Development of molecular breeding resources for increased pro-vitamin A carotenoids in sorghum grain

Includes bibliographical references.2022 Fall.Vitamin A deficiency (VAD) affects millions of people in countries in Africa and South-East Asia, contributing to decreased immune response and increased morbidity and mortality from common infections. Sorghum [Sorghum bicolor L. (Moench)] is a staple cereal crop in these regions, thus, sorghum carotenoid biofortification is a potential method to improve the vitamin A status of these communities. The overall aim of this research was to determine the feasibility of biofortification breeding for sorghum grain carotenoids, and to develop genomic tools to assist in molecular breeding. Global sorghum germplasm collections were evaluated for pro-vitamin A carotenoids, and concentrations were found to be below target values. Due to the low number of accessions with above average pro-vitamin A content, the genetic diversity of the high carotenoid lines in the global germplasm was assessed. High carotenoid accessions were found to be highly related, hence, to increase genetic diversity for breeding, a genomic prediction model was used to identify additional germplasm with potentially high concentrations of pro-vitamin A carotenoids. Through a genome-wide association study, it was confirmed that carotenoid variation in sorghum grain is oligogenic, but there was also evidence of a polygenic component. Therefore both marker-assisted selection (MAS) and genomic selection (GS) may be effective in accelerating breeding efforts. KASP markers in linkage with genomic regions associated with carotenoid concentrations were developed and validated in six F2:3 populations. Two markers in the intronic region of the carotenoid pathway β-OH gene were identified as good candidates to use for MAS due to their predictive ability. A marker inside the coding sequence of the carotenoid pathway ZEP gene was also identified as a good marker for MAS. An RNA-seq experiment identified additional genes in the MEP, carotenoid biosynthesis and carotenoid degradation pathways that could be used for MAS. The results of these studies provide a foundation for vitamin A biofortification through genomics-assisted breeding

Hunting for carotenoid-derived retrograde signals that regulate plastid development

In plants, carotenoids are essential for photosynthesis and photoprotection. However, carotenoids are not the end-products of the pathway: apocarotenoids are produced by carotenoid cleavage dioxygenases (CCDs) or non-enzymatic processes. Apocarotenoids are more soluble or volatile than carotenoids, but they are not simply breakdown products as there can be modifications post cleavage and functions include hormones, volatiles or signals. Evidence is emerging for a class of apocarotenoids herein referred to as Apocarotenoid Signals (ACSs) that have regulatory roles throughout plant development beyond those ascribed to ABA and strigolactone. In the present study, we provide evidence that ACS2, a cis-carotenoid-derived retrograde signal, regulates plastid development during both skotomorphogenesis and photomorphogenesis. cis-carotenoids produced early in the carotenoid pathway may serve as substrates for the production of novel ACSs that regulate nuclear gene expression, metabolic homeostasis and leaf development. When and where they accumulate and what physiological functions they may serve in higher plants remain unclear. cis-carotenoids are not easily detected in most plant tissues, except in the absence of carotenoid isomerase (CRTISO) activity when photoisomerisation rate-limits the isomerisation of tetra-cis to all-trans-lycopene. The accumulation of cis-carotenoids in Arabidopsis crtiso mutant (carotenoid and chloroplast regulation 2, ccr2) tissues was observed in plant tissues grown under extended darkness (i.e. shorter photoperiod) and coincided with a perturbation in chloroplast development that caused leaf yellowing. A forward genetic screen identified an epistatic interaction between the ζ-carotene isomerase (ziso) and ccr2 which could restore plastid development, and revealed that di-cis-ζ-carotene, tri-cis-neurosporene and tetra-cis-lycopene are likely substrates for the generation of an ACS, named ACS2. Transcriptomics analysis of ccr2 ziso mutant tissues revealed that photosynthesis associated nuclear gene expression (PhANG) was activated through the down-regulation of genes involved in repressing photomorphogenesis. We identified an alternative splice mutant of det1, a repressor of photomorphogenesis, which could restore PLB formation and cotyledon greening following de-etiolation in ccr2. Chemical inhibition of carotenoid cleavage dioxygenase activity provided evidence that ACS2 posttranscriptionally maintains protochlorophyllide oxidoreductase (POR) protein levels acting downstream of DET1 to control PLB formation and plastid development. Phytoene synthase (PSY) is a major rate-controlling enzyme that catalyses the initial step of carotenoid biosynthesis and is hence under multi-level regulation. Alteration of PSY gene expression, protein levels or enzyme activity can exert profound effects on carotenoid composition and plant development. Here we show that four mutants of PSY: psy-4, psy-90, psy-130 and psy-145 reduced cis-carotenoids to levels below a threshold and suppressed ACS2 which negatively regulates plastid development in ccr2. The restoration of plastid development in the four ccr2 psy double mutants was caused by decreased PSY activity and reduced protein levels due to altered PSY-AtOR (ORANGE) interaction, but not by changed localization of PSY. This study reveals a novel role of PSY, modulating carotenoid-derived retrograde signals and regulating plastid development

Photochemical efficiency correlated with candidate gene expression promote coffee drought tolerance.

The aim of this study was to identify the correlation between photochemical efficiency and candidate genes expression to elucidate the drought tolerance mechanisms in coffee progenies (Icatu Vermelho IAC 3851-2 x Catimor UFV 1602-215) previously identified as tolerant in field conditions. Four progenies (2, 5, 12 and 15) were evaluated under water-deficit conditions (water deficit imposed 8 months after transplanting seedlings to the pots) and under irrigated system. Evaluations of physiological parameters and expression of candidate genes for drought tolerance were performed. Progeny 5 showed capacity to maintain water potential, which contributed to lower qP variation between irrigated and deficit conditions. However, the increases of qN and NPQ in response to stress indicate that this progeny is photochemically responsive to small variations of am protecting the photosystem and maintaining qP. Data obtained for progeny 12 indicated a lower water status maintenance capacity, but with increased qN and NPQ providing maintenance of the PSII and ETR parameters. A PCA analysis revealed that the genes coding regulatory proteins, ABA-synthesis, cellular protectors, isoforms of ascorbate peroxidase clearly displayed a major response to drought stress and discriminated the progenies 5 and 12 which showed a better photochemical response. The genes CaMYB1, CaERF017, CaEDR2, CaNCED, CaAPX1, CaAPX5, CaGolS3, CaDHN1 and CaPYL8a were up-regulated in the arabica coffee progenies with greater photochemical efficiency under deficit and therefore contributing to efficiency of the photosynthesis in drought tolerant progenie

Fruit Pigment Biogenesis in Raspberry Cultivars: Characterisation of Anthocyanin and Carotenoid Biosynthesis

Raspberry (Rubus idaeus L.) is a nutrient-rich fruit crop containing high yields of natural bioactive compounds, such as flavonoids and carotenoids, which are known to have potential health benefits in humans. Various colored raspberry fruits offer a unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of colorless leucoanthocyanidins to colored anthocyanidins, a key step in biosynthesis of anthocyanins. The current study revealed that reduced anthocyanins in yellow raspberry (“Anne”) were due to loss of function mutation or inactive ans allele. A 5-bp insertion (ans+5) in the coding region creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids of wild type ANS of red raspberry “Tulameen”. Apparently, the mutated ans gene transcripts are suppressed as a secondary effect because of nonsense-mRNA mediated decay (NMD). Functional characterization and complementation of Ans/ans alleles in planta provide strong proof of inactive ANS protein of “Anne” as compared to the functional protein of “Tulameen”. Further, molecular screening of various colored raspberries for Ans/ans alleles indicated that most of the yellow and orange fruiting raspberries contain various types of ans mutations that cause frameshifts and initiate premature stop codons leading to loss of function of the ANS proteins. In anthocyanin-free varieties, yellow/orange fruit pigmentation seems to exist as a net result of accumulation/degradation of specific carotenoids at ripe stage. The putative carotenoid pathway genes from Rubus “Anne” inserted in standard expression cassettes along with plasmids capable of generating different carotenoid precursors resulted in the successful characterization of the pathway genes via complementation in a bacterial host. It suggests that accumulation of β-branch carotenoids like β-carotene and xanthophylls (lutein) are the principal components that provide yellow coloration to anthocyanin-free raspberry fruits. Taken together, molecular and functional characterization of the carotenoid pathway genes helped to predict a preliminary pathway map for pigmentation in non-red (yellow, orange) fruiting raspberries

新規花色のためのカロテノイド代謝の遺伝子工学に関する研究

この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201

SCREENING AND CHARACTERIZATION OF ARABIDOPSIS THALIANA MUTANTS WITH ALTERED CAROTENOID PROFILE

Carotenoids are organic pigments that are mainly found in the chloroplasts and chromoplasts of plants and other photosynthetic organisms. Carotenoid molecules containing oxygen, such as lutein, violaxanthin and zeaxanthin are called xanthophylls and the rest containing un-oxygenated carotenoids are known as carotenes. Carotenoids form the integral part of the photosystem II LHC. Xanthophylls mainly aid in light harvesting and dissipation of harmful excess energy from excited chlorophyll molecules, thereby protecting chlorophyll from photo-degradation. The biosynthesis of carotenoids has been widely studied using plant and algae. However, the regulatory mechanisms involved in carotenoid metabolism need better understanding. This thesis identified novel regulatory mechanisms involved in the carotenoid biosynthetic pathway using activation-tagged Arabidopsis mutants. Two screening methods, red seed coat screening and norflurazon resistance screening, were used in this study. Fourteen mutants were screened using red seed coat screening but a successful mutant characterization could not be performed due to the unavailability of mutants with a single copy T-DNA insertion. Norflurazon screening identified eight mutants, out of which two mutants, KN203 and KN231, were characterized. The KN203 mutant had a defective keto-acyl CoA synthase 19 gene. KN203 mutant had lower carotenoid levels in the leaves and increased carotenoid levels in the mature seeds; the mutant was able to revert back to wild type phenotype after complementation of a functional KCS19 gene copy driven by native promoter. The fatty acid analysis indicated that the mutant KN203 had decreased MGDG and increased lysoPG, lysoPC and lysoPE content. Reduced carotenoid content in KN203 leaves was attributed to changes in fatty acid composition of chloroplast envelope membrane. Mutant KN231 had a T-DNA insertion in a gene encoding a RNA binding protein (RBP47C). KN231 leaf carotenoid levels were similar to wild type but their levels were significantly higher in their seeds. Two allelic mutants were selected to characterize the mutants. Overexpression of functional RBP47C in the mutants reverted to wild type phenotype in some overexpression mutants. A tandem repeat homologue of RBP47C, RBP47C’was identified. In-silico analysis predicted RBP47C to be a potential candidate for chloroplast localization

A global perspective on carotenoids: metabolism, biotechnology, and benefits for nutrition and health.

Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic bacteria and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits

Control of Wheat Tillering by Nitrogen Availability: The Case of Strigolactones

Primary plant responses to nutrient-deficient conditions include changes in root and shoot architecture. Above-ground plant architecture is shaped by modulating tillering patterns. Tillering is known to be regulated by the interaction between three classes of phytohormones: auxin, cytokinins (CKs) and strigolactones (SLs). Gene expression analysis showed that nitrogen (N) limitation systematically induced the SL biosynthetic genes in the root and the basal nodes of wheat, whereas N resupply quickly reversed the induction of SL biosynthetic genes. This observation raised questions about the functionality of SLs under N-limiting conditions. Although many studies have focused on the transcriptional and hormonal changes that govern N limitation response in roots, fewer studies have focused on the molecular pathways involved in tillering modulation by N limitation during vegetative plant growth in wheat. RNA-sequencing and phytohormonal analysis in basal nodes of N-limited wheat plants showed that N limitation strongly induced bud dormancy and affected many metabolic and hormonal pathways, including changes in the expression of many N-response master regulators, strong suppression of CK biosynthesis and changes in sugar partitioning and utilization. In addition, the SL metabolic pathway was among the top enriched pathways under N limitation, implying that SLs may be involved in coordinating morphological, physiological, and transcriptional changes in response to N status. To test this hypothesis, a Tad17 SL-deficient mutant was generated using lines from the hexaploid wheat TILLING population. The phenotypic response of Tad17 mutants and transcriptomic analysis in the basal nodes showed that SLs are required but are not necessary for tiller inhibition by N limitation. SLs affected CK metabolic genes and CK levels in the basal nodes, however, the lack of SLs was not sufficient to suppress the N limitation mediated decline in CK levels, which contributed to tiller suppression under N limitation. However, lack of SL biosynthesis and imbalance in tillering regulation affected plant adaptation to N-limiting conditions. Tad17 mutant showed changes in resource allocation between root and shoot, N remobilization and the regulation of master regulators of N-response, suggesting that SLs are required for the fine-tune regulation of the N limitation transcriptional network. The genetic information and the results presented regarding the role of SLs in wheat growth and development set the foundation for and highlighted the potential of manipulation of SL metabolism in order to improve wheat architecture or nutrient use efficiency for increasing wheat crop productivity