Acetyl coenzyme A biosynthesis in the chloroplast

Acetyl coenzyme A (CoA) biosynthesis in spinach chloroplasts has been investigated by following the incorporation of bicarbonate and acetate into fatty acids under a variety of conditions. Both substrates were readily incorporated into fatty acids in a light-dependent manner by intact photosynthesising chloroplasts, but when the concentrations of these substrates were adjusted to those found in vivo, i.e. 200 M acetate, 10 M bicarbonate, then acetate was found to supply carbon atoms for fatty acids biosynthesis via acetyl CoA at forty times the rate of bicarbonate. It is proposed that extra-chloroplastic free acetate is the pricipal substrate for chloroplasts acetyl CoA biosynthesis in spinach.Abbreviations ACP acyl carrierprotein - CoASH coenzyme A     

Use of mitochondrial DNA as a sensitive and specific marker for localization of mitochondria in fractionated plant extracts

Upon subfractionation of certain plant seed homogenates on sucrose density gradients, we encountered problems in defining the location and amount of mitochondria using marker enzymes. In order to overcome the inherent limitations of enzyme assays, we utilized a heterologous DNA probe specific foratp6 in maize orBrassica tournefortii to detect mitochondria. The samples were treated with SDS, proteinase K, and RNase A followed by agarose gel electrophoresis, and blotting. The immobilized DNA was detected with [³²P]-labelled probes, and quantified using a phosphor imager. The assay is specific, sensitive, and independent of species, cell type, and developmental stage, thus circumventing the need for expressed protein to assay enzyme activity.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.     

Acetyl coenzyme A carboxylase in species of Triticum of different ploidy

The cellular amounts and cellular activities of acetyl CoA carboxylase (ACC; EC 6.4.1.2.) were determined in the first leaves of diploid, tetraploid and hexaploid species of Triticum (wheat). Per leaf the ACC activities were very similar in T. monococcum (2 ), T. dicoccum (4 ) and T. aestivum (6 ). The ACC activity per chloroplast also showed little variation between species of different ploidy but since chloroplast number increases with ploidy, the ACC activities and ACC amounts per cell also increased with ploidy. These cellular increases in ACC amounts associated with increases in gene dosage were highly co-ordinated in the diploids T. monococcum and T. tauschii and their respective autotetraploids so the specific activity of ACC was highly conserved in these plants. The relevance of these findings to attempts to genetically manipulate lipid biosynthesis in chloroplasts is discussed.Abbreviation ACC acetyl CoA carboxylase We are very grateful to Dr. Kevin Pyke and Miss Jo Marrison for many helpful discussions and to Dr. Collin Law for the generous gift of seeds.     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

蓝桉凋落叶分解对菠菜生长及光合特性的影响

通过盆栽试验,模拟蓝桉(Eucalyptus globulus)凋落叶在土壤中分解对受体植物菠菜(Spinacia oleracea)生长及光合特性的影响,设置L30(30 g/盆)、L60(60 g/盆)、L90(90 g/盆)和L120(120 g/盆)4个凋落叶施用水平,对照(CK)不加凋落叶。结果表明:(1)在分解30 d时,低量的凋落叶(L30)未抑制菠菜生物量的积累,而中量和高量处理(L60、L90和L120)下其生物量显著降低(P0.05);在凋落叶分解50 d后,蓝桉凋落叶的化感抑制效应已经减弱,仅L120仍然抑制菠菜生物量的积累;(2)一定量的凋落叶(L30和L60)对菠菜叶绿素的合成有促进作用,而凋落叶超过一定量时(L120)转为抑制作用,在凋落叶分解28 d时相对不明显,40 d后逐渐显现;(3)经凋落叶处理的菠菜叶片胞间CO2浓度(Ci)、气孔导度(Gs)及蒸腾速率(Tr)均显著高于对照(CK)(P0.05),而净光合速率(Pn)在L30处理下最高,L60和L90处理与CK差异不显著,仅L120处理显著低于CK(P0.05);(4)光响应与CO2响应曲线的特征参数表观量子效率(AQY)、最大净光合速率(Pmax)、光饱和点(Lsp)、光补偿点(Lcp)、暗呼吸速率(Rd)、Ru BP表观羧化效率(CE)和光呼吸速率(Rp)在L30处理下高于CK,其余凋落叶处理均低于CK,而CO2饱和点(Csp)、CO2补偿点(Ccp)随凋落叶量的增加而升高。综合各指标可见,少量的蓝桉凋落叶分解(L30)可通过增加叶绿素含量,提高对光和CO2的利用能力促进菠菜的光合作用,进而促进其生长,而凋落叶超过一定量时(L90—L120)则起到相反的作用。     

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene -glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and -amylase inhibitors.     

Effects of supplementary UV-B radiation on development of damping-off in spinach caused by the soil-borne fungusFusarium oxysporum

The effects of UV-B radiation (290–320 nm) on development of damping-off of spinach (Spinacia oleracea) caused by the fungusFusarium oxysporum were examined in a growth cabinet. The incidence of disease greatly increased when experimental plants were grown in visible radiation with supplementary UV-B radiation. This increase was suppressed by increasing the irradiation of visible radiation.Fusarium oxysporum was isolated from the roots of all damping-off plants and the roots of some unwilted plants, indicating that spinach infected with the pathogen did not necessarily suffer from damping-off in 15d. Supplementary UV-B radiation suppressed the increase in growth components such as the number of leaves, the plant height and the fresh weight of aboveground plant parts, but did not affect the fresh weight of roots. The ratio of the number of plants infected with pathogen to the total number of plants was over 80% irrespective of light conditions. It was suggested that the defense response of spinach to this pathogen was greatly influenced by the physiological state of aboveground plant parts resulting from supplementary UV-B radiation.     

蓖麻RcACA基因家族鉴定及非生物胁迫下表达模式分析

自抑制Ca^(2+)-ATPase酶(auto-inhibited Ca2+-ATPase,ACA)作为Ca2+-ATPase的亚家族之一,在植物细胞内维持Ca2+浓度平衡发挥着重要的作用。为探究蓖麻(Ricinus communis)RcACA基因家族的功能及基因表达模式,文中采用生物信息学手段鉴定蓖麻RcACA基因家族成员,预测分析了其基础的理化性质、亚细胞位置、蛋白的二级和三级结构、保守域、保守基序、基因结构、染色体位置及共线关系、进化特征、启动子顺式作用元件,并通过蓖麻转录组数据中的表达量(fragments per kilobase of exon model per million mapped fragments,FPKM)分析RcACA基因在非生物胁迫下的表达模式。结果表明,在蓖麻中共鉴定到8个RcACA基因家族成员,均是酸性蛋白且定位在细胞质膜;所有蛋白的二级和三级结构中α-螺旋和不规则卷曲较多;RcACA基因被聚为3类,同一类别中基因的结构与保守基序相似;均有典型的4个结构域RcACA3–RcACA8,还有1个Ca^(2+)-ATPase N端自抑制结构域(N-terminal autoinhibitory domain);RcACA基因多位于染色体长臂,拥有2对共线关系。RcACA基因编码区上游拥有较多的光响应作用元件,激素诱导类作用元件较少。种间聚类显示ACA基因在物种间的进化是保守的。组织表达模式分析显示,RcACA基因拥有明显的组织表达特异性,且多数基因在雄花中表达量最高;非生物胁迫表达分析表明,RcACA2–RcACA8在高盐和干旱胁迫下均上调表达,RcACA1在低温胁迫的0–24 h上调表达,表明RcACA基因积极地响应非生物胁迫。上述结果为探究RcACA基因在蓖麻生长发育和逆境胁迫中的作用提供了理论参考。     

Characterization of a gene highly expressed in the Brassica napus pistil that encodes a novel proline-rich protein

Pis 30, a gene highly expressed in Brassica napus pistils and encoding a novel proline-rich protein was isolated and characterized. Sequences homologous to the Brassica Pis 30 gene were found only in Arabidopsis thaliana. The Pis 30 gene encodes a mature protein of 8.4 kDa with no previously characterized protein domains and whose function remains unknown. PIS 30 contains especially high levels of Pro (33%), but also of Leu (14%), Phe (10%) and Ser (6%). Although it is a proline-rich protein, PIS 30 shows only limited similarity to previously characterized plant proline-rich proteins. When compared to the stigma-specific activity of the B. napus SLR1 gene promoter in pistils of transgenic Arabidopsis, an 808 bp Pis 30 promoter fragment directed -glucuronidase expression primarily in the ovary, as well as in the stigma.     

Determination of endogenous and supplied deuterated abscisic acid in plant tissues by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring

A specific, sensitive, and accurate method for determination of abscisic acid (ABA) in plant tissues is described. The method employs reversed-phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry for multiple reaction monitoring of underivatized ABA and deuterated ABA analogs. Specific analogs were used to study the mechanism of ABA fragmentation, to select appropriate standards, and to identify compounds suitable for metabolic studies involving the supply of differentially labeled ABA. Limits of detection and quantification of 1.9 and 4.7 pg, respectively, were obtained over a linear calibration range of 0-1.5 ng ABA (on-column injected) using 5.8', 8', 8'-d(4) ABA as the internal standard. Accuracy and precision were within 15% for routine quality control samples. The method of standard additions, as applied to Arabidopsis thaliana seed extracts, was also used to validate the method for analysis of plant tissue samples. The utility of the method was further demonstrated by determining levels of ABA in western white pine seeds and of ABA and supplied 8', 8', 8', 9', 9', 9'-d(6) ABA in Brassica napus tissues, using 5.8', 8', 8'-d(4) ABA or 8', 8', 8'-d(3) ABA as the internal standard. Limits of quantification as low as 0.89 ng/g were achieved by optimizing the extraction procedure for each type of plant tissue.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Overexpression of formate dehydrogenase inArabidopsis thaliana resulted in plants tolerant to high concentrations of formate

The potential role played by formate dehydrogenase (FDH) in formate metabolism has been examined by the overexpression of FDH in Arabidopsis thaliana. Three independent transgenic lines were selected and shown to produce elevated amounts of FDH protein with a corresponding elevated FDH activity (2.5-5 fold) over wild-type (WT) plants. Under normal growth conditions, no altered phenotype was observed in these transgenic plants; in growth media supplied with formate, however, significant differences in shoot and root growth, compared to that of WT plants, were observed. WT plants were severely injured if grown in the presence of 16 mmol/L formate, while the transgenic plants were able to grow well. Formate delayed germination of both WT and transgenic seeds at concentrations above 4 mmol/L, but both types of seeds were eventually able to complete more than 95 % germination even at 32 mmol/L formate. Formate markedly inhibited primary root elongation, and its inhibitory action on WT was much stronger than on transgenic plants. Different formate salts affected root elongation similarly, indicating that the formate ion was the major factor inhibiting root growth. Sodium acetate (NaAc), an analogue of formate, also inhibited root elongation, but its action on WT and transgenic plants was the same, indicating that tolerance of transgenic plants to formate toxicity was specific. Transgenic plants showed no significant tolerance to the toxicity of two other one-carbon metabolites, methanol and formaldehyde. A role for FDH in detoxifying formate is proposed.     

Inhibition of stem elongation in spinach by theobroxide

In the current study, we investigated the influences of theobroxide on stem elongation in spinach (Spinacia oleracea). Our results showed that stem elongation and flower formation were inhibited by spraying spinach plants with theobroxide under inductive, long day conditions (16 h light/8 h dark), while application of exogenous applied GA3 prevented the effect of theobroxide. Quantitative analysis showed that theobroxide suppressed GA1 biosynthesis, whereas the endogenous content of jasmonic acid was unchanged. However, under short day conditions (10 h light/14 h dark), there were no differences in stem length between treated and untreated plants. These results suggest that the inhibition of stem elongation by theobroxide is probably due to the suppression of gibberellin biosynthesis.     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.