The ASYMMETRIC LEAVES2 (AS2) gene in Arabidopsis thaliana, a member of the AS2/LOB gene family, is involved in the development of a symmetrical expanded lamina, with the rachis as the axis. On flowering, transcripts of AS2 were detected in inflorescence meristems, floral meristems, and primordia of all floral organs. Levels of accumulation of AS2 transcripts were then decreased in the late stage of the floral organ primordia. β-glucuronidase (GUS) activities were examined in transgenic plants that had been transformed with the GUS gene controlled by the AS2 promoter and were detected in early stages of flower development. We quantified the length of floral organs at each developmental stage. Our results showed that the ratios of lengths of stamen to sepal and carpel to sepal in the as2-1 and as1-1 mutants were larger than those seen in wild-type plants in stages 12 to 14. Furthermore, the changes in the ratios of the lengths of floral organs in as2-1 and as1-1 mutants were suppressed by the mutations of class 1 KNOX genes. These results indicate that AS2 and AS1 genes would control the balance of the floral organ lengths via repressing class 1 KNOX genes in reproductive stages.

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Seed germination is the initial step of seedling development in plants. Seed priming with salts has been used to synchronise seed germination. In general, a long-term treatment with a relatively high salt concentration, such as 1 M NaCl, is employed. To improve the efficiency of this treatment, we examined the effect of seed priming with a lower NaCl concentration than conventional method in tomato (Solanum lycopersicum). Tomato seeds were soaked for 24 h at 25°C in the dark in 100–1000 mM of NaCl solution (NaCl-priming) or distilled water (hydro-priming). To estimate the effect of NaCl-priming on seed germination and subsequent seedling growth, the germination rate, seedling emergence, plant height, and hypocotyl and root length were investigated under NaCl-, hydro- and non-priming treatments. At 4 d after sowing, the seedling emergence was markedly promoted by 300 mM of NaCl-priming. The NaCl-priming also significantly enhanced the seed germination rate at 48 h after sowing. Seedling growth, as indicated by plant height, stem diameter and hypocotyl and root length, was promoted by NaCl-priming. These results suggest that priming with low saline has similar effects as conventional priming methods. A comprehensive gene expression analysis showed that the genes related to seedling growth and stress responses were up-regulated by NaCl-priming at 144 h after the start of the treatment, followed by advanced and uniform seed germination. The seedlings exhibited an increased tolerance to Ralstonia solanacearum, the causative agent of bacterial wilt of tomato, compared with the hydro-primed and non-primed seedling.

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Phytohormones have vigorous crosstalk relationships. For example, abscisic acid (ABA), a hormone involved in abiotic stress responses, has antagonistic interactions with plant hormones that play pivotal roles in defense responses, including salicylic acid (SA) and methyl-jasmonic acid (MeJA). Evidence indicates that the relationships among these plant hormones extend beyond simple antagonism. To explore the interplay between hormones in detail, we analyzed the effects of double hormone treatment on gene expression. By contrast to the antagonistic effects reported previously, our data indicates that ABA interacts with SA and MeJA cooperatively as well. Particularly many genes responded only to double hormone treatment, and, interestingly, the loci that responded to ABA+SA also responded to ABA+MeJA. The expression of early-response genes following double hormone treatment did not fit the linear superposition of individual hormone treatments, in contrast to mammalian and prokaryotic cell responses to multiple chemical stimuli. Thus, synergies in these plant hormone signalings are not simply the sum of individual responses. ABA and SA collaboratively down-regulated the expression of genes involved in cell cycle progression at G2/M phase. Presumably, plants interpret combined hormone signals differently from individual signals in order to respond appropriately to their environmental conditions.

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In tobacco (Nicotiana tabacum), several transcription factors belonging to the IXa group of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) cluster at the regulatory NIC2 locus for the leaf nicotine level, and directly activate structural genes required for nicotine biosynthesis and transport. Solanaceous Atropa and Hyoscyamus plants synthesize medicinal tropane alkaloids by utilizing enzymes that partially overlap with the nicotine pathway. We tested whether the tobacco NIC2-locus ERF189 binds to potential ERF binding sites in the promoters of various structural genes involved in biosynthesis of nicotine and tropane alkaloids by an electrophoresis mobility shift assay. ERF189 bound four new GCC-box-like sequences in three nicotine structural genes, but did not recognize other candidate binding sites. This binding preference was used to optimize the consensus binding sequence of ERF189 as 5′-(A/C)GC(A/C)NNCC(A/T)-3′. Five group IXa ERFs (tobacco ERF189, tobacco ERF163, Catharanthus ORCA3, Arabidopsis AtERF13, and Arabidopsis AtERF1) were examined whether they bind to the ERF189-recognition site in the promoter of the tobacco putrescine N-methyltransferase gene in vitro, and transactivate the promoter in a transient expression assay using cultured tobacco cells. The more the protein sequences were similar to ERF189, the more effective these ERFs were in these functional assays. The transient expression assay also identified transactivation domains in an N-terminal acidic region of ERF189, and in a C-terminal Ser-rich region of AtERF13.

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Untranslated regions (UTRs) of mRNA are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA had a 5′ UTR of more than 500 nucleotides (nt), containing a CU-rich region and three upstream open reading frames (uORFs) preceding the downstream ORF. The expected GFP-fusion protein was detected at the cell membrane, indicating the occurrence of translation of the downstream ORF. The 5′ UTR contained three splicing variants that were generated by alternative splicing. A reporter analysis using β-glucuronidase indicated that only the longest one, UTRc, showed a significant ability for the efficient translation of the downstream ORF, whereas other splicing variants showed low level of translational efficiency of the corresponding ORF. These results suggested that the additional 38-nt sequence unique to UTRc localized between the CU-rich region and the uORFs may be involved in the increased translational efficiency of the downstream ORF located after the long 5′ UTR.

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Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional activation of ER-resident molecular chaperones and folding enzymes to maintain cellular homeostasis. This process is known as the ER stress response or the unfolded protein response. We have identified tunicamycin induced 1 (TIN1), a plant-specific ER stress-inducible Arabidopsis thaliana gene. The TIN1 protein is localized in the ER; however, its molecular function has yet to be clarified. In this study, we performed functional analysis of TIN1 in planta. RT-PCR analysis showed that TIN1 is highly expressed in pollen. Analysis using the β-glucuronidase reporter gene demonstrated that the TIN1 promoter is active throughout pollen development, peaking at the time of flowering and in an ovule of an open flower. Although a T-DNA insertion mutant of TIN1 grows normally under ambient laboratory conditions, abnormal pollen surface morphology was observed under a scanning electron microscope. Based on the current and previous observations, a possible physiological function of TIN1 during pollen development is discussed.

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Glutamate dehydrogenase (GDH) catalyzes the reversible amination of 2-oxoglutarate with ammonium to form glutamate. GDH functions in nitrogen assimilation in microorganisms, such as Aspergillus nidulans. However, in plants, glutamine synthetase, not GDH, carries out nitrogen assimilation. Here, we report the effects of introduction of the gdhA gene, encoding NADP(H)-dependent glutamate dehydrogenase, from A. nidulans into potato. We analyzed the resulting changes of photosynthesis, biomass, carbon and nitrogen contents under control and low-nitrogen conditions at the flowering stage and the tuber-bulking stage. There were higher NADP(H)-GDH activities in GDH potato leaves than in the wild type. Regardless of nitrogen conditions, photosynthetic rates and soluble protein concentrations of leaves increased in GDH potatoes at the flowering stage. High photosynthetic rates remained at the tuber-bulking stage in GDH potatoes. The number and dry weight of tubers also increased in GDH potatoes. Under the low-nitrogen condition in particular, carbon and nitrogen contents of GDH potato tubers increased compared with those of the wild type. This resulted from higher rates of carbon and nitrogen redistribution to tuber in GDH potatoes than in wild-type potatoes. Our findings show that the gdhA gene is a powerful tool to increase tuber dry matter and improve efficiency of nitrogen use of potato.

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Previously we reported that three basic helix–loop–helix (bHLH) protein dimers are involved in regulating grain length of rice. Two atypical bHLH proteins named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1) and PGL2 positively regulate grain length in transgenic rice when they are overexpressed in lemma/palea. Their interaction partner ANTAGONIST OF PGL1 (APG), a typical bHLH, is assumed to have opposite function. Suppression of APG expression level by RNAi increased grain length significantly. Here, we report further evidence that APG is a negative regulator of grain length in rice. Overexpression of APG resulted in short grain. Inner epidermal cell layer of lemma from APG overexpression lines showed reduced cell length. Taken together, the results illustrated the biological role of a typical bHLH APG as a negative regulator of rice grain length that controls cell elongation.

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The effect of ionic composition in medium on proliferation of shoot tip-derived PLBs of two Calanthe species, C. aristulifera and C. striata, was investigated by using the media with 12 different ionic compositions, which were systematically modified according to the method of Ichihashi (1992) based on the composition of modified Gamborg’s B5 medium. These two species showed almost the same growth response to the ionic compositions and the growth of PLBs was inhibited on media with higher ratios of NH₄⁺ and Ca²⁺, but promoted with higher ratios of both K⁺ and H₂PO₄⁻. Based on the results of PLB growth, optimum cationic compositions calculated for C. aristulifera and C. striata were NH₄⁺ : K⁺ : Ca²⁺ : Mg²⁺=10.4 : 70.0 : 9.6 : 10.0 and 10.9 : 68.1 : 11.0 : 10.0, respectively, whereas optimum anionic compositions were NO₃⁻ : H₂PO₄⁻ : SO₄²⁻=42.1 : 43.7 : 14.2 and 43.0 : 42.9 : 14.1, respectively. The percentages of NO₃⁻ and H₂PO₄⁻ in the optimum medium thus obtained were about half and 12-hold of the control B5 medium, respectively. Among the 100 micropropagated C. striata plants examined, no obvious variation in flower shape and color were observed except two plants which had larger and thicker flowers than original plants. Those two variants were confirmed to be chromosome doubled tetraploids by flow cytometric analysis.

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The interspecific hybrids were successfully obtained from different cross combinations between 12 cultivars (8 diploids and 4 tetraploids) of Begonia semperflorens (section Begonia) and B. pearcei (section Eupetalum) by in vitro culture of mature and immature seeds on MS medium (Murashige and Skoog 1962) containing 30 g l⁻¹ sucrose, 0.1 mg l⁻¹ α-naphthylacetic acid, 0.1 mg l⁻¹ 6-benzyladenine, 2.5 g l⁻¹ gellan gum, and 10 mg l⁻¹ gibberellic acid. Although all the cross combinations yielded plantlets at the plantlet yielding efficiency of 0.1–2.6/ovary in the culture of mature seeds, some cross combinations gave higher plantlet yields up to 5.7/ovary when immature seeds of 12–16 days after pollination were cultured. The plantlets obtained were confirmed their hybridity through flow cytometric analysis and random amplified polymorphic DNA analysis. In the crosses using diploid B. semperflorens hybrids with normal genome combinations (SP genome) and chromosome doubled hybrids with SSPP genome were obtained, whereas only normal triploid hybrids (SSP genome) were produced from the cross with tetraploid B. semperflorens. Some flowered hybrids of SP and SSP genomes showed intermediate flower colors between the parents such as apricot, cream and light yellow colors, suggesting the expression of yellow flower color of B. pearcei. Although the pollen fertility of those interspecific hybrids was sterile, they are expected to utilize as the important breeding materials to produce yellow flower cultivars of B. semperflorens after restoring the fertility by chromosome doubling treatments.

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Five novel thionin genes have been isolated from three Brassicaceae species, Brassica oleracea var. acephala, Nasturtium officinale, and Barbarea vulgaris. Comparison of the deduced amino acid sequences shows that these thionin proteins share seven highly conserved cysteine residues. These five thionin genes were, respectively, inserted between cauliflower mosaic virus 35S promoter and nopaline synthase terminator in a binary vector pEKH2. Transgenic potato plants were generated through Agrobacterium-mediated transformation methods. Southern blot analysis of transgenic potato plants indicated successful integration with varying copy number of the thionin genes into the plant genomes. Expressions of thionin transgenes in the transgenic plants were confirmed by RT-PCR and their protein products were immunologically detected by western blot analyses. Antifungal assay using detached leaves from transgenic lines expressing either of the thionin genes showed similar levels of enhanced resistance to Botrytis cinerea as compared to those of non-transformed control plants. Novel thionin genes isolated in this study are probably useful tools to produce transgenic plants resistant to various phytopathogens.

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An antifungal protein fraction against Botrytis cinerea was purified from the leaves of Gentiana triflora. Polypeptides with molecular weight ca. 8.0 K (8.0 KP) and 7.0 K (7.0 KP) were detected by Tricine-SDS PAGE under reducing conditions. The deduced amino acid sequence of 8.0 KP cDNA showed amino acid identities with lipid transfer proteins from Apium graveolens (32.4%), Lycopersicon pennellii (31.5%), L. esculentum (31.5%), Nicotiana tabacum (30.7%), non-specific lipid transfer protein from L. esculentum (28.0%), and an unknown protein from Prosopis juliflora (30.7%) (GtLTP1). The deduced amino acid sequence of 7.0 KP showed amino acid identities with putative lipid transfer proteins from Arabidopsis thaliana (26.3% and 22.9%), Gossypium hirsutum (21.0%), Tamarix hispida (19.5%), Populus trichocarpa (15.9%) and an unknown protein from Vitis vinifera (17.2%) (GtLTP2). Both GtLTP1 and GtLTP2 were present as a multi-gene family in the genome and were strongly expressed in the roots and stems. Overexpression of GtLTP1 in tobacco plants improved tolerance against B. cinerea, demonstrating that GtLTP1 is a useful molecular tool for genetic engineering of disease-resistant plants.

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Food plants are important sources of iodine, an essential nutrient required for the synthesis of thyroid hormone in humans. Understanding iodine metabolism in plants is important for tackling iodine deficiency, a serious micronutrient deficiency in the world. Elucidation of iodine metabolism in plants is also important for understanding biogeochemical iodine cycling because plants absorb iodine from the soil and emit considerable amounts of gaseous iodine compounds into the atmosphere. HAMLESS TO OZONE LAYER (HOL) family proteins found in plants have been reported to synthesize methyl halides, including methyl iodide, from halide ions and S-adenosyl-L-methionine (SAM). Here, we report the kinetic analysis of two rice HOL proteins, named OsHOL1 and OsHOL2. Recombinant OsHOL1 and OsHOL2 proteins synthesized methyl iodide from an iodide ion and SAM. Kinetic analyses of the recombinant proteins showed that methyltransferase activities toward iodide ions were highest among the examined substrates—bromide, chloride, and thiocyanate ions. These results suggested that OsHOL1 and OsHOL2 are involved in iodine metabolism in rice and contribute to methyl iodide emissions from rice.

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Several software programs that facilitate processing of data from multiple spectrograms obtained by liquid chromatography-mass spectrometry are available; these programs align features of the ions identified in separate samples into a matrix for further computational analysis. Generally, most alignments within a given matrix are correct, but some alignments seem incorrect, and incorrect alignments must be revised manually. Here, we developed a software tool, MatchedIonsFinder, that revises alignments using a defined algorithm. This program was used to revise an aligned matrix of tomato fruit metabolite ions produced by metabolome analysis software. The matrix revised using the program was comparable to the matrix that was revised manually.

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Introns have an enhancing effect on gene expression known as intron-mediated enhancement in various organisms, including plants. Although the mechanism of the enhancement is largely unknown, most enhancing introns are first introns. In this study, we examined whether the first intron of rice superoxide dismutase sodCc2 gene has an enhancing effect in rice and other plant species. A transient expression assay revealed that the sodCc2 intron elevated reporter gene expression in rice, wheat, and maize, but not in Arabidopsis and tobacco, indicating that the sodCc2 intron has an enhancing effect in monocot but not in dicot plants. To find the putative signal sequences responsible for the enhancement, we carried out an in silico search and found two motifs conserved among the sodCc2 intron and 11 enhancing introns previously known in rice. The motifs contain a consensus sequence, GATCTG, which also exists in the conserved motif found in Arabidopsis enhancing introns. Our results suggest that common motifs are conserved between rice and Arabidopsis enhancing introns.

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