木薯品种(系)类胡萝卜素代谢相关基因和蛋白表达水平分析

以白心木薯华南6068、华南9号、紫叶黄心木薯BGM019和粉红木薯Mirasol为材料,探究木薯块根膨大期和成熟期与类胡萝卜素代谢通路相关的14个基因和4种蛋白质表达水平变化。用HPLC检测块根β-胡萝卜素含量的变化,分别用qRT-PCR和Western blot方法对类胡萝卜素代谢通路相关基因和蛋白酶的表达水平进行分析。以华南6068为对照,研究结果表明:华南9号和紫叶黄心木薯BGM019成熟期中的类胡萝卜素合成途径关键基因PSY2、LCYB基因显著高于膨大期,而降解相关的关键基因CCD1、NCED3在成熟期的表达量显著低于膨大期(P0.05)。粉红木薯Mirasol成熟期中PSY2、LCYB的显著下调与CCD1、NCED3的显著上调(P0.05)是造成β-胡萝卜素含量差异的原因之一。通过分析不同木薯品种(系)在膨大期和成熟期块根类胡萝卜素代谢途径相关基因的表达水平,有助于解析β-胡萝卜素积累的分子机理。此外,Western blot结果显示抗坏血酸过氧化物酶、谷胱甘肽还原酶、超氧化物歧化酶和HSP70虽然和块根类胡萝卜素代谢途径没有直接关联,但它们在木薯膨大期和成熟期块根表达水平有显著差异(P0.05)。     

Carotenoid Content and Root Color of Cultivated Carrot: A Candidate-Gene Association Study Using an Original Broad Unstructured Population

Accumulated in large amounts in carrot, carotenoids are an important product quality attribute and therefore a major breeding trait. However, the knowledge of carotenoid accumulation genetic control in this root vegetable is still limited. In order to identify the genetic variants linked to this character, we performed an association mapping study with a candidate gene approach. We developed an original unstructured population with a broad genetic basis to avoid the pitfall of false positive detection due to population stratification. We genotyped 109 SNPs located in 17 candidate genes – mostly carotenoid biosynthesis genes – on 380 individuals, and tested the association with carotenoid contents and color components. Total carotenoids and β-carotene contents were significantly associated with genes zeaxanthin epoxydase (ZEP), phytoene desaturase (PDS) and carotenoid isomerase (CRTISO) while α-carotene was associated with CRTISO and plastid terminal oxidase (PTOX) genes. Color components were associated most significantly with ZEP. Our results suggest the involvement of the couple PDS/PTOX and ZEP in carotenoid accumulation, as the result of the metabolic and catabolic activities respectively. This study brings new insights in the understanding of the carotenoid pathway in non-photosynthetic organs.     

过表达AtchyB基因提高洋桔梗光胁迫耐受性的研究

类胡萝卜素尤其是叶黄素循环类物质在植物抵抗由强光照引起的非生物胁迫中发挥着重要的作用,为了提高洋桔梗对强光照的抗性,从拟南芥中克隆了类胡萝卜素生物合成途径中参与叶黄素循环关键酶——β-胡萝卜素羟化酶基因(AtchyB),利用农杆菌介导法将其转入洋桔梗中,最终得到遗传转化植株2个株系,研究发现,转基因植株中总类胡萝卜素含量高于对照组,且叶黄素循环池被不同程度地放大。在不同光照强度下,转基因洋桔梗植株对光照耐受性明显强于对照组,且转基因植株生物量也明显提高。表明过表达AtchyB基因使洋桔梗光胁迫耐受性有所增强。     

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.     

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48 h after wounding and remained elevated through 96 h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8′-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3 h of wounding and remained elevated through 96 h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.     

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g(-1) and 13 μg g(-1) fresh weight (FW), respectively, and for BS 19 μg g(-1) and 0.27 μg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2

Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.     

Cloning and functional characterization of the maize carotenoid isomerase and β-carotene hydroxylase genes and their regulation during endosperm maturation

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and β-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9′-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert β-carotene into β-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form β-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.     

Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes

Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44,000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene alpha-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode alpha-copaene synthase. Other potential 'flavour genes', identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5'-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.     

反义酸性转化酶基因低温贮藏马铃薯块茎还原糖和淀粉含量的影响

对2个含有酸性转化酶(AcInv)反义基因的转基因马铃薯品系及对照品种进行低温贮藏(4℃)及室温还暖处理.随低温贮藏时间的延长,供试材料均表现出还原糖含量升高,总淀粉含量下降的趋势.低温处理40 d时,"Ac转Atlantic"和"Ac转甘农薯2号"的还原糖含量比未转基因品种低23%和18%.总淀粉含量分别比未处理前下降约1.0%和1.3%,支链淀粉含量分别下降约1.4%和1.7%,淀粉直/支比明显低于对照,分别为0.29和0.38.块茎的石蜡切片显示,转基因块茎中深蓝色淀粉颗粒明显少于未转基因对照.另外,对低温贮藏的块茎室温还暖后,2个转基因品系的还原糖含量仍低于对照品种.实验结果证明反义AcInv基因对低温贮藏下块茎还原糖和淀粉含量具有下向调节作用.     

Genetic architecture controlling variation in grain carotenoid composition and concentrations in two maize populations

Key message

Genetic control of maize grain carotenoid profiles is coordinated through several loci distributed throughout three secondary metabolic pathways, most of which exhibit additive, and more importantly, pleiotropic effects.

Abstract

The genetic basis for the variation in maize grain carotenoid concentrations was investigated in two F_2:3 populations, DEexp × CI7 and A619 × SC55, derived from high total carotenoid and high β-carotene inbred lines. A comparison of grain carotenoid concentrations from population DEexp × CI7 grown in different environments revealed significantly higher concentrations and greater trait variation in samples harvested from a subtropical environment relative to those from a temperate environment. Genotype by environment interactions was significant for most carotenoid traits. Using phenotypic data in additive, environment-specific genetic models, quantitative trait loci (QTL) were identified for absolute and derived carotenoid traits in each population, including those specific to the isomerization of β-carotene. A multivariate approach for these correlated traits was taken, using carotenoid trait principal components (PCs) that jointly accounted for 97 % or more of trait variation. Component loadings for carotenoid PCs were interpreted in the context of known substrate-product relationships within the carotenoid pathway. Importantly, QTL for univariate and multivariate traits were found to cluster in close proximity to map locations of loci involved in methyl-erythritol, isoprenoid and carotenoid metabolism. Several of these genes, including lycopene epsilon cyclase, carotenoid cleavage dioxygenase1 and beta-carotene hydroxylase, were mapped in the segregating populations. These loci exhibited pleiotropic effects on α-branch carotenoids, total carotenoid profile and β-branch carotenoids, respectively. Our results confirm that several QTL are involved in the modification of carotenoid profiles, and suggest genetic targets that could be used for the improvement of total carotenoid and β-carotene in future breeding populations.