A community resource for exploring and utilizing genetic diversity in the USDA pea single plant plus collection

Globally,pea(Pisum sativum L.)is an important temperate legume crop for food,feed and fodder,and many breeding programs develop cultivars adapted to these end-uses.In order to assist pea development efforts,we assembled the USDA Pea Single Plant Plus Collection(PSPPC),which contains 431 P.sativum accessions with morphological,geographic and taxonomic diversity.The collection was characterized genetically in order to maximize its value for trait mapping and genomics-assisted breeding.To that end,we used genotyping-by-sequencing—a cost-effective method for de novo single-nucleotide polymorphism(SNP)marker discovery—to generate 66591 high-quality SNPs.These data facilitated the identification of accessions divergent from mainstream breeding germplasm that could serve as sources of novel,favorable alleles.In particular,a group of accessions from Central Asia appear nearly as diverse as a sister species,P.fulvum,and subspecies,P.sativum subsp.elatius.PSPPC genotypes can be paired with new and existing phenotype data for trait mapping;as proof-of-concept,we localized Mendel’s A gene controlling flower color to its known position.We also used SNP data to define a smaller core collection of 108 accessions with similar levels of genetic diversity as the entire PSPPC,resulting in a smaller germplasm set for research screening and evaluation under limited resources.Taken together,the results presented in this study along with the release of a publicly available SNP data set comprise a valuable resource for supporting worldwide pea genetic improvement efforts.

A novel histone methyltransferase gene Cg SDG40 positively regulates carotenoid biosynthesis during citrus fruit ripening

The flesh color of pummelo(Citrus maxima)fruits is highly diverse and largely depends on the level of carotenoids,which are beneficial to human health.It is vital to investigate the regulatory network of carotenoid biosynthesis to improve the carotenoid content in pummelo.However,the molecular mechanism underlying carotenoid accumulation in pummelo is not fully understood.In this study,we identified a novel histone methyltransferase gene,CgSDG40,involved in carotenoid regulation by analyzing the flesh transcriptome of typical white-fleshed pummelo,red-fleshed pummelo and extreme-colored F1 hybrids from a segregated pummelo population.Expression of CgSDG40 corresponded to flesh color change and was highly coexpressed with CgPSY1.Interestingly,CgSDG40 and CgPSY1 are located physically adjacent to each other on the chromosome in opposite directions,sharing a partially overlapping promoter region.Subcellular localization analysis indicated that CgSDG40 localizes to the nucleus.Overexpression of CgSDG40 significantly increased the total carotenoid content in citrus calli relative to that in wild type.In addition,expression of CgPSY1 was significantly activated in overexpression lines relative to wild type.Taken together,our findings reveal a novel histone methyltransferase regulator,CgSDG40,involved in the regulation of carotenoid biosynthesis in citrus and provide new strategies for molecular design breeding and genetic improvement of fruit color and nutritional quality.

Carotenoid metabolism and regulation in horticultural crops

Carotenoids are a diverse group of pigments widely distributed in nature.The vivid yellow,orange,and red colors of many horticultural crops are attributed to the overaccumulation of carotenoids,which contribute to a critical agronomic trait for flowers and an important quality trait for fruits and vegetables.Not only do carotenoids give horticultural crops their visual appeal,they also enhance nutritional value and health benefits for humans.As a result,carotenoid research in horticultural crops has grown exponentially over the last decade.These investigations have advanced our fundamental understanding of carotenoid metabolism and regulation in plants.In this review,we provide an overview of carotenoid biosynthesis,degradation,and accumulation in horticultural crops and highlight recent achievements in our understanding of carotenoid metabolic regulation in vegetables,fruits,and flowers.

Carotenoid Pigment Accumulation in Horticultural Plants

Carotenoids are a group of widely distributed natural pigments.They give many horticultural plants the bright red,orange,and yellow colors,as well as the aroma and flavor.Carotenoids enhance the health value and represent an essential quality trait of horticultural products.Significant efforts have been made to correlate specific carotenoid production with pathway gene expression.Some transcription factors that directly regulate transcription of the pathway genes have been identified.Horticultural crops have evolved with complicated and multifaceted regulatory mechanisms to generate the enormous diversity in carotenoid content and composition.However,the diverse and complex control of carotenoid accumulation is still not well understood.In this review,we depict carotenoid accumulation pathways and highlight the recent progress in the regulatory control of carotenoid accumulation in horticultural plants.Because of the critical roles of chromoplasts for carotenoid hyperproduction,we evaluate chromoplast ultrastructures and carotenoid sequestrations.A perspective on carotenoid research in horticultural crops is provided.

Comparative transcriptome analyses shed light on carotenoid production and plastid development in melon fruit

Carotenoids,such asβ-carotene,accumulate in chromoplasts of various fleshy fruits,awarding them with colors,aromas,and nutrients.The Orange(CmOr)gene controlsβ-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressingβ-carotene turnover in chromoplasts.Carotenoid isomerase(CRTISO)isomerizes yellow prolycopene into red lycopene,a prerequisite for further metabolism intoβ-carotene.We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants,low-β(Cmor)and yofi(Cmcrtiso).The Cmor mutation in low-βcaused a major transcriptomic change in the mature fruit.In contrast,the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages.These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion,but minimally by carotenoid composition in the ripe fruit.Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts.Moreover,large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-β.CmOR family proteins were found to physically interact with light-harvesting chlorophyll a–b binding proteins,suggesting a new role of CmOR for chloroplast maintenance in melon fruit.This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.

Breeding Sorghum Using Induced Mutations: Future Prospect for Namibia

In arid and semi-arid regions of the world sorghum stands out as a climate change-ready crop with high potential for the production of food, feed, fodder, fiber and fuel in the face of increasing human population. The present review highlights induced mutation breeding technique as a potential tool for improving sorghum in Namibia. The review discussed the following issues;crop improvement using mutagens, mutant screening, selection and evaluation, impact of induced mutation breeding, factors for declining production and future implication of sorghum mutation breeding. In Namibia, severe drought stress resulting in total crop failure has become frequent. This is partly a consequence of farmers growing crop varieties which cannot withstand impact of drought. As such Namibia has limited drought tolerant varieties available for the diverse agro-ecologies. Farmers keep growing the familiar landraces which performs well in good rainfall years but fails to produce stable yield with irregular and erratic rainfall. Thus, breeding new sorghum varieties of high yield and quality combined with multiple agronomic traits including pest and disease resistance and high efficiency in nutrient and water use is needed. Induced mutation is one of the breeding methods utilized worldwide to supplement conventional breeding for developing superior varieties with desirable traits in different crops. Development of high yielding, drought tolerant, and dwarf sorghums with early maturity enables effective utilization of available soils moisture and in optimizing plant density for achieving higher yield in farmers’ fields. Recombination breeding through exploitation of natural genetic variability and mutation breeding to reduce the plant height without disturbing agronomic superiority of elite lines is recommended for sorghum improvement in Namibia.

Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants

Chlorophylls and carotenoids are essential photosynthetic pigments.Plants spatiotemporally coordinate the needs of chlorophylls and carotenoids for optimal photosynthesis and fitness in response to diverse environmental and developmental cues.However,how the biosynthesis pathways of these two pigments are coordinated,particularly at posttranslational level to allow rapid control,remains largely unknown.Here,we report that the highly conserved ORANGE(OR)family proteins coordinate both pathways via posttranslationally mediating the first committed enzyme in each pathway.We demonstrate that OR family proteins physically interact with magnesium chelatase subunit I(CHLI)in chlorophyll biosynthesis pathway in addition to phytoene synthase(PSY)in carotenoid biosynthesis pathway and concurrently stabilize CHLI and PSY enzymes.We show that loss of OR genes hinders both chlorophyll and carotenoid biosynthesis,limits light-harvesting complex assembly,and impairs thylakoid grana stacking in chloroplasts.Overexpression of OR safeguards photosynthetic pigment biosynthesis and enhances thermotolerance in both Arabidopsis and tomato plants.Our findings establish a novel mechanism by which plants coordinate chlorophyll and carotenoid biosynthesis and provide a potential genetic target to generate climate-resilient crops.

Genotype-by-environment interaction for grain yield among novel cowpea(Vigna unguiculata L.) selections derived by gamma irradiation

This study determined the effects of genotype-by-environment(G × E) interaction and stability of yield among elite cowpea(Vigna unguiculata L.) selections derived by gamma irradiation. The study was conducted in Namibia at three selected sites: Bagani, Mannheim,and Omahenene, during 2014/2015 and 2015/2016. Thirty-four newly developed mutant genotypes and three local checks were evaluated using a randomized complete block design with three replications. Grain yield data were analyzed using the additive main effects and multiplicative interaction(AMMI) and the genotype main effect plus genotype-by-environment interaction(GGE) biplot methods. The AMMI and GGE biplot models explained 77.49% and 75.57% of total observed genotypic variation, respectively.Bagani and Omahenene were the environments best discriminating the test genotypes during 2014/2015 and 2015/2016, respectively. Four promising mutant genotypes: G9(Sh L3 P74), G10(Sh R3 P4), G12(Sh R9 P5), and G4(Sh L2 P4), showed wide adaptation and grain yields of 2.83, 2.06, 1.99, and 1.95 t ha^(-1), respectively. The novel mutant lines are useful genetic resources for production or future cowpea breeding programs in Namibia or similar environments.

A QTL associated with leaf trichome traits has a major influence on the abundance of the predatory mite Typhlodromus pyri in a hybrid grapevine population

The abundance of predatory phytoseiid mites,Typhlodromus pyri,important biological control agents of spider mite pests in numerous crops,is positively influenced by the density of leaf trichomes and tuft-form domatia in vein axils.Identification of the genetic regions controlling both trophic levels could facilitate the improvement of predatory mite habitat in breeding programs.The abundance of T.pyri and non-glandular trichomes was measured in a segregating F1 family derived from the cross of the complex Vitis hybrid,‘Horizon’,with Illinois 547-1(V.rupestris B38×V.cinerea B9),finding positive correlation among traits.High density genetic maps were used to localize one major quantitative trait locus(QTL)on chromosome 1 of Illinois 547-1 associated with both predatory mite abundance and leaf trichomes.This QTL explained 23%of the variation in phytoseiid abundance and similar amounts of variance in domatia rating(21%),domatia size(16%),leaf bristle density(37%in veins and 33%in blades),and leaf hair density(20%in veins and 15%in blades).Another nine QTL distributed among chromosomes 1,2,5,8,and 15 were associated solely with trichome density,and explained 7–17%of the phenotypic variation.Combined,our results provide evidence of the genetic architecture of non-glandular trichomes in Vitis,with a major locus influencing trichome densities,domatia size and predatory mite abundance.This information is relevant for breeding grapevines with a more favorable habitat for biological control agents.

A next-generation marker genotyping platform(AmpSeq)in heterozygous crops:a case study for marker-assisted selection in grapevine

Marker-assisted selection(MAS)is often employed in crop breeding programs to accelerate and enhance cultivar development,via selection during the juvenile phase and parental selection prior to crossing.Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery.To bridge the gap between marker development and MAS implementation,this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery,low-cost genotyping through amplicon sequencing(AmpSeq)and decision making.The results document the development of a MAS package derived from genotyping-by-sequencing using three traits(flower sex,disease resistance and acylated anthocyanins)in grapevine breeding.The vast majority of sequence reads(⩾99%)were from the targeted regions.Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data,most amplicons(83 to 87%)had<10%missing data,and read depth had a median of 220–244×.Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy,flexibility,speed,high-throughput,low-cost and easily automated analysis.

Plant carotenoids: recent advances and future perspectives

Carotenoids are isoprenoid metabolites synthesized de novo in all photosynthetic organisms.Carotenoids are essential for plants with diverse functions in photosynthesis,photoprotection,pigmentation,phytohormone synthesis,and signaling.They are also critically important for humans as precursors of vitamin A synthesis and as dietary antioxidants.The vital roles of carotenoids to plants and humans have prompted significant progress toward our understanding of carotenoid metabolism and regulation.New regulators and novel roles of carotenoid metabolites are continuously revealed.This review focuses on current status of carotenoid metabolism and highlights recent advances in comprehension of the intrinsic and multi-dimensional regulation of carotenoid accumulation.We also discuss the functional evolution of carotenoids,the agricultural and horticultural application,and some key areas for future research.

Induced Mutation for Developing Mutant Rice Lines Tolerant to the Parasitic Weed Striga asiatica (L.) Kuntze

This work aims to screen mutant rice lines tolerant to Striga asiatica.Two rainfed sensitive rice varieties B22 and F154 were used.Plants survival rates of the two parents were significantly lower respectively(9.74a and 11.83a)than those of mutant lines(55.36c to 74.36b);Striga plants emergence/pot were significantly higher for the parents(13.96c and14.89c)compared to the mutants(0.12a to 1.5b);the infection rate of parents(7.37b;7.86b)was higher compared to the mutants(2.27a to 2.74a);fertility rate/plant of parents was lower(20.98%b;22.29%b)but much higher than mutants(72.19%b to 78.35%b);the average panicle number/plant of parents was significantly lower(0.5a;1a)than those of mutants(1.5b to 2.4bc)and the 100 g grain weight of parents are lower(2.35a;2.56a)than those of mutants(3.19b to 3.23b).The culture of those mutant lines may increase rice production and contribute to enhancing food security in Madagascar.

Carotenoid Metabolism in Plants： The Role ol Plastids

Carotenoids are indispensable to plants and critical in human diets. Plastids are the organelles for carotenoid biosynthesis and storage in plant cells. They exist in various types, which include proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. These plastids have dramatic differences in their capacity to synthesize and sequester carotenoids. Clearly, plastids play a central role in governing carotenogenic activity, carotenoid stability, and pigment diversity. Understanding of carotenoid metabolism and accumulation in various plastids expands our view on the multifaceted regulation of carotenogenesis and facilitates our efforts toward developing nutrient-enriched food crops. In this review, we provide a comprehensive overview of the impact of various types of plastids on carotenoid biosynthesis and accumulation, and discuss recent advances in our understanding of the regulatory control of carotenogenesis and metabolic engineering of carotenoids in light of plastid types in plants.

Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis

Phytoene synthase （PSY） is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits （i.e., ClpS1, CIpC1, and CIpD）. High levels of PSY and several other carotenogenic enzyme proteins overac- cumulate in the clpcl, clpp4, and clprl-2 mutants. The overaccumulated PSY was found to be partially enzy- matically active. Impairment of Clp activity in clpcl results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE （OR） protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpcl, counterbalancing CIp-mediated proteol- ysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the qual- ity control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants.

OR^His, a Natural Variant of OR, Specifically Interacts with Plastid Division Factor ARC3 to Regulate Chromoplast Number and Carotenoid Accumulation

Chromoplasts are colored plastids that synthesize and store massive amounts of carotenoids.Chromoplast number and size define the sink strength for carotenoid accumulation in plants.However,nothing is known about the mechanisms controlling chromoplast number.Previously,a natural allele of Orange(OR),OR^His,was found to promote carotenoid accumulation by activating chromoplast differentiation and increasing carotenoid biosynthesis,but cells in orange tissues in melon fruit and cauliflower OR mutant have only one or two enlarged chromoplasts.In this study,we investigated an OR^His variant of Arabidopsis OR,genetically mimicking the melon OR^His allele,and found that it also constrains chromoplast number in Arabidopsis calli.Both in vitro and in vivo experiments demonstrate that OR^His specifically interacts with the Membrane Occupation and Recognition Nexus domain of ACCUMULATION AND REPLICATION OF CHLOROPLASTS 3(ARC3),a crucial regulator of chloroplast division.We further showed that OR^His interferes with the interaction between ARC3 and PARALOG OF ARC6(PARC6),another key regulator of chloroplast division,suggesting a role of OR^His in competing with PARC6 for binding to ARC3 to restrict chromoplast number.Overexpression or knockout of ARC3 in Arabidopsis OR^His plants significantly alters total carotenoid levels.Moreover,overexpression of the plastid division factor PLASTID DIVISION 1 greatly enhances carotenoid accumulation.These division factors likely alter carotenoid levels via their influence on chromoplast number and/or size.Taken together,our findings provide novel mechanistic insights into the machinery controlling chromoplast number and highlight a potential new strategy for enhancing carotenoid accumulation and nutritional value in food crops.

Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality

Nutrient deficiencies in crops are a serious threat to human health,especially for populations in poor areas.To overcome this problem,the development of crops with nutrient-enhanced traits is imperative.Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components.Compared with agronomic practices and conventional plant breeding,plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients,phytonutrients,and/or bioactive components in crops.In this review,we discuss recent progress in the field of plant synthetic metabolic engineering,specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways,with a focus on improving traits related to micronutrients,phytonutrients,and bioactive components.Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.

Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification.However,carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains.In this study,we utilized Arabidopsis as a model to establish car-otenoid biofortification strategies in seeds.We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase(PSY)increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage,consistent with previous studies of provitamin A biofortified grains.In contrast,stacking with Orange(OR^(His)),a gene that initiates chromopl ast biogenesis,dramatically enhances provitamin A and total carotenoid content and stability.Up to 65-and 10-fold increases of β-carotene and total car-otenoids,res pectively,with provitamin A carotenoids composing over 63%were observed in the seeds containing OR^(His) and PSY.Co-expression of Homogen tisate geranylgeranyl transferase(HGGT)with OR^(His) and PSY further increases carotenoid accumulation and stability during seed maturation and storage.Moreover,knocking-out of B-carotene hydroxylase 2(BCH2)by CRISPR/Cas9 not only potentially facilitates β-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germi nation.Our findings provide new insights into various processes on carotenoid accu-mulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis,turnover,and stable storage for carotenoid biofortification in crop seeds.

Involvement of cytokinins in STOP1-mediated resistance to proton toxicity

STOP1(sensitive to proton rhizotoxicity1)is a master transcription factor that governs the expression of a set of regulatory and structural genes involved in resistance to aluminum and low pH(i.e.,proton)stresses in Arabidopsis.However,the mechanisms and regulatory networks underlying STOP1-mediated resistance to proton stresses are largely unclear.Here,we report that low-pH stresses severely inhibited root growth of the stop1 plants by suppressing root meristem activities.Interestingly,the stop1 plants were less sensitive to exogenous cytokinins at normal and low pHs than the wild type.Significantly,low concentrations of cytokinins promoted root growth of the stop1 mutant under low-pH stresses.Moreover,lateral and adventitious root formation was stimulated in stop1 and by low-pH stresses but suppressed by cytokinins.Further studies of the expression patterns of a cytokinin signaling reporter suggest that both the loss-of-function mutation of STOP1 and low-pH stresses suppressed cytokinin signaling outputs in the root.Furthermore,the expression of critical genes involved in cytokinin biosynthesis,biodegradation,and signaling is altered in the stop1 mutant in response to low-pH stresses.In conclusion,our results reveal a complex network of resistance to low-pH stresses,which involves coordinated actions of STOP1,cytokinins,and an additional low-pH-resistant mechanism for controlling root meristem activities and root growth upon proton stresses.