A manipulation of carotenoid metabolism influence biomass partitioning and fitness in tomato

Improving yield, nutritional value and tolerance to abiotic stress are major targets of current breeding and biotechnological approaches that aim at increasing crop production and ensuring food security. Metabolic engineering of carotenoids, the precursor of vitamin-A and plant hormones that regulate plant growth and response to adverse growth conditions, has been mainly focusing on provitamin A biofortification or the production of high-value carotenoids. Here, we show that the introduction of a single gene of the carotenoid biosynthetic pathway in different tomato cultivars induced profound metabolic alterations in carotenoid, apocarotenoid and phytohormones pathways. Alterations in isoprenoid- (abscisic acid, gibberellins, cytokinins) and non-isoprenoid (auxin and jasmonic acid) derived hormones together with enhanced xanthophyll content influenced biomass partitioning and abiotic stress tolerance (high light, salt, and drought), and it caused an up to 77% fruit yield increase and enhanced fruit's provitamin A content. In addition, metabolic and hormonal changes led to accumulation of key primary metabolites (e.g. osmoprotectants and antiaging agents) contributing with enhanced abiotic stress tolerance and fruit shelf life. Our findings pave the way for developing a new generation of crops that combine high productivity and increased nutritional value with the capability to cope with climate change-related environmental challenges.     

Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.     

Carotene Hydroxylase Activity Determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots

The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels of α-carotene in addition to β-carotene. We found similarly increased α-carotene levels in leaves of orange carrots compared with white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those found in white-rooted cultivars and strongly reduced α-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to 50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux. Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping analysis using a large carrot population revealed a significant association of this polymorphism with both α-carotene content and the α-/β-carotene ratio and explained a large proportion of the observed variation in carrots.     

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.     

Cassava Breeding: Current Status, Bottlenecks and the Potential of Biotechnology Tools

Cassava is an important energy source in the diets of millions of people in tropical and subtropical regions of the world. It is a key subsistence crop, and its industrial uses are steadily growing. In spite of its economic and social relevance, relatively little investment has been made for research on cassava. However, conventional breeding resulted in more stable production through enhanced tolerance to biotic and abiotic stresses; increased productivity, both in fresh root production and increased dry matter content; and, more recently, improvements in qualitative traits such as starch quality and increased carotenoids content. The inbreeding of cassava has been identified as a key step for more efficient genetic improvement of the crop, therefore, research is underway to develop protocol(s) for the production of doubled haploids. Marker-assisted selection has been successfully applied to cassava, but in a more modest scale compared with other crops. More support and emphasis is needed on practical applications of molecular marker technology in cassava improvement. The availability of more efficient genotyping approaches and the cassava genome sequence promise to increase the impact of biotechnology tools on cassava improvement. Efficient and reliable phenotyping of cassava remains a challenging goal to achieve in the near future.     

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 Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background

As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.

Methology/Principal Findings

In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.

Conclusions

The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.     

Isozyme Diversity in Cassava Cultivars (Manihot esculenta Crantz)

Isoenzyme electrophoresis was used as a method to determine genetic diversity in various M. esculenta cultivars collected in the Southwestern (SW) and Northwestern (NW) regions of the State of Parana, in the South region of Brazil, and in cultivars produced at the Agronomic Institute of Campinas (IAC), S~ao Paulo State, Southeastern region of Brazil. The cultivars have been maintained by vegetative propagation for 5 years and are useful in production programs. A total of 28 loci in the acid phosphatase (ACP; EC 3.1.3.2), esterases (EST; EC 3.1.1.1), malate dehydrogenase (MDH; EC 1.1.1.37), and shikimate dehydrogenase (SKDH; EC 1.1.1.15) isozymes was analyzed. The proportion of polymorphic loci for NW, SW, and IAC cultivars was 57.14, 50.0, and 53.6%, respectively. Genetic diversity calculated by Nei's genetic identity (I) showed high I values for the three M. esculenta subpopulations. The high degree of polymorphism expressed by cassava cultivars is highly relevant to stimulate breeding programs with M. esculenta species.     

类胡萝卜素生物合成相关基因的克隆及其遗传工程的研究进展

类胡萝卜素具有重要的生物学功能,尤其对人体健康有着更重要的作用,近年来一直是研究的热点。综述了类胡萝卜素生物合成途径及相关基因的分离,以及运用这些基因提高微生物和植物中类胡萝卜素含量的遗传工程研究进展。     

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub‐organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live‐cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid‐based phenotypes.     

Cassava genetic transformation and its application in breeding

As a major source of food, cassava (Manihot esculenta Crantz) is an important root crop in the tropics and subtropics of Africa and Latin America, and serves as raw material for the production of starches and bioethanol in tropical Asia. Cassava improvement through genetic engineering not only overcomes the high heterozygosity and serious trait separation that occurs in its traditional breeding, but also quickly achieves improved target traits. Since the first report on genetic transformation in cassava in 1996, the technology has gradually matured over almost 15 years of development and has overcome cassava genotype constraints, changing from mode cultivars to farmer-preferred ones. Significant progress has been made in terms of an increased resistance to pests and diseases, biofortification, and improved starch quality, building on the fundamental knowledge and technologies related to planting, nutrition, and the processing of this important food crop that has often been neglected. Therefore, cassava has great potential in food security and bioenergy development worldwide.     

Generation of transgenic maize with enhanced provitamin A content

Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as beta-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of 'Golden Rice 2' which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and zeta-carotene desaturase in plants), under the control of a 'super gamma-zein promoter' for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of beta-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high beta-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of beta-carotene is due to an up-regulation of the endogenous lycopene beta-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD.