Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants1

To confer abscisic acid (ABA) and/or stress-inducible gene expression, an ABA-response complex (ABRC1) from the barley (Hordeum vulgare L.) HVA22 gene was fused to four different lengths of the 5′ region from the rice (Oryza sativa L.) Act1 gene. Transient assay of β-glucuronidase (GUS) activity in barley aleurone cells shows that, coupled with ABRC1, the shortest minimal promoter (Act1–100P) gives both the greatest induction and the highest level of absolute activity following ABA treatment. Two plasmids with one or four copies of ABRC1 combined with the same Act1–100P and HVA22(I) of barley HVA22 were constructed and used for stable expression of uidA in transgenic rice plants. Three Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. Northern analysis indicated that uidA expression is induced by ABA, water-deficit, and NaCl treatments. GUS activity assays in the transgenic plants confirmed that the induction of GUS activity varies from 3- to 8-fold with different treatments or in different rice tissues, and that transgenic rice plants harboring four copies of ABRC1 show 50% to 200% higher absolute GUS activity both before and after treatments than those with one copy of ABRC1.

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA1 and GA4 levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA3 was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA3-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.

Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue1

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.

Effects of Hypoxia on 13NH4+ Fluxes in Rice Roots1 : Kinetics and Compartmental Analysis

Techniques of compartmental (efflux) and kinetic influx analyses with the radiotracer 13NH4+ were used to examine the adaptation to hypoxia (15, 35, and 50% O2 saturation) of root N uptake and metabolism in 3-week-old hydroponically grown rice (Oryza sativa L., cv IR72) seedlings. A time-dependence study of NH4+ influx into rice roots after onset of hypoxia (15% O2) revealed an initial increase in the first 1 to 2.5 h after treatment imposition, followed by a decline to less than 50% of influx in control plants by 4 d. Efflux analyses conducted 0, 1, 3, and 5 d after the treatment confirmed this adaptation pattern of NH4+ uptake. Half-lives for NH4+ exchange with subcellular compartments, cytoplasmic NH4+ concentrations, and efflux (as percentage of influx) were unaffected by hypoxia. However, significant differences were observed in the relative amounts of N allocated to NH4+ assimilation and the vacuole versus translocation to the shoot. Kinetic experiments conducted at 100, 50, 35, and 15% O2 saturation showed no significant change in the Km value for NH4+ uptake with varying O2 supply. However, Vmax was 42% higher than controls at 50% O2 saturation, unchanged at 35%, and 10% lower than controls at 15% O2. The significance of these flux adaptations is discussed.

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit 1

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF1) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF1. Both the 64-kD protein and the 61-kD CF1 α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF1 α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m−2 s−1) and declined in response to low irradiance (80 μmol photons m−2 s−1) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF1 α-subunit found only in monocots. Analysis of purified CF1 complexes showed that the 64-kD protein represented up to 15% of the total CF1 α-subunit.

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots1

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 107 bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Products of Proline Catabolism Can Induce Osmotically Regulated Genes in Rice1

Many plants accumulate high levels of free proline (Pro) in response to osmotic stress. This imino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The present study provides evidence that the synthesis of Pro may have an additional effect. We found that intermediates in Pro biosynthesis and catabolism such as glutamine and Δ1-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated genes in rice (Oryza sativa L.), including salT and dhn4. One millimolar P5C or its analog, 3,4-dehydroproline, produced a greater effect on gene expression than 1 mm l-Pro or 75 mm NaCl. These chemicals did not induce hsp70, S-adenosylmethionine synthetase, or another osmotically induced gene, Em, to any significant extent. Unlike NaCl, gene induction by P5C did not depend on the normal levels of either de novo protein synthesis or respiration, and did not raise abscisic acid levels significantly. P5C- and 3,4-dehydroproline-treated plants consumed less O2, had reduced NADPH levels, had increased NADH levels, and accumulated many osmolytes associated with osmotically stressed rice. These experiments indicate that osmotically induced increases in the concentrations of one or more intermediates in Pro metabolism could be influencing some of the characteristic responses to osmotic stress.

Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes.

Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species. However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations. Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH. Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture. BAC clones as small as 40 kb were successfully mapped. To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis. The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis.

Zinco para arroz e soja: doses, aproveitamento pelas culturas, fitodisponibilidade e formas desse elemento no solo

A deficiência de Zn no solo causa efeitos indesejáveis na produção agrícola, pois a baixa disponibilidade deste micronutriente para as plantas promove a diminuição da atividade enzimática, além da deficiência deste elemento na alimentação, que pode levar ao estado de subnutrição. Tendo em vista a problemática do Zn no sistema solo-planta e suas variações nos compartimentos do solo, é importante a avaliação de sua fitodisponibilidade e as frações do solo que este elemento está associado. O objetivo deste trabalho foi avaliar a fitodisponibilidade e a compartimentalização de Zn no solo, para as culturas de arroz (Oryza sativa L.) e soja (Glycine max L. Merrill) e avaliar o efeito das doses de Zn sobre a nutrição e exportação deste nutriente pela cultura. Utilizou-se como plantas teste as culturas de arroz e soja para avaliar o efeito das doses de Zn sobre a nutrição e translocação deste nutriente até os grãos. Para tanto, uma amostra de um Latossolo Vermelho, textura argilosa da região de Piracicaba (SP) foi utilizada e ZnCl2 (marcado com 65Zn) como fonte. O experimento foi conduzido em casa de vegetação em DIC, com cinco doses de Zn (0, 1, 2, 4 e 8 mg kg-1 de solo), com quatro repetições. O experimento foi conduzido até a formação de grãos e foi realizada determinação de Zn por Espectrômetria de Absorção Atômica após digestão nitroperclórica e contagem do 65Zn nas partes da planta: parte aérea (PA) e panícula (P), para arroz e PA, vagem (V) e grão (G), para soja. Calculou-se a quantidade de Zn proveniente da fonte (Znpf) nas partes das plantas e o aproveitamento do Zn da fonte pelas culturas (Ap). Nas amostras de solo foram realizadas extrações por DTPA (ZnDTPA) e Mehlich-1 (ZnM1) em duas subamostragens (t1 e t2), antes da semeadura e florescimento, respectivamente. O fracionamento de Zn foi realizado em amostras de t2 nas frações: trocável (ZnTroc); ligado a carbonatos (ZnCarb); a matéria orgânica (ZnMO); a óxidos (ZnOxi) e residual (Znres). Adicionalmente, foi realizada análise do teor pseudo-total de Zn (ZnPST). Os dados obtidos foram submetidos à análise de variância pelo teste-F a 95 % de probabilidade, ajuste das variáveis em função das doses por regressões e teste de média e análises de correlações entre as principais variáveis respostas. O Zn acumulado total na planta se ajustou à regressão linear em função do aumento das doses, entretanto ao analisar as partes separadamente, só houve diferença entre as doses para a variável PA em ambas as culturas. O Znpf total nas plantas apresentou incremento com a adição das doses crescentes de Zn ao solo, entretanto, eu aproveitamento foi baixo, 12 e 8,75 % para arroz e soja, respectivamente. As doses de ZnCl2 adicionadas ao solo, aumentaram a concentração de Zn presente nas frações ZnTroc > ZnMO > ZnCarb, em ordem decrescente. O Zn total acumulado nas plantas de arroz e soja apresentam correlações crescentes para os extratores DTPA e M1 nas duas subamostragens (t1 e t2), em função das doses avaliadas. O Zn extraído pelo DTPA ou M1, apresentaram correlação significativa com o Zn extraído nas frações, na ordem decrescente, ZnTroc > ZnCarb > ZnMO

Etr1-1 gene expression alters regeneration patterns in transgenic lettuce stimulating root formation

We have evaluated the transformation efficiency of two lettuce ( Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens- mediated gene transfer. Six- day- old cotyledons were co- cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the beta- glucuronidase gene ( GUS) under the control of the cauliflower mosaic virus 35S promoter ( CaMV 35S), while the second construct contained the ethylene mutant receptor etr1- 1, which confers ethylene insensitivity, under the control of a leaf senescence- specific promoter ( sag12). Tissues co- cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were transgenic. Very different results were observed when cotyledon explants were co- cultivated with Agrobacteria carrying the etr1- 1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed in 10% of cotyledons and some organogenic shoots were obtained ( 2.86%). These results indicate that the ethylene insensitivity conferred by the etr1- 1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating root formation during regeneration.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Identifying and discriminating among lucerne cultivars using plant morphological characters

The use of morphological data obtained from field (plot test) and glasshouse trials to identify and discriminate among four Iranian and two New Zealand lucerne (Medicago sativa L.) cultivars was investigated, following guidelines established by the International Union for the Protection of New Varieties of Plants (UPOV) for cultivar registration and the Organisation for Economic Co-operation and Development (OECD) for seed certification. Data were collected for terminal leaflet length, width and ratio, angle of stem growth, date of first flowering, stem height at first flowering, flower colour, cutting recovery height, and disease scores. None of these characters were sufficient to identify or discriminate among the six cultivars. The results indicate a need to find cost-effective and efficient laboratory techniques to enhance the assessment of distinctness of lucerne cultivars (UPOV) and for determining cultivar purity for lucerne seed certification (OECD).

Rice morphogenesis and plant architecture: Measurement, specification and the reconstruction of structural development by 3D architectural modelling

Background and Aims The morphogenesis and architecture of a rice plant, Oryza sativa, are critical factors in the yield equation, but they are not well studied because of the lack of appropriate tools for 3D measurement. The architecture of rice plants is characterized by a large number of tillers and leaves. The aims of this study were to specify rice plant architecture and to find appropriate functions to represent the 3D growth across all growth stages. Methods A japonica type rice, 'Namaga', was grown in pots under outdoor conditions. A 3D digitizer was used to measure the rice plant structure at intervals from the young seedling stage to maturity. The L-system formalism was applied to create '3D virtual rice' plants, incorporating models of phenological development and leaf emergence period as a function of temperature and photoperiod, which were used to determine the timing of tiller emergence. Key Results The relationships between the nodal positions and leaf lengths, leaf angles and tiller angles were analysed and used to determine growth functions for the models. The '3D virtual rice' reproduces the structural development of isolated plants and provides a good estimation of the fillering process, and of the accumulation of leaves. Conclusions The results indicated that the '3D virtual rice' has a possibility to demonstrate the differences in the structure and development between cultivars and under different environmental conditions. Future work, necessary to reflect both cultivar and environmental effects on the model performance, and to link with physiological models, is proposed in the discussion.

The interaction of nitrogen application and temperature during reproductive stage on spikelet sterility in field-grown rice

Increased grain yield in response to high rates of application of nitrogen (N) fertiliser is often limited by increased spikelet sterility, particularly under low temperature conditions in the New South Wales ( NSW) rice industry. In 3 field experiments, different N rates were applied for different sowing dates to investigate the interaction between N rate and temperature during microspore development on spikelet sterility and grain yield. In one experiment the effect of water depth on spikelet sterility was also investigated. Engorged pollen production, spikelet sterility, and yield and its components were recorded. Application of N affected a few different processes that lead into spikelet sterility. Application of N at both pre-flood (PF) and panicle initiation ( PI) significantly reduced the number of engorged pollen grains per anther, which was negatively correlated with spikelet sterility. Application of N and low temperature during microspore development with the absence of deep water also decreased pollen engorgement efficiency ( the percentage of pollen grains that were engorged). Application of N further increased spikelet density, which, in turn, increased both spikelet sterility and grain yield. The combined effect of spikelet density and low temperature during microspore development explained the 44% of variation in the number of engorged pollen grains per anther. Grain yield was decreased by low temperature during microspore development in the shallow water when N was applied. Spikelet sterility as a result of late sowing was strongly correlated with minimum temperature during flowering. It is concluded that N application reduced pollen number per anther as a result of increased spikelet density, and this made the spikelets more susceptible to low temperature, causing increased spikelet sterility.