Oscillating flower colour changes of Causonis japonica (Thunb.) Raf. (Vitaceae) linked to sexual phase changes.

Flower colour change may represent an 'honest signal' for pollinators, denoting flowers with good conditions for rewards and pollination. All previously reported flower colour changes are unidirectional, except for an incomplete case in one Fabaceae species. In this study, we discovered a very rare example of complete oscillating flower colour change associated with sexual phase changes in Causonis japonica (Vitaceae). More specifically, flower discs of C. japonica exhibit an orange colour in the initial male phase then soon fade into pink with desorption of the stamens. Several hours later in the daytime of the same or the following day, with stigma maturation and style elongation, the orange flower disc colour is recovered before fading into pink again. Importantly, we found that the colour change is caused by the accumulation and the degradation of carotenoids. Moreover, nectar secretion was roughly correlated with the abovementioned colour changes. This is the first example of an apparent oscillating colour change mediated by carotenoid content alteration in flowers.

Infrared thermography and odour composition of the Amorphophallus gigas (Araceae) inflorescence: the cooling effect of the odorous liquid.

During the second blooming of a cultivated Amorphophallus gigas Teijsm and Binnend in the Botanical Gardens of the University of Tokyo, the surface temperature of the inflorescence was measured using an infrared camera. Contrary to studies of other species in the genus Amorphophallus, the surface of the inflorescence showed only very faint thermogenesis and had a lower temperature than that of the background. This cooling effect appeared to be due to a loss of heat through evaporation, which was caused by the secretion of a very large amount of odorous liquid. Chemical analysis revealed that the major components of this liquid were acetic acid, propionic acid, butyric acid and valeric acids. The composition of the odorous liquid was slightly different between the spathe surface and the sterile appendix. The major component(s) of the odorous material from the spathe was butyric acid, and from the sterile appendix was valeric acids. These components would play dual roles of adding the characteristic smell to the inflorescence and cooling the inflorescence.

Morphological variation in leaf shape in Ainsliaea apiculata with special reference to the endemic characters of populations on Yakushima Island, Japan.

We analyzed leaf shape variations in Ainsliaea apiculata Sch. Bip. to evaluate the uniqueness of morphological characters in populations on Yakushima Island, Kagoshima Prefecture, Japan. Leaf size and shape from populations on Yakushima Island (n = 300) were compared with those from populations in other areas of Japan (n = 300). A considerable amount of variation occurred in leaf size in A. apiculata populations both on Yakushima Island and elsewhere, but clear discontinuities in leaf size were not detected. Some variants previously thought to be endemic to Yakushima Island, i.e., A. apiculata var. acerifolia and A. apiculata var. rotundifolia, were also found in other locations in Japan. Moreover, these leaf types were found to be continuous with the typical leaf shape of A. apiculata var. apiculata via various intermediate types, suggesting the need for future revision of these taxa. Based on these results, we reevaluated the uniqueness of the Yakushima populations of A. apiculata in terms of leaf variation. The uniqueness of the Yakushima populations was defined by a more diverse leaf shape than found in populations from other areas.

The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves.

The asymmetric leaves2 (as2) mutant of Arabidopsis thaliana generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner. Both the delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants. A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves. However, several parallel veins of very similar thickness were evident in such leaves. The complexity of venation patterns in all leaf-like organs of as2 plants was reduced. The malformed veins were visible before the development of asymmetry of the leaf lamina and were maintained in mature as2 leaves. In vitro culture on phytohormone-free medium of leaf sections from as2 mutants and from the asymmetric leaves1 (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells. Analysis by the reverse transcription-polymerase chain reaction showed that transcripts of the KNAT1, KNAT2 and KNAT6 (a recently identified member of the class 1 knox family) genes accumulated in the leaves of both as2 and as1 plants but not of wild type. Transcripts of the STM gene also accumulated in as1 leaves. These findings suggest that, in leaves, the AS2 and AS1 genes repress the expression of these homeobox genes, which are thought to maintain the indeterminate cell state in the shoot apical meristem. Taken together, our results suggest that AS2 and AS1 might be involved in establishment of a prominent midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of class 1 knox homeobox genes in leaves.

Heteroblasty in Arabidopsis thaliana (L.) Heynh.

Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific beta-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emfl lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position.

Cell cycling and cell enlargement in developing leaves of Arabidopsis.

Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level.

Changes in the shapes of leaves and flowers upon overexpression of cytochrome P450 in Arabidopsis.

In Arabidopsis, the two-dimensional expansion of leaves is regulated via the polarized elongation of cells. The ROTUNDIFOLIA3 (ROT3) protein, a member of the family of cytochromes P450, is involved in this process and regulates leaf length. Transgenic plants that overexpressed a wild-type ROT3 gene had longer leaves than parent plants, without any changes in leaf width. The shapes of floral organs were also altered, but elongation of the stem, roots, and hypocotyls was unaffected. To our knowledge, no similar specific regulation of leaf length has been reported previously. Transgenic plants overexpressing the rot3-2 gene had enlarged leaf blades but leaf petioles of normal length. Morphological alterations in such transgenic plants were associated with changes in shape of leaf cells. The ROT3 gene seems to play an important role in the polar elongation of leafy organs and should be a useful tool for the biodesign of plant organs.

Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana.

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild-type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L-Pro but not by D-Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana.

The CURLY LEAF (CLF) gene in Arabidopsis thaliana (L). Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which tell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia.

The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells.

The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. We reported previously that the rotundifolia3 (rot3) mutant of Arabidopsis has a defect in the polar elongation of leaf cells. In the present study, we isolated two additional alleles with mutations in the ROT3 gene. The ROT3 gene was cloned by a T-DNA-tagging method and isolation of the gene was confirmed by a molecular analysis of three rot3 mutant alleles obtained from different mutagenesis. The ROT3 gene encodes a cytochrome P-450 (CYP90C1) with domains homologous to regions of steroid hydroxylases of animals and plants. Expression of the ROT3 gene was detected in all major plant organs. Especially, higher expression was detected in the tissues that had high activity of cell division. We confirmed that the ROT3 gene controls polar elongation specifically in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments. Our results imply that the ROT3 protein is a member of a new class of cytochrome P-450 encoding putative steroid hydroxylases, which is required for the regulated polar elongation of cells in leaves of Arabidopsis.

Genetic analyses of the formation of the serrated margin of leaf blades in Arabidopsis: combination of a mutational analysis of leaf morphogenesis with the characterization of a specific marker gene expressed in hydathodes and stipules.

Developmental control of the formation of the serrated margin of leaf blades was investigated. First, the expression was characterized of a marker gene encoding beta-glucuronidase in strain #1-35-38, a transgenic strain of Arabidopsis thaliana (L.) Heynh, derived by the use of a previously described transposon-tagging system. In strain #1-35-38, expression of the marker gene was tissue-specific, being restricted to stipules and the toothed margins of laminae. Using this transgenic marker gene, we examined the development of leaf blade margins in Arabidopsis. We compared the pattern of expression of the marker gene in the leaves of the wild-type plant with that in plants carrying the asymmetric leaves1 (as1) mutation, which causes dramatic changes in leaf-blade morphology in Arabidopsis. The as1 mutant showed normal morphology of early leaf primordia. The mutation affected the development of leaf segmentation in Arabidopsis without any change in the number or morphology of cells in laminae. The as1 mutation affected leaf morphology independently of mutations in other genes known to affect leaf morphogenesis, such as the acaulis1 mutation and the angustifolia mutation. Based upon these results, the development of the morphology of leaf margins in Arabidopsis is discussed.

Determination of the unequal fate of cotyledons of a one-leaf plant, Monophyllaea.

One-leaf plants, belonging to the family Gesneriaceae, were described for the first time more than 150 years ago. One such unusual plant, Monophyllaea, has only one leaf at maturity. Only one of the two cotyledons grows continuously, without the formation of true leaves, and this feature, known as anisocotyledonous development, has been repeatedly mentioned in textbooks of plant morphology. However, the mechanism for the determination of the one-leaf phenotype remains to be ascertained. In this study, meristematic regions were identified, by monitoring DNA synthesis, at the base of both cotyledons just after germination, while no such regions were found in the shoot apex. Surgical experiments with seedlings and analysis of the anisocotyledonous development revealed that the fate of the cotyledons is determined during their growth. Anisocotyledonous development seems to be the result of competition between the two cotyledons. The mechanism that governs the development of the shoot in the genus Monophyllaea is discussed in relation to apical dominance, which is the common mechanism that regulates shoot development in many plants.

Morphological, physiological and molecular genetic characterization ofArabidopsis himalaica, with reference toA. thaliana.

Arabidopsis himalaica (Edgeworth) O.E. Schulz, a poorly characterized species typical of HimalayanArabidopsis, was analyzed in terms of its morphology, physiology, chromosome number and molecular genetics, in comparison withA. thaliana which is the standard species in the genusArabidopsis. From view point of developmental genetics, several features which are specific toA. himalaica seem not to be derived by single-gene mutations inA. thaliana. Phylogenetic analyses based onrbcL sequences suggested that genusArabidopsis is not monophyletic. The detailed characterization ofA. himalaica should provide clues to understand the trait of evolution of particular features of Himalayan species ofArabidopsis and their genetic basis.

An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.

Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh.

For genetic analysis of mechanisms of leaf morphogenesis, we chose Arabidopsis thaliana (L.) Heynh. as a model for leaf development in dicotyledonous plants. Leaves of the angustifolia mutant were the same length as but narrower and thicker than wild-type leaves. The total number of cells in leaf blades of angustifolia plants was the same as in the wild type. At the cellular level in the angustifolia mutant it was found that the cells were smaller in the leaf-width direction and larger in the leaf-thickness direction than in wild type, revealing the function of the ANGUSTIFOLIA gene, which is to control leaf morphology by regulating polarity-specific cell elongation. The existence of similar genes that regulate leaf development in the length direction was, therefore, predicted. Three loci and several alleles associated with short-leaved mutants were newly isolated as rotundifolia mutants. The rotundifolia3 mutant had the same number of cells as the wild type, with reduced cell elongation in the leaf-length direction. The features of the angustifolia rotundifolia3 double mutant indicated that ANGUSTIFOLIA and ROTUNDIFOLIA3 genes act independently. We propose that leaf expansion in Arabidopsis involves at least two independent developmental processes: width development and length development, with the ANGUSTIFOLIA and ROTUNDIFOLIA3 genes playing different polarity-specific roles in cell elongation.

Characterization and subcellular localization of a small GTP-binding protein (Ara-4) from Arabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1.

Small GTP-binding proteins belonging to the rab/YPT family play key roles at various steps in intracellular transport pathways in yeast and mammalian cells. Many members of rab/YPT family have been isolated from plants to date. However, detailed information about the localization and function of the gene products remains limited, even though intracellular transport is likely to be involved in important phenomena such as cell elongation, transport of storage proteins, determination and maintenance of cell polarity and intercellular signal transduction. We have attempted to establish transgenic Arabidopsis plants that overexpress ARA-4, a rab/YPT homologue in order to analyze the function and the localization of the gene product. For overexpression and also for regulation of the expression of this gene, the promoter of the gene for HSP81-1 was employed to drive the transcription of ARA-4 in transgenic plants. The response of the introduced genes to heat shock was analyzed. Upon heat-shock treatment, the ARA-4 gene was efficiently transcribed and translated. The induction of ARA-4 by heat shock was transient, and at least two distinct forms of this protein were found in membrane and cytosolic fractions from transgenic plants. Prolonged incubation after heat shock reduced the amount of the cytosolic form of the induced protein, and the cytosolic form of the protein thus probably represents the unprocessed precursor. Using transgenic plants, we determined the subcellular localization of the product of ARA-4. The protein was predominantly localized on Golgi-derived vesicles, Golgi cisternae and the trans-Golgi network.

Floral organ-specific and constitutive expression of an Arabidopsis thaliana heat-shock HSP18.2::GUS fusion gene is retained even after homeotic conversion of flowers by mutation.

Organ-specific and constitutive expression of the Arabidopsis HSP18.2 gene under normal growth conditions (22 degrees C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for beta-glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.

Analysis of intergenic spacer regions in the nuclear rDNA of Pharbitis nil.

The nucleotide sequence of a 4.2-kb EcoRI fragment from the intergenic region between the genes for 25S and 18S ribosomal RNA of Pharbitis nil Choisy was determined. The region contained a unique repetitive family of DNA sequences, called the RsaI family, composed of 32-bp units. The 32-bp unit was tandemly repeated in the intergenic region, and four subfamilies of repeating units were clustered as discrete blocks. The RsaI family of repeats was shown to be specific to the genus Pharbitis by Southern blot hybridization.