Chorismate mutase isoenzymes from Sorghum bicolor: immunological characterization

Highly purified fractions of chorismate mutase 1 and 2 from etiolated seedlings of Sorghum bicolor were used as the antigen for antibody production in BALB/c mice. Tests for antigen-antibody complex formation were made by immunodiffusion, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). These tests indicated the presence of specific antibodies for each isoenzyme in their antisera. However, in the same tests, no cross-reaction was found between chorismate mutase 1 and 2 and their antisera. This indicates no immunological similarity between the two isoenzymes of chorismate mutase from sorghum.     

Chorismate mutase isoenzymes from Sorghum bicolor: purification and properties

Two forms of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, have been detected in etiolated seedlings of Sorghum bicolor after DEAE-cellulose chromatography. CM-1 and CM-2 contained 44 and 56%, respectively, of the total activity measured after DEAE-cellulose chromatography. CM-1 was activated by tryptophan and inhibited by phenylalanine and tyrosine. In contrast, CM-2 was insensitive to all three aromatic amino acids. CM-1 and CM-2 were purified 1389- and 1018-fold, respectively, by anion exchange, hydrophobic, and dye matrix chromatography. The molecular weights estimated by gel filtration on Sephacryl S-200 were 56,000 for CM-1 and 48,000 for CM-2. Subunit molecular weights of the two forms were estimated by sodium dodecyl sulfate-gel electrophoresis at 36,000 and 51,000 for CM-1 and CM-2, respectively. Tryptophan was required for the stability of CM-1 at all stages of purification. Both isoenzymes were stable at 0 or -20 degrees C and had broad pH optima (6-10 for CM-1 and 7.5-9.5 for CM-2).     

橡胶树白粉菌分支酸变位酶基因克隆及蛋白表达纯化

由橡胶树白粉菌引起的橡胶树白粉病是橡胶树的重要的叶部病害之一,严重影响橡胶的产量。然而目前对橡胶树白粉菌的致病机理研究匮乏。分支酸变位酶是莽草酸途径的关键酶,能够将分支酸转化为预苯酸,为植物提供氨基酸及大量代谢产物,在植物抗病中起到非常重要的作用。而植物病原物在致病过程当中能够分泌分支酸变位酶影响植物莽草酸途径,从而抑制植物的防卫反应。因此研究橡胶树白粉菌分支酸变位酶在其致病过程中的功能具有一定的意义。试验利用同源比对分析在橡胶树白粉菌基因组中获得一个分支酸变位酶同源蛋白,并用PCR克隆获得橡胶树白粉菌分支酸变位酶基因,命名为OHCmu。后续构建GST-OHCmu融合原核表达载体,筛选最优诱导条件,并利用GST亲和层析柱对蛋白进行纯化。结果表明橡胶树白粉菌OHCmu基因大小843 bp,具有1个内含子,编码263个氨基酸;具有d5csma_结构域,属于Chorismate mutaseⅡ蛋白家族;GST-OHCmu融合蛋白外源诱导表达在供试条件下(IPTG:0.8 mmol/L, 16℃)可以有较好的表达,获得融合蛋白大小约为56 kD。经过GST亲和层析柱纯化、切割GST标签后,顺利获得浓度较高、较纯的橡胶树白粉菌OHCmu蛋白。研究结果为后续OHCmu蛋白的特性及致病机理研究提供参考。     

Purification and Characterization of Chorismate Synthase from Euglena gracilis: Comparison with Chorismate Synthases of Plant and Microbial Origin

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.     

Effects of Ring D-Modified Gibberellins on Gibberellin Levels and Development in Selected Sorghum bicolor Maturity Genotypes

Plants of early flowering mutant and wild type genotypes of Sorghum bicolor were treated with ring D-modified gibberellins (GAs), and the effects on endogenous GA levels were determined. The growth and timing of floral initiation in 58M plants grown under 18-h days (which significantly delays floral initiation in this short day plant) following treatment with these compounds, relative to GA₃ and GA₅ treatments, were also investigated. Application of the endo-isomer of C16,17-dihydro-GA₅ (endo-DiHGA₅), the exo-isomer of C16,17-dihydro-GA₅ (exo-DiHGA₅), and C16α,17-dichloromethanodihydro-GA₅ (DMDGA₅) altered GA levels in both genotypes. Each ring D-modified GA significantly inhibited shoot growth while significantly decreasing levels of GA₁ and increasing levels of its immediate precursor, GA₂₀. Gibberellin A₈ levels also decreased. Tillering was not affected by any treatment. For the early flowering genotype 58M, grown under noninductive long days, both dihydro-GA₅ isomers promoted floral initiation while shoot growth was strongly inhibited, and floral development was strongly advanced beyond floral stage 4. Gibberellin A₃ and GA₅, applied under the same conditions, promoted shoot growth slightly and gave ``floral-like' apical meristems that did not develop past floral stage 1. These results suggest that the reduced shoot growth of sorghum, which follows application of those ring D-modified GAs, is due to their inhibiting the 3β hydroxylation of GA₂₀ to GA₁, thereby reducing the GA₁ content. That floral initiation was hastened and floral development promoted in genotype 58M by application of both isomers of DiHGA₅ are in contrast to the effects of other GA biosynthesis inhibitors, which act earlier in the GA biosynthesis pathway, but are consistent with results seen for long day grasses. This suggests that endo-DiHGA₅ and exo-DiHGA₅ may be acting directly in promoting floral initiation and subsequent floral apex development of this short day plant under long day conditions. Received October 3, 1996; accepted January 22, 1997     

Growth and Photosynthesis in Sorghum bicolor Infected with Striga hermonthica

Sorghum plants were grown in the laboratory with the root systemof each plant split between two pots. Three split pot treatmentswere established: (– –) treatment, where both halvesof the root were free from Striga; (– +) treatment, wherethe soil in one half of the pot had been inoculated with Strigaseed; (+ +) treatment, where the soil in both halves of thepot had been inoculated with Striga. Seed, stem and leaf weight were reduced by 82, 60 and 26 percent respectively in (+ +) plants compared to (– –)plants. Partially infected plants (– +) behaved similarlyto (+ +) plants. Rates of light saturating carbon dioxide fixation in (+ +) and(– +) plants were only 60 per cent of those measured in(– –) plants. This reduction was independent ofchanges in stomatal conductance. The effects of Striga on the growth and photosynthesis of sorghumappear to be independent of the degree of parasitism to whichthe host is subjected. The difference in production betweeninfected and uninfected plants was greater than could be accountedfor in term of competition with the parasite for resources,and Striga appears to have a pathological effect on the host. Sorghum, Striga, parasitic angiosperm, growth, photosynthesis     

高粱LEA3蛋白基因和启动子的克隆及序列分析

根据禾本科LEA3基因保守序列设计简并引物,同时结合RACE方法获得高粱LEA3基因全长cDNA序列1032bp,该序列含有一个612bp的阅读框,编码203个氨基酸,包含7个串联的LEA3蛋白的基元序列。通过与玉米、小麦、水稻、大麦的LEA3蛋白序列比较,氨基酸序列同源性分别为73.8%、53.77%、45.63%和53.99%;其编码蛋白理论相对分子量为21.22kD,等电点pI=8.79;蛋白质二级结构预测表明2段α螺旋结构占主导,与目前已知的多种植物的LEA3蛋白具有相似的结构功能域。通过热不对称交错PCR(TAIL PCR)技术获得LEA3基因启动子749bp的DNA序列,该区域包含ABA应答元件、干旱胁迫应答元件、以及胚胎和胚乳特异表达元件;通过PHyML软件构建了禾本科植物LEA3基因ML系统树。这些研究结果为深入了解该基因的功能和高粱抗旱的分子机理提供了基础数据。     

Control of the Biosynthesis of Phenylalanine and Tyrosine in Plant Tissue Cultures: Lack of Repression of Chorismate Mutase

Tobacco, rice, carrot and tomato tissue cultures were grown in liquid media containing l-phenylalanine or l-tyrosine, or both together. The addition of these amino acids increased their respective cellular levels (4–20 fold), but did not lower the level of chorismate mutase, an enzyme in the biosynthetic pathway of phenylalanine and tyrosine. These results indicate that the biosynthesis of phenylalanine and tyrosine in cultured plant cells is not regulated by repression of the synthesis of chorismate mutase by phenylalanine or tyrosine.     

Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor

 A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62–72% homology and the dimers were 79–82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6–1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres. Received: 11 November 1997 / Accepted: 17 November 1997     

Genotype Dependent Somatic Embryogenesis and Regeneration from Leaf Base Cultures of Sorghum bicolor

A simple and reproducible protocol for callus induction and regeneration of plantlets from leaf base cultures of agronomically important Indian cultivars of Sorghum bicolor(L.) Moench (296 Band RS 585) has been developed. A strong genotype dependent response was observed and the genotype 296 B was found to be the most responsive as compared to the other genotypes tested. Cultures were raised from the III, IV and V leaf bases, excised from 12-day-old in vitro raised plantlets and cultured on Callus Induction Medium (CM). Callus initiation took place after 10-14 days of culture. The explants were maintained on this medium for 30- 35 days, after which they were transferred to Regeneration Medium (RM). Histological examination indicated that somatic embryogenesis was prevalent in the leaf base cultures and the embryos started to germinate after 15-20 days of transfer to RM. Plantlets with complete shoot and root system have been raised with as many as 30 plantlets regenerating from a single explant. These plantlets could be easily separated from one another and transferred to culture tubes for faster growth and development. Later, individual plants numbering more than 50 were transferred to pots containing soil: soilrite (1:1) for hardening. A high regeneration frequency of up to 40 % could be obtained in the genotype 296 B followed by 10.8 % in the genotype RS 585 and 7.8 % in C 43.     

Nucleotide sequence of the split tRNAleu(UAA) gene from Sorghum bicolor chloroplasts

The nucleotide (nt) sequence of the split tRNAleu(UAA) gene and 328 nt of its flanking regions from sorghum chloroplasts (cp) has been determined. This gene is located in the BamHI-6 fragment in a map position very similar to that of maize. The exon of sorghum tRNAleu gene has an identical nt sequence to its counterpart in maize. Although the 450 nt of intron in sorghum is 8 nt shorter than that of maize, the nt sequence between them shows 97% homology. Like maize and broad bean, the intron from sorghum cp tRNAleu gene could be folded into a secondary structure which is similar to the postulated structure of the intron from the auto-spliceable rRNA precursor of Tetrahymena. Both introns from sorghum and maize contain open reading frames (ORFs) which are conserved at the N terminus. The putative AUG initiation codon for both ORFs is located in the stem region of a 12-bp secondary structure of highly A + T-rich sequences.     

Viability and Germination of the Pollen of Sorghum [Sorghum bicolor (L.) Moench]

Previous studies have demonstrated that pollen of sorghum [Sorghumbicolor (L.) Moench] loses capacity to both germinate in vitroand to set seed in vivo soon after being shed. The current studyevaluates the capacity for dehydrated pollen to effect in vitrogermination, reduce tetrazolium chloride, and set seed on cytoplasmicmale sterile plants. Morphological changes during pollen germinationwere examined by scanning electron microscopy (SEM). Close to70% of the pollen germinated in 5 min, or less, when collectedat 80% relative humidity (RH) and stored in sealed glass vials.Pollen tubes elongated autotropically with atmospheric humidityapparently being a controlling factor in the process. Pollendehydrated at 50% RH and 25°C for 15-30 min neither germinatedin vitro, reduced tetrazolium chloride, nor set seed on malesterile plants. Rehydrating the pollen did not restore the capacityfor germination. SEM micrographs demonstrated that elongatingpollen tubes encircled the pollen grain and were contiguousto the surface. A fibrillar-like material existed on the exineof separated pollen grains at the point where the grains hadbeen previously attached.Copyright 1994, 1999 Academic Press Sorghum pollen, germination, seed-set, viability, scanning electron microscopy, Sorghum bicolor (L.) Moench     

Endodermal Silicon Deposits and Their Linear Distribution in Developing Roots of Sorghum bicolor (L.) Moench.

Seedlings of Sorghum bicolor (L.) Moench. were cultured in nutrientsolution containing 100 p.p.m. SiO₂. Over periods up to 14 days,the progressive accumulation of Si deposits was recorded bymeans of the electron-probe microanalyser and the scanning electronmicroscope along the seminal root length. An acropetal, linear gradient of Si deposits developed in theendodermis, beginning at the proximal end of the root after1 day, and subsequently extended distally until after 7 days,the total silicified endodermal zone occupied the proximal 75per cent of the root length, a value which remained relativelyconstant thereafter, in spite of root extension. No Si was detectedbelow this zone towards the apex. This result is believed tobe related directly to the asynchronous gradient of cell maturationexhibited by the endodermis behind the apex, and specificallyto the degree of wall development therein. Opaline silica was deposited only in the endodermis, initiallyon the inner tangential wall (ITW) surface after only 1 day,as spherical masses of coalesced, primary particles for whichthe term ‘silica aggregate’ is proposed. A thinlayer of silica over the wall surface was formed as a secondaryphase. The aggregates reached mature size after approximately7 days. Conditions favourable to the inception of silica depositionare discussed including the significance of the chemical compositionof the aggregates, and the importance of the degree of cellulosicthickening, as well as the surface characteristics, of the ITW.     

Effect of Silicon and Phosphate-Solubilizing Bacteria on Improved Phosphorus (P) Uptake Is Not Specific to Insoluble P-Fertilized Sorghum (Sorghum bicolor L.) Plants

In the research, the single-and dual effects of phosphate-solubilizing bacteria (PSB) (B0, Pseudomonas sp. FA1, and Bacillus simplex UT1) and silicon (Si) (0, 150, 300, and 600 mg kg⁻¹ used as silicic acid) on P uptake by sorghum (Sorghum bicolor L.) plant fertilized with soluble or insoluble P (rock phosphate—RP) were studied via a perlite-potted experiment. Moreover, the effects of various treatments on morphological (shoot and root dry weight), nutritional (the uptake of Si and K) and physiological parameters (activity of catalase, superoxide dismutase, and peroxidase enzymes) of this plant were also measured. When grown in RP-fertilized medium compared with those grown in soluble P-fertilized medium, both shoot biomass and root biomass of sorghum plants were noticeably diminished. The PSB strains and Si levels independently improved all the aforementioned parameters. Use of Si and PSB strains to sorghum plants grown in soluble P or insoluble P medium significantly augmented P use efficiency. Silicon not only augmented the uptake of P from sparingly soluble-P source (RP), but also augmented uptake of P from water-soluble P source. Both B. simplex UT1 and Pseudomonas sp. FA1 indicated a meaningful betterment in sorghum plant dry matter and uptake of P (and K and Si) under both soluble and insoluble P fertilization conditions with Pseudomonas sp. FA1 being more efficacious than B. simplex UT1. But, the dual use of the PSB with Si resulted in the greatest increase in sorghum plant P uptake and other measured growth indices. Application of 600 mg Si kg⁻¹ and Pseudomonas sp. FA1 significantly augmented the P shoot concentration of sorghum plant fertilized with RP to an sufficient level (> 0.3%) in the range of P-fertilized sorghum plants. Therefore, in addition to PSB utilization, Si should be considered as soil amendment in agricultural soils inadequate in plant-available Si as a means of sustainable agriculture with respect to possible savings of scarce P resources.

     

Partially Fertile Line with Apospory Obtained from Tissue Culture of Male Sterile Plant of Sorghum (Sorghum bicolor L. Moench)

A partial restoration of male fertility has been revealed inthree among 20 regenerants from callus cultures obtained fromthe panicle fragments of the sorghum plant with cytoplasmicmale sterility. Induced fertility is retained under self-pollinationfor eight generations. The pollen fertility level in every generationvaries in different plants (0-70%) and depends on the environmentalfactors, rather than on plant genotype. A high positive correlation(r = 0·90; P < 0·01) has been revealed betweenthe pollen fertility level and the total rain precipitationduring the microgametophytogenesis. Cytoembryological investigationof this line has shown the presence of structures similar toaposporous embryo sacs (ESs), which have developed in the ovulesalong with the sexual ESs. The ovules with aposporous formationshave been observed almost in all of the studied plants in severalgenerations, however, their frequency varied (2-53%). Parthenogeneticproembryos and endosperm nuclei without signs of pollen tubepenetration have been found in some of such ESs. The natureof the instability observed in a number of generations in maleand female generative structures is discussed.Copyright 1995,1999 Academic Press Cytoplasmic male sterility, fertile revertants, apomixis, somaclonal variability, Sorghum bicolor L. (Moench)     

Genetic mapping of QTLs associated with greenbug resistance and tolerance in Sorghum bicolor

Ninety three recombinant inbreds of Sorghum bicolor (L. Moench) were derived from a cross between two sorghum lines GBIK and Redlan. This population was used to identify quantitative trait loci (QTLs) for resistance and tolerance to greenbug (Schizaphids graminum Rondani) Biotypes I and K. One hundred and thirteen loci (38 SSRs and 75 RAPDs) were mapped in 12 linkage groups covering 1,530 cM. In general, nine QTLs were detected affecting both resistance and tolerance to greenbug (GB) Biotypes I and K. The phenotypic variance explained by each QTL ranged from 5.6% to 38.4%. Four SSRs and one RAPD marker were associated with the expression of all resistance and tolerance traits. These markers appear to be linked to biotype non-specific resistance and tolerance genes. Four additional markers were associated with biotype-specific resistance or tolerance traits. The detection of more than one locus for each biotype supports the hypothesis that several regions, which represent different genes, control the expression of resistance and tolerance to greenbug in sorghum. The results can be used for marker-assisted selection and the breeding of greenbug-tolerant sorghum cultivars.     

X-ray Microanalysis of the Seminal Root of Sorghum bicolor with Particular Reference to Silicon

Sorghum bicolor (L.) Moench. cv. P508.GB plants were grown inwater culture for 1 week, when the seminal roots were harvestedand sampled at five positions starting from the base: 0.0, 0.25,0.50 and 0.75 of the axis length, and a sub-apical position,11 mm behind the tip. Mineral distribution in bulk frozen rootsegments was investigated using SEM and X-ray microanalysis.The elements detected were potassium, chlorine, sulphur, sodium,phosphorus, calcium and silicon. The first four occurred inall root zones. Phosphorus was ubiquitous, but appeared to accumulatein the pericycle protoplasm. Calcium and silicon exhibited themost variation along the seminal axis. Calcium was present inall tissues at the base, but decreased acropetally, being detectedin only the outer cortical and epidermal walls of the subapicalzone. Silicon was present at low levels in protoplasts and wallsof most root tissues, and accumulated in the endodermal protoplastand walls. Deposition in walls is initiated coincident withthe earliest stages of secondary wall thickening. Silicon contentof the inner tangential wall of the endodermis exhibits a decreasingacropetal gradient along the axis length. It is absent frommost cell walls of the sub-apical zone. Silicon pathways inthe root, and silica aggregate formation in relation to thesurrounding ionic environment, are discussed. Sorghum bicolor (L). Moench, seminal root, cryostage, SEM, X-ray microanalysis, ion localization, silicon, endodermis     

Purification and Properties of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Dark-Grown Seedlings of Sorghum bicolor

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent K_m values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (K_i = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.     

Isolation from the Sorghum bicolor Mycorrhizosphere of a Bacterium Compatible with Arbuscular Mycorrhiza Development and Antagonistic towards Soilborne Fungal Pathogens

A gram-positive bacterium with antagonistic activity towards soilborne fungal pathogens has been isolated from the mycorrhizosphere of Sorghum bicolor inoculated with Glomus mosseae. It has been identified as Paenibacillus sp. strain B2 based on its analytical profile index and on 16S ribosomal DNA analysis. Besides having antagonistic activity, this bacterium stimulates mycorrhization.