dde I, suggesting that a rapid authentication system for green tea cultivars grown in different regions in Korea is available.Keywords : Green tea, STS-RFLP, CAPS, polymorphism

Comparative genomic analysis of Korean and Japanese green tea trees by using molecular markers

Although green tea is one of the most popular beverages in many countries, the evolutionary origin of Korean and Japanese green tea trees has not been extensively elucidated in a molecular level. Lineages of the five Korean green tea populations and cultivars growing in Hadong area were examined in comparison with the six Japanese and one Chinese cultivars using phylogenetic analysis and restriction fragment length polymorphism (RFLP) analysis with cleaved amplified polymorphic sequence (CAPS) markers. Molecular phylogenetic analyses using the non-transcribed region (NTS) region of the 5S rRNA suggested that the Korean Hadong cultivar was a minor variant of the Korean Hadong Cheon-nyeon wild tea plant, which has grown in the Hadong area more than 800 years. RFLP analysis with CAPS markers of the genes in phenylpropanoid biosynthetic pathway showed that all of the Korean Hadong wild tea populations and cultivar had unique polymorphism patterns, when compared with those of the six Japanese and one Chinese c...

DRL1 regulates adaxial leaf patterning and shoot apical meristem activity inArabidopsis

Leaf shape is controlled early on by initiation at the shoot apical meristem (SAM), as well as by changes in the rates and planes of cell division and the polarity-dependent differentiation of leaf cells. To elucidate the regulation of this differentiation by signal(s) from the SAM, we screened for mutations in genes that might be involved in these early processes. A novel recessive mutant, 356-2 [identified as a new allele of thedeformed root and leaf1 (drl1) mutant], was isolated from a collection ofDs transposon insertion lines. The356- 2/drl1- 101 mutant produces narrow, filamentous leaves and defective mer-istems. Its palisade cells have a spongy cell-like structure and are fewer in number, indicating that the leaves are abaxialized. Interestingly, some of those filament-like leaves have no vascular tissues inside their blades.DRL1 encodes a protein similar to the yeast elongator-associated protein (EAP) KTI12. The amino acid sequence of DRL1 is universally conserved in prokaryotes and eukaryotes. These facts suggest that DRL1 might positively regulate leaf polarity and SAM activity by controlling cell proliferation and differentiation.

Regulation of cell size and cell number by LANCEOLATA1 gene in Arabidopsis

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.

MACROPHYLLA/ROTUNDIFOLIA3 gene of Arabidopsis controls leaf index during leaf development

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.