Regulation of seed germination and seedling growth by an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytocystatin isoform, <Emphasis Type="Italic">AtCYS6</Emphasis>

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin₄₊₇ (GA₄₊₇) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

Fungi from leaves of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis>)

In spite of the self-cleaning property of its leaves called the lotus effect, leaves of lotus (Nelumbo nucifera) provide a habitat for an unknown fungal diversity. The aim of this study was to detect and identify fungi from leaves of N. nucifera, including ectophytic, parasitic and endophytic fungi, in Taiwan using different collection strategies, as well as morphological and diverse molecular markers established in the different systematic groups of fungi. Among ectophytic and parasitic fungi, a new species of Dissoconium and of Pseudocercospora are described, respectively. Phyllosticta nelumbonis Sawada is transferred to Diaporthe. Among plant parasitic fungi, Erysiphe takamatsui and Ps. nymphaeacea are recorded in Taiwan for the first time. Euryale is recorded as a new host genus for Ps. nymphaeacea. The basidiomycetous yeast Fereydounia khargensis is recorded for the first time from living plants and in East Asia. Endophytic fungi from lotus were studied for the first time. From 1002 plant segments, 476 endophytic isolates were produced in culture, comprising 33 typical terrestrial species mainly belonging to the genera Colletotrichum (mainly C. siamense), Diaporthe (D. tulliensis and D. ueckerae) and Fusarium (F. solani species 6, hitherto known from clinical samples), as well as to Xylariaceae, but no Ingoldian fungi. Most isolates were from leaf laminas (71%) compared to those from petioles (29%). From this observation, we conclude that the fungi of the aquatic lotus plant appear to have terrestrial origin and, after dispersal by wind and in spite of the lotus effect, may enter the plant from the lamina. Only three species isolated as endophytes were also found as ectophytic or parasitic fungi.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Characterization of a field-grown transgenic pineapple clone containing the genes <Emphasis Type="Italic">chitinase</Emphasis>, <Emphasis Type="Italic">AP24</Emphasis>, and <Emphasis Type="Italic">bar</Emphasis>

We previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. The present report focuses on the evaluation of the first vegetative generation of a transgenic clone containing these genes. Three materials were compared: macropropagated controls (non-transformed), micropropagated controls (non-transformed), and micropropagated transformed plants. From each group, 50% of the plants were sprayed with FINALE^® 3 mo after the experiment initiation. The characterization was performed after 1 yr of field growth. FINALE^® killed all non-transgenic plants. Plants that survived the herbicide application showed 2n?=?50 chromosomes in their roots after 1 yr in the field. Micropropagated transformed plants sprayed with FINALE^® did not show phenotype differences from micropropagated transformed plants not sprayed with the herbicide. Between the micropropagated transformed plants sprayed with FINALE^® and the micropropagated control plants, the following differences were observed: modifications in levels of cell wall-linked, free and total phenolics, and total proteins. Moreover, changes of the fruit mass without crown were also recorded. Between the micropropagated transformed plants sprayed with FINALE^® and the macropropagated control plants, levels of chlorophyll b, total chlorophyll pigments, and proteins were different. Furthermore, activities of phenylalanine ammonia-lyase, superoxide dismutase, and glutamine synthetase were dissimilar. The plant height and diameter, and the crown height were also different. Until now, we have evaluated transformed pineapple plants during hardening and field growth. Although some unexpected variations were recorded, we believe they are not relevant enough to justify rejection of transgenesis as an important tool for pineapple genetic improvement.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Factors influencing shoot multiplication of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis>)

Effect of plant growth regulators, explant size, season of explant collection, temperature (20, 25 and 30 °C) and photoperiod on in vitro lotus (Nelumbo nucifera Gaertn.) shoot formation and growth were examined. Shoots formation was greatly influenced by growth regulators, explant size and season of explant collection. The maximum number of shoots were induced from bud explants on Murashige and Skoog (MS) medium containing 4.44 μM benzyladenine (BA) + 0.54 μM α-naphthalene acetic acid (NAA). Explants formed by bud of one expanded and one unexpanded leaf, which was collected in spring gave encouraging results of shoot production. Higher temperature favoured shoot induction and subsequent growth was much better at 25 °C compared to that at 20 and 30 °C.     

Ectopic over-expression of two apple <Emphasis Type="Italic">Flowering Locus T</Emphasis> homologues, <Emphasis Type="Italic">MdFT1</Emphasis> and <Emphasis Type="Italic">MdFT2</Emphasis>, reduces juvenile phase in <Emphasis Type="Italic">Arabidopsis</Emphasis>

To get insight into mechanism by which apple tree (Malus domestica Borkh.) regulates flowering, two apple flowering locus T (FT) homologues, MdFT1 and MdFT2, were isolated from the leaf cDNAs of cultivar Gala. The open reading frames (ORFs) of two MdFTs encoded 174 amino acids. The deduced amino acid sequence of MdFT1 and MdFT2 showed 94.3 % similarity to each other, while 72.6 and 76.0 % to AtFT protein, respectively. Semi-quantitative RT-PCR indicated their specific expression in leaves. Visualization of MdFT2-GFP fusion protein demonstrated its localization on membrane. Ectopic overexpression of either MdFT1 or MdFT2 in Arabidopsis significantly induced early flowering by activating the downstream flowering-related genes.     

Propagation of <Emphasis Type="Italic">Haemaria discolor</Emphasis> via <Emphasis Type="Italic">in vitro</Emphasis> seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm⁻³ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm⁻³ α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.This research was supported by grant (91AS-3.1.1-CI-C3) from the Council of Agriculture, Executive Yuan of Taiwan and grants (NSC-89-2317-B055-002 and NSC-91-2317-B324-001) from the National Science Council of Taiwan. This paper is Agricultural Research Institute Contribution No. 2158.