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The Elongator complex in eukaryotes has conserved tRNA modification functions and contributes to various physiological processes such as transcriptional control, DNA replication and repair, and chromatin accessibility. ARABIDOPSIS ELONGATOR PROTEIN 4 (AtELP4) is one of the six subunits (AtELP1–AtELP6) in Arabidopsis Elongator. In addition, there is an Elongator-associated protein, DEFORMED ROOTS AND LEAVES 1 (DRL1), whose homolog in yeast (Kti12) binds tRNAs. In this study, we explored the functions of AtELP4 in plant-specific aspects such as leaf morphogenesis and evolutionarily conserved ones between yeast and Arabidopsis. ELP4 comparison between yeast and Arabidopsis revealed that plant ELP4 possesses not only a highly conserved P-loop ATPase domain but also unknown plant-specific motifs. ELP4 function is partially conserved between Arabidopsis and yeast in the growth sensitivity toward caffeine and elevated cultivation temperature. Either single Atelp4 or drl1-102 mutants and double Atelp4 drl1-102 mutants exhibited a reduction in cell proliferation and changed the adaxial–abaxial polarity of leaves. In addition, the single Atelp4 and double Atelp4 drl1-102 mutants showed remarkable downward curling at the whole part of leaf blades in contrast to wild-type leaf blades. Furthermore, our genetic study revealed that AtELP4 might epistatically act on DRL1 in the regulation of cell proliferation and dorsoventral polarity in leaves. Taken together, we suggest that AtELP4 as part of the plant Elongator complex may act upstream of a regulatory pathway for adaxial–abaxial polarity and cell proliferation during leaf development.

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin's action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them. [ABSTRACT FROM AUTHOR]

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIOPSlS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CK forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 over-expression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis. [ABSTRACT FROM AUTHOR]

A full-length cDNA of the OgPAE1 gene encoding the α5 subunit of the 20S proteasome was isolated from wild rice ( Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism. [ABSTRACT FROM AUTHOR]

Abstract  Homeobox genes are essential regulators of plant development. ATHB23, a class I homeodomain leucine zipper gene of Arabidopsis, was found to be induced by treatment with the phytohormone gibberellin (GA). In order to clarify its role in development, we performed a histochemical analysis of transgenic plants containing a construct with a GUS::GFP reporter under the control of the 1.5 kb upstream region of ATHB23. The construct was mainly expressed in young leaves and the styles of flowers but not in mature leaves. Microscopic examination of young leaves revealed that it was expressed in the adaxial domain of leaf primordia and the rib meristem. Expression of ATHB23, like that of GA5 encoding GA 20-oxidase, was reduced in mutants related to adaxial–abaxial leaf polarity (phb-1d, se-2, and kan1 kan2). Reduced expression of the GUS::GFP reporter gene was also observed in an se-2 background. These results indicate that ATHB23 is under the control of GA and other activators such as PHB, and is involved in establishing polarity during leaf development. [ABSTRACT FROM AUTHOR]

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to ∼31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H2O2- and α-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H2O2/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexo kinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis. [ABSTRACT FROM AUTHOR]

Plant morphogenesis is controlled by the integration of endogenous genetic programs and responses to exogenous signals. The leaf is a good subject for studying plant morphogenesis, the diversity of which is reflected in leaf shape. Early control of leaf shape relies on controlling leaf initiation at the shoot apical meristem (SAM), the rates and planes of cell division, and the polarity-dependent differentiation of leaf cells. Final leaf form involves coordination of the rates of division, enlargement, and differentiation of leaf cells. In addition, recent genetic studies have revealed a different mechanism that plays an important role in regulating leaf shape: the control of spatial and temporal expression by microRNA and programmed cell death. This brief review focuses on the genetic regulation of leaf shape, from the perspective of the spatial and temporal balance between cell division, cell enlargement, and cell differentiation, with special emphasis on our own studies. doi:10.1300/J411v17n01_09 [ABSTRACT FROM PUBLISHER]

Leaves are major photosynthetic organs, and their diverse shapes and sizes allow adaptation to the natural environment. The early control of leaf shape and size depends on the control of the rate and plane of cell division at the shoot apical meristem and the polarity-dependent cell differentiation in the leaf primordium. In this review, we first summarize knowledge regarding several genes that control the initial stages of leaf formation and leaf polarity (e.g. adaxial–abaxial polarity, symmetry, and flat morphology). Formation of the lateral leaf morphology involves co-ordination of the rates of division and enlargement of leaf cells. Thus, we also summarize information on a number of genes that control these stages of two-dimensional lateral leaf growth (e.g. polarized cell expansion, specific control of cell proliferation, and integration of cell proliferation and expansion). In addition, we discuss several recently identified microRNAs, which are important factors affecting the development of leaf shape via control of spatial and temporal expression of target gene families. We focus on the genetic regulation of leaf shape in the model plant Arabidopsis thaliana from the perspective of spatial and temporal balance among cell proliferation, enlargement, and differentiation, with special emphasis on the results of our own studies. [ABSTRACT FROM AUTHOR]

Suppression subtractive hybridization was used to construct a subtracted library ( ogfw) from plants of a wild rice species, Oryza grandiglumis, subjected to a fungal elicitor and physical wounding. To screen the differentially expressed transcripts in the library, we applied a reverse Northern blot analysis to a cDNA microarray containing 1,152 random clones. Based on the average expression ratio, we selected 156 clones showing an elevated expression level. The elevated expression levels and overall expression profiles over time were verified by Northern blot analysis. A comparative functional categorization of the subtracted expressed sequence tags (ESTs) of the ogfw library against ESTs isolated from blast-infected O. sativa showed that the functional categories of cell rescue, defense and virulence, transcription, and cellular transport and transport mechanism of the ogfw library were threefold higher in the former than in the latter. These subtracted ESTs can be presumed to be related to the defense/resistance system and will be used to investigate the defense mechanisms of wild rice and to provide new insights into the genome of wild rice, which in turn will assist molecular breeding strategies of cultivated rice. [ABSTRACT FROM AUTHOR]

The development of the flat morphology of leaf blades is dependent on the control of cell proliferation as well as cell expansion. Each process has a polarity with respect to the longitudinal and transverse axes of the leaf blade. However, only a few regulatory components of these processes have been identified to date. We have characterized two genes from Arabidopsis thaliana: ANGUSTIFOLIA3 ( AN3), which encodes a homolog of the human transcription coactivator SYT, and GROWTH-REGULATING FACTOR5 ( AtGRF5), which encodes a putative transcription factor. AN3 is identical to GRF-INTERACTING FACTOR1 ( AtGIF1). The an3 and atgrf5 mutants exhibit narrow-leaf phenotypes due to decreases in cell number. Conversely, cell proliferation in leaf primordia is enhanced and leaves grow larger than normal when AN3 or AtGRF5 is overexpressed. Both genes are expressed in leaf primordia, and in the yeast two-hybrid assay, the gene products were found to interact with each other through their N-terminal domains. These results suggest that AN3 and AtGRF5 act together and are required for the development of appropriate leaf size and shape through the promotion and/or maintenance of cell proliferation activity in leaf primordia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

During the shade-avoidance response, leaf blade expansion is inhibited and petiole elongation is enhanced. In this study, we examined the roles of photoreceptors and sugar on the differential growth of the leaf blade and petiole in shade conditions. Under the conditions examined, cell expansion, not cell division, played a major role in the differential leaf growth. The enhanced cell expansion in the leaf blade is associated with an increase in the ploidy level, whereas cell elongation was stimulated in the petiole in dark conditions without an increase in the ploidy level. Analysis of phytochrome, cryptochrome and phototropin mutants revealed that phytochromes and cryptochromes specifically regulate the contrasting growth patterns of the leaf blade and petiole in shade. Examination of the effects of photo-assimilated sucrose on the growth of the leaf blade and petiole revealed growth-promotional effects of sucrose that are highly dependent on the light conditions. The leaf blades of abscisic acid-deficient and sugar-insensitive mutants did not expand in blue light, but expanded normally in red light. These results suggest that both the regulation of light signals and the modulation of responses to sugar are important in the control of the differential photomorphogenesis of the leaf blade and petiole. [ABSTRACT FROM AUTHOR]

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-I 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. [ABSTRACT FROM AUTHOR]

Plants have diversified their leaf morphologies to adapt to diverse ecological niches. The molecular components responsible for regulating leaf morphology, however, have not been fully elucidated. By screening Arabidopsis activation-tagging lines, we identified a dominant mutant, which we designated longifolia1-1D (lng1-1D). lng1-1D plants were characterized by long petioles, narrow but extremely long leaf blades with serrated margins, elongated floral organs, and elongated siliques. The elongated leaves of the mutant were due to increased polar cell elongation rather than increased cell proliferation. Molecular characterization revealed that this phenotype was caused by overexpression of the novel gene LNG1, which was found to have a homolog, LNG2,in Arabidopsis. To further examine the role of the LNG genes, we characterized lng1 and lng2 loss-of-function mutant lines. In contrast to the elongated leaves of lng1-1D plants, the lng1 and lng2 mutants showed slightly decreased leaf length. Furthermore, the lng1-3 lng2-1 double mutant showed further decreased leaf length associated with less longitudinal polar cell elongation. The leaf widths in lng1-3 lng2-1 mutant plants were similar to those in wild type, implying that the role of LNG1 and LNG2 on polar cell elongation is similar to that of ROTUNDIFOLIA3 (ROT3). However, analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D rot3-1 double mutant indicated that LNG1 and LNG2 promote longitudinal cell elongation independently of ROT3. Taken together, these findings indicate that LNG1 and LNG2 are new components that regulate leaf morphology by positively promoting longitudinal polar cell elongation independently of ROT3 in Arabidopsis. [ABSTRACT FROM AUTHOR]