2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ～144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.     

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke,and its expression upon UV-C stress

Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3′-hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with p-coumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps. Nucleotide sequence data reported are available in the GenBank database under accession number: FJ225121     

Construction of a reference molecular linkage map of globe artichoke (<Emphasis Type="Italic">Cynara cardunculus</Emphasis> var. <Emphasis Type="Italic">scolymus</Emphasis>)

The genome organization of globe artichoke (Cynara cardunculus var. scolymus), unlike other species belonging to Asteraceae (=Compositae) family (i.e. sunflower, lettuce and chicory), remains largely unexplored. The species is highly heterozygous and suffers marked inbreeding depression when forced to self-fertilize. Thus a two-way pseudo-testcross represents the optimal strategy for linkage analysis. Here, we report linkage maps based on the progeny of a cross between globe artichoke (C. cardunculus var. scolymus) and cultivated cardoon (C. cardunculus var. altilis). The population was genotyped using a variety of PCR-based marker platforms, resulting in the identification of 708 testcross markers suitable for map construction. The male map consisted of 177 loci arranged in 17 major linkage groups, spanning 1,015.5 cM, while female map was built with 326 loci arranged into 20 major linkage groups, spanning 1,486.8 cM. The presence of 84 loci shared between these maps and those previously developed from a cross within globe artichoke allowed for map alignment and the definition of 17 homologous linkage groups, corresponding to the haploid number of the species. This will provide a favourable property for QTL scanning; furthermore, as 25 mapped markers (8%) correspond to coding regions, it has an additional value as functional map and might represent an important genetic tool for candidate gene studies in globe artichoke.     

Roles of PprA,IrrE, and RecA in the resistance of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> to germicidal and environmentally relevant UV radiation

To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation.     

The effects of salicylic acid on pigment contents in ultraviolet radiation stressed pepper plants

Pepper (Capsicum annuum L.) plants were sprayed with salicylic acid (SA) and treated with ultraviolet radiation UV-A (320–390 nm), UV-B (312 nm), and UV-C (254 nm) of 6.1, 5.8, and 5.7 W m⁻², respectively. UV significantly reduced contents of chlorophyll (Chl) a and b, and carotenoids (Car). SA treatment moderated Chl and Car reduction in plants treated with UV-B and UV-C. The quantity of antocyanins, flavonoids, rutin, and UV-absorbing compounds in plants that were treated with UV-B, UV-C, and SA were significantly increased. Foliar spray of SA counteracted the UV effects on pepper.     

Phenolic compounds and antioxidant properties in the snow alga Chlamydomonas nivalis after exposure to UV light

The snow alga Chlamydomonas nivalis was collected from the Sierra Nevada, California, USA, and examined for its ability to produce phenolic compounds, free proline, and provide antioxidant protection factor in response to UV-A and UV-C light. Exposure of C. nivalis cells to UV-A light (365nm) for 5 days resulted in a 5–12% increase in total phenolics, where as exposure to UV-C light (254 nm) resulted in a 12–24% increase in phenolics after 7 days of exposure. Free proline was not affected by UV-A, but increased markedly after UV-C exposure. A three-fold increase in free proline occurred within two days after exposure to UV-C, but then dropped as cells became bleached. Antioxidant protection factor (PF) increased after treatment of cells with UV-A and remained constant throughout UV-C exposure. Spectral analysis of algal extracts revealed a decrease in absorption in the 215–225 nm region, short-term (2day) stimulation of pigment at 280 nm, and an increase in carotenoids (473 nm), after exposure to UV-A. Snow alga exposed to UV-C light had a different spectrum from that of UV-A exposed cells, i.e. an enhancement of three major peaks at 220, 260, and 280 nm, and loss of absorption in the carotenoid region.We report that UV light exposure, especially in the UV-C range, can stimulate phenolic-antioxidant production in aplanospores of C. nivalis effecting biochemical pathways related to proline metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic mapping and annotation of genomic microsatellites isolated from globe artichoke

Cynara cardunculus includes three taxa, the globe artichoke (subsp. scolymus L. Hegi), the cultivated cardoon (var. altilis) and their progenitor, the wild cardoon (var. sylvestris). Globe artichoke is an important component of the Mediterranean rural economy, but its improvement through breeding has been rather limited and its genome organization remains largely unexplored. Here, we report the isolation of 61 new microsatellite loci which amplified a total of 208 alleles in a panel of 22 C. cardunculus genotypes. Of these, 51 were informative for linkage analysis and 39 were used to increase marker density in the available globe artichoke genetic maps. Sequence analysis of the 22 loci associated with genes showed that 9 are located within coding sequence, with the repetitive domain probably being involved in DNA binding or in protein–protein interactions. The expression of the genes associated with 9 of the 22 microsatellite loci was demonstrated by RT-PCR.     

萌芽菊芋块茎对盐碱土壤胁迫的生理响应

土壤盐碱化是影响全球农业生产和生态环境的重要问题。在农田、轻度盐碱草地和重度盐碱草地设置样地以块茎种植菊芋,次年5月块茎萌发阶段取块茎样品测定丙二醛、游离脯氨酸、可溶性糖含量以及抗氧化酶活性并进行蛋白质组学分析,分析了萌芽菊芋块茎对盐碱土壤胁迫的生理响应。0—20 cm土层的电导率(表征土壤可溶盐含量)表明从农田到轻度、重度盐碱草地土壤盐碱胁迫逐渐增强,丙二醛含量变化反映出菊芋块茎受害程度逐渐增加,并且基于游离脯氨酸的渗透调节能力也在逐渐增强。蛋白质组学分析结果显示与遗传信息加工相关的差异蛋白数量最多(占28.75%)且多为表达上调,意味着DNA复制和转录、蛋白质合成和折叠的相关蛋白在响应盐碱胁迫中发挥关键作用。碳水化合物及多糖代谢(占15%)、氨基酸代谢(占11.25%)以及能量代谢(占7.5%)相关的差异蛋白数量也较多,说明调节物质代谢平衡在萌芽菊芋块茎应对盐碱土壤胁迫过程中有重要作用。这些结果为揭示萌芽菊芋块茎适应盐胁迫的生理机制奠定了基础。     

Characterization of Three Sorbitol Transporter Genes in Micropropagated Apple Plants Grown under Drought Stress

Sorbitol, a major end-product of photosynthesis in many species of the Rosaceae family, accumulates in response to abiotic stressors. However, the relationship that arises between the expression of sorbitol transporters and sorbitol accumulation under abiotic stress remains unclear. In this study, micropropagated ‘Fuji’ apple plants (Malus domestica Borkh. ‘Fuji’) were exposed to two varying degrees of osmotic stress and compared relative to an unstressed control. The osmotic stress was generated by adding PEG 6000 into full-strength Hoagland solution and adjusted the osmotic potential to either −0.75 MPa (mild drought stress [MIS]) or −1.5 MPa (severe drought stress [SES]). Analysis of sorbitol levels via high performance liquid chromatography (HPLC) showed that the sorbitol concentration was elevated in roots, phloem tissues and leaves in both the MIS and SES treatments compared to controls for the entire duration of the experiment. Three cDNA sequences, encoding sorbitol transporters (MdSOT3, MdSOT4 and MdSOT5), were isolated from leaves. Real-time quantitative PCR (RT-qPCR) data suggests that the expression levels of MdSOT3 and MdSOT5 were higher under MIS and SES in roots, phloem tissues and leaves compared to unstressed controls. The average mRNA levels of MdSOT4 in phloem tissues declined under both drought treatments (with the exception being at 2 h of SES). In roots and leaves under SES, mRNA production was increased. These results indicate that the up-regulation of MdSOT3 and MdSOT5 expression is consistent with the accumulation of sorbitol under conditions of osmotic stress in apple plants. They enhanced drought tolerance in vegetative tissues. Increased MdSOT4 mRNA enhanced drought tolerance under SES.     

Jasmonic acid,polyamine, and antioxidant levels in apple seedlings as affected by Ultraviolet-C irradiation

The effect of UV-C irradiation on jasmonates, abscisic acid (ABA), polyamines and antioxidant activities was investigated in apple plants (Malus domestica Borkh.). The EC₅₀ values of O₂ ^–-scavenging activity in the UV-C treated leaves were lower than those in the untreated control. In addition, total ascorbic acid and polyphenolic concentrations in the UV-C treated leaves were generally higher than those in the untreated control. Although the endogenous ABA concentrations were not significantly different between the untreated control and UV-C treatment, the endogenous jasmonic acid, putrescine, and spermine concentrations in the UV-C treated plants were higher than those in the untreated control. However, the application of PDJ before UV treatment inhibited the rise of endogenous jasmonate and putrescine concentrations. The increase of ascorbic acid was also inhibited by PDJ application before UV treatment. These facts suggest that the increase of jasmonates and polyamines may be associated with UV-C stimulation, and UV-C irradiation may be effective for increasing antioxidant activity in apple leaves.     

Divergent low water potential response in Arabidopsis thaliana accessions Landsberg erecta and Shahdara

The Arabidopsis thaliana accession Shahdara (Sha) differs from Landsberg erecta (Ler) and other accessions in its responses to drought and low water potential including lower levels of proline accumulation. However, Sha maintained greater seedling root elongation at low water potential and a higher NADP/NADPH ratio than Ler. Profiling of major amino acids and organic acids found that Sha had reduced levels of all glutamate family amino acids metabolically related to proline, but increased levels of aspartate‐derived amino acids (particularly isoleucine), leucine and valine at low water potential. Although Sha is known for its different abiotic stress response, RNA sequencing and co‐expression clustering found that Sha differed most from Ler in defence/immune response and reactive oxygen‐related gene expression. HVA22B and Osmotin34 were two of the relatively few abiotic stress‐associated genes differentially expressed between Ler and Sha. Insensitivity to exogenous glutamine and a different expression profile of glutamate receptors were further factors that may underlie the differing metabolism and low water potential phenotypes of Sha. These data define the unique environmental adaptation and differing metabolism of Sha including differences in defence gene expression, and will facilitate further analysis of Sha natural variation to understand metabolic regulation and abiotic/biotic stress interaction.     

Insect pollinators improve seed production in globe artichoke (Cynara cardunculus var. scolymus)

The role of many insect species in crop pollination has been widely studied. The use of entomophilous pollination, commonly adopted for other crops, may be of key importance in order to improve seed yields in seed-propagated globe artichoke (Cynara cardunculus var. scolymus) cultivars. In this regard, in a 2-year field experiment, the role of two honeybees (Apis mellifera ligustica and A. mellifera siciliana) and one bumblebee (Bombus terrestris) was evaluated on the seed production of two globe artichoke cultivars in caged-protected environments. Overall, the number and weight of seeds plant⁻¹ and head⁻¹, 1,000 seed weight and fruit setting were higher in caged plants with imposed pollination than open field plants. The pollination efficiency was both insect and cultivar dependent. Both honeybee species performed better on the Mediterranean cultivar ‘Violetto di Sicilia’, while the bumblebee performed better on the Brazilian cultivar ‘NP4’. These results could be very useful to modernise the agronomic management of globe artichoke and reduce the costs of cultivation. In addition, such knowledge can be used to improve the seed production in globe artichoke seed-propagated cultivars and cardoon. This will benefit the bioenergy, nutraceutical, cosmetic and ornamental applications.     

Physiological responses of two Jerusalem artichoke cultivars to drought stress induced by polyethylene glycol

Physiological responses of two Jerusalem artichoke (Helianthus tuberosus L.) cultivars with different drought sensitivity to drought stress induced by polyethylene glycol (PEG) 6000 were investigated by characterizing water status, membrane lipid peroxidation, key antioxidant enzymes activity, and proline accumulation. It was observed that the drought-tolerant Jerusalem artichoke cv. Xiuyan maintained a relatively higher water status than the drought-sensitive cv. Yulin upon drought treatments. Meanwhile, lower levels of malondialdehyde (MDA) as well as higher levels of free proline occurred in cv. Xiuyan after 36 h drought treatments. Moreover, the activities of catalase (CAT) and superoxide dismutase (SOD) in cv. Xiuyan were higher than cv. Yulin after drought stress. These results indicated that drought sensitivities actually differ between Jerusalem artichoke cv. Xiuyan and cv. Yulin, and the cv. Xiuyan was more tolerant to drought stress caused by polyethylene glycol.     

Production and fingerprinting of virus-free clones in a reflowering globe artichoke

Most of the 24 viruses which infect globe artichoke are detrimental to the crop’s performance and hamper the development of a nursery activity in the respect of current EU legislation. We describe a procedure to sanitize globe artichoke “Brindisino” from Artichoke Italian latent virus (AILV) and Artichoke latent virus (ArLV), while preserving its valuable early flowering trait. ArLV was successfully eliminated by meristem-tip culture, while AILV was removed when two rounds of meristem-tip culture were spaced out with in vitro thermotherapy. In vivo thermotherapy, followed by meristem-tip culture, was also successful in producing virus-free material but was less efficient in terms of the number of plants recovered post treatment. Due to the multi-clonal composition of the populations at present in cultivation, the selected and sanitised clones were fingerprinted by applying microsatellite and AFLP (amplified fragment length polymorphism) markers. One AFLP primer combination produced 28 informative fragments used to evaluate genetic relatedness among the clones in study. Our results demonstrates that AFLP-based molecular fingerprinting enables to verify the true to clone correspondence in nurseries, ensure the effective correspondence between the real and the declared identity of a clone, so that to avoid commercial frauds, and might represents a valuable tool for assessing somaclonal variation occuring during ‘in vitro’ propagation.     

Aroma volatiles of Cynara scolymus and Helianthus tuberosus

Samples of the aroma volatiles of globe artichoke and Jerusalem artichoke were obtained by well established methods and were analysed by routine GC and GC/MS. Eight sesquiterpene hydrocarbons afforded the major group of components (over 42%) in globe artichoke samples, with β-selinene (ca 32%) as the main constituent. Previously reported caryophyllene could not be detected. α-Cedrene was found to have globe artichoke aroma characteristics on odour evaluation of separated components at an odour port at the exit of the GC column. Jerusalem artichoke samples contained one major component (β-bisabolene, ca 51%) and a range of saturated long-chain hydrocarbons (ca 22%). The sesquiterpene presumably contributes appreciably to the characteristic flavour of Jerusalem artichoke. Both types of artichoke gave a low concentration of total volatiles.     

谷子PAL基因家族全基因组的鉴定和表达分析

苯丙氨酸解氢酶(phenylalanine ammonia-lyase,PAL)基因家族参与苯丙烷类代谢过程,通过调控植物抗病次生物质的合成在植物抗逆反应中发挥重要作用。为明确谷子PAL基因家族在逆境胁迫下的表达规律,该研究利用生物信息学方法对谷子PAL基因家族进行鉴定和表达分析。结果表明:谷子具有11个PAL基因,在进化树中可分为3个亚家族,SiPAL7独自进化为一支。通过构建蛋白结构域发现PAL基因家族成员均含有保守的PAL结构域。启动子分析显示,PAL基因含有应答激素、逆境胁迫等多种因子的顺式作用元件,说明PAL基因广泛参与不同生物学调控过程。RT-qPCR结果显示,谷子PAL基因家族多为诱导型表达,不同光照条件下PAL基因表达量变化明显,不同基因具有不同响应模式,说明谷子PAL基因家族在参与光调节反应中发挥重要作用。谷子PAL基因高度保守,广泛响应不同非生物胁迫,具有表达特异性。该研究结果为揭示PAL基因家族在调节谷子抗性及胁迫应答过程中的作用提供了参考。