The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding

Grapevine is one of the most economically important crops worldwide.However,the previous versions of the grapevine reference genome tipically consist of thousands of fragments with missing centromeres and telomeres,limiting the accessibility of the repetitive sequences,the centromeric and telomeric regions,and the study of inheritance of important agronomic traits in these regions.Here,we assembled a telomere-to-telomere(T2T)gap-free reference genome for the cultivar PN40024 using PacBio HiFi long reads.The T2T reference genome(PN_T2T)is 69 Mb longer with 9018 more genes identified than the 12X.v0 version.We annotated 67%repetitive sequences,19 centromeres and 36 telomeres,and incorporated gene annotations of previous versions into the PN_T2T assembly.We detected a total of 377 gene clusters,which showed associations with complex traits,such as aroma and disease resistance.Even though PN40024 derives from nine generations of selfing,we still found nine genomic hotspots of heterozygous sites associated with biological processes,such as the oxidation–reduction process and protein phosphorylation.The fully annotated complete reference genome therefore constitutes an important resource for grapevine genetic studies and breeding programs.

Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci

Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent,circulative manner by homopteran insects.Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability,we recently discovered a panel of protein biomarkers that predict vector competency.Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects.S.graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci.Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species.The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop.

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.

Kiwifruit Genome Database(KGD):a comprehensive resource for kiwifruit genomics

Kiwifruit(Actinidia spp.)plants produce economically important fruits containing abundant,balanced phytonutrients with extraordinarily high vitamin C contents.Since the release of the first kiwifruit reference genome sequence in 2013,large volumes of genome and transcriptome data have been rapidly accumulated for a handful of kiwifruit species.To efficiently store,analyze,integrate,and disseminate these large-scale datasets to the research community,we constructed the Kiwifruit Genome Database(KGD;http://kiwifruitgenome.org/).The database currently contains all publicly available genome and gene sequences,gene annotations,biochemical pathways,transcriptome profiles derived from public RNA-Seq datasets,and comparative genomic analysis results such as syntenic blocks and homologous gene pairs between different kiwifruit genome assemblies.A set of user-friendly query interfaces,analysis tools and visualization modules have been implemented in KGD to facilitate translational and applied research in kiwifruit,which include JBrowse,a popular genome browser,and the NCBI BLAST sequence search tool.Other notable tools developed within KGD include a genome synteny viewer and tools for differential gene expression analysis as well as gene ontology(GO)term and pathway enrichment analysis.

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.

Tomato fruit as a model for tissue-specific gene silencing in crop plants

Use of CRISPR-Cas9(Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)-CRISPR-associated 9)-mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression,usually in the context of either transient or stably inherited genetic alternations.While extremely useful in many applications,modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism.Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models,and recently in Arabidopsis.We developed an in planta GFP(green fluorescent protein)assay system to demonstrate fruit-specific gene editing in tomato using a phosphoenolpyruvate carboxylase 2 gene promoter.We then targeted a SET-domain containing polycomb protein,SlEZ2,previously shown to yield pleiotropic phenotypes when targeted via ^(35)S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations.Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development.

The genome and transcriptome analysis of snake gourd provide insights into its evolution and fruit development and ripening

Snake gourd(Trichosanthes anguina L.),which belongs to the Cucurbitaceae family,is a popular ornamental and food crop species with medicinal value and is grown in many parts of the world.Although progress has been made in its genetic improvement,the organization,composition,and evolution of the snake gourd genome remain largely unknown.Here,we report a high-quality genome assembly for snake gourd,comprising 202 contigs,with a total size of 919.8 Mb and an N50 size of 20.1 Mb.These findings indicate that snake gourd has one of the largest genomes of Cucurbitaceae species sequenced to date.The snake gourd genome assembly harbors 22,874 protein-coding genes and 80.0%of the genome consists of repetitive sequences.Phylogenetic analysis reveals that snake gourd is closely related to sponge gourd but diverged from their common ancestor~33–47 million years ago.The genome sequence reported here serves as a valuable resource for snake gourd genetic research and comparative genomic studies in Cucurbitaceae and other plant species.In addition,fruit transcriptome analysis reveals the candidate genes related to quality traits during snake gourd fruit development and provides a basis for future research on snake gourd fruit development and ripening at the transcript level.

Towards an open grapevine information system

Viticulture,like other fields of agriculture,is currently facing important challenges that will be addressed only through sustained,dedicated and coordinated research.Although the methods used in biology have evolved tremendously in recent years and now involve the routine production of large data sets of varied nature,in many domains of study,including grapevine research,there is a need to improve the findability,accessibility,interoperability and reusability(FAIR-ness)of these data.Considering the heterogeneous nature of the data produced,the transnational nature of the scientific community and the experience gained elsewhere,we have formed an open working group,in the framework of the International Grapevine Genome Program(www.vitaceae.org),to construct a coordinated federation of information systems holding grapevine data distributed around the world,providing an integrated set of interfaces supporting advanced data modeling,rich semantic integration and the next generation of data mining tools.To achieve this goal,it will be critical to develop,implement and adopt appropriate standards for data annotation and formatting.The development of this system,the GrapeIS,linking genotypes to phenotypes,and scientific research to agronomical and oeneological data,should provide new insights into grape biology,and allow the development of new varieties to meet the challenges of biotic and abiotic stress,environmental change,and consumer demand.

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.

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.

Transcriptome analysis provides insights into the regulation of metabolic processes during postharvest cold storage of loquat(Eriobotrya japonica)fruit

Loquat(Eriobotrya japonica)fruit accumulates lignin during postharvest storage under chilling conditions(0℃),while low-temperature conditioning(LTC;5℃for 6 days followed by transfer to 0℃)or heat treatment(HT;40℃for 4 h followed by transfer to 0℃)can alleviate lignification.Here we compared transcriptome profiles of loquat fruit samples under LTC or HT to those stored at 0℃at five time points from day 1 to day 8 after treatment.High-throughput transcriptome sequences were de novo assembled into 53,319 unique transcripts with an N50 length of 1306 bp.A total of 2235 differentially expressed genes were identified in LTC,and 1020 were identified in HT compared to 0℃.Key genes in the lignin biosynthetic pathway,including EjPAL2,EjCAD1,EjCAD3,4CL,COMT,and HCT,were responsive to LTC or HT treatment,but they showed different expression patterns during the treatments,indicating that different structural genes could regulate lignification at different treatment stages.Coexpression network analysis showed that these candidate biosynthetic genes were associated with a number of transcription factors,including those belonging to the AP2,MYB,and NAC families.Gene ontology(GO)enrichment analysis of differentially expressed genes indicated that biological processes such as stress responses,cell wall and lignin metabolism,hormone metabolism,and metal ion transport were significantly affected under LTC or HT treatment when compared to 0℃.Our analyses provide insights into transcriptome responses to postharvest treatments in loquat fruit.

A key‘foxy’aroma gene is regulated by homologyinduced promoter indels in the iconic juice grape‘Concord’

‘Concord’,the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L.,possesses a special‘foxy’aroma predominantly resulted from the accumulation of methyl anthranilate(MA)in berries.This aroma,however,is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V.vinifera.Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key‘foxy’aroma gene,anthraniloyl-CoA:methanol acyltransferase(AMAT),that is responsible for MA biosynthesis.We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in‘Concord’and other V.labrusca-derived grapes.These promoter variants,all with direct and inverted repeats,were further confirmed in more than 1,300 Vitis germplasm.Moreover,functional impact of these indels was validated in transgenic Arabidopsis.Superimposed on the promoter regulation,large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V.vinifera grapes.Elucidation of the AMAT genetic regulation advances our understanding of the‘foxy’aroma trait and makes it genetically trackable and amenable in grapevine breeding.

A chromosome-scale genome assembly of Artemisia argyi reveals unbiased subgenome evolution and key contributions of gene duplication to volatile terpenoid diversity

Artemisia argyi Le´vl.et Vant.,a perennial Artemisia herb with an intense fragrance,is widely used in traditional medicine in China and many other Asian countries.Here,we present a chromosome-scale genome assembly of A.argyi comprising 3.89 Gb assembled into 17 pseudochromosomes.Phylogenetic and comparative genomic analyses revealed that A.argyi underwent a recent lineage-specificwhole-genomeduplication(WGD)event after divergence fromArtemisia annua,resulting in two subgenomes.Wedeciphered the diploid ancestral genome of A.argyi,and unbiased subgenome evolution was observed.The recent WGD led to a large number of duplicated genes in the A.argyi genome.Expansion of the terpene synthase(TPS)gene family through various types of gene duplication may have greatly contributed to the diversity of volatile terpenoids in A.argyi.In particular,we identified a typical germacrene D synthase gene cluster within the expanded TPS gene family.The entire biosynthetic pathways of germacrenes,(+)-borneol,and(+)-camphor were elucidated in A.argyi.In addition,partial deletion of the amorpha-4,11-diene synthase(ADS)gene and loss of function of ADS homologs may have resulted in the lack of artemisinin production in A.argyi.Our study provides newinsights into the genome evolution of Artemisia and lays a foundation for further improvement of the quality of this important medicinal plant.

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.

The role of aluminum sensing and signaling in plant aluminum resistance

As researchers have gained a better understanding in recent years into the physiological, molecular, and genetic basis of how plants deal with aluminum （AI） toxicity in acid soils prevalent in the tropics and sub-tropics, it has become clear that an important component of these responses is the triggering and regulation of cellular pathways and processes by AI. In this review of plant AI signaling, we begin by summarizing the understanding of physiological mechanisms of AI resistance, which first led researchers to realize that AI stress induces gene expression and modifies protein function during the activation of AI resistance responses. Subsequently, an overview of AI resistance genes and their function provides verification that AI induction of gene expression plays a major role in AI resistance in many plant species. More recent research into the mechanistic basis for Al-induced transcrip- tional activation of resistance genes has led to the identifica- tion of several transcription factors as well as cis-elements in the promoters of AI resistance genes that play a role in greater Al-induced gene expression as well as higher constitutive expression of resistance genes in some plant species. Finally, the post-transcriptional and translational regulation of AI resistance proteins is addressed, where recent research has shown that AI can both directly bind to and alter activity of certain organic acid transporters, and also influence AI resistance proteins indirectly, via protein phosphorylation.

Karyotype Stability and Unbiased Fractionation in the Paleo-Allotetraploid Cucurbita Genomes

The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Here, we report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetraploidization event in Cucurbita. We are able to partition the genome into two homoeologous subgenomes based on different genetic distances to melon, cucumber, and watermelon in the Benincaseae tribe. We estimate that the two diploid progenitors successively diverged from Benincaseae around 31 and 26 million years ago （Mya）, respectively, and the allotetraploidization happened at some point between 26 Mya and 3 Mya, the estimated date when C. maxima and C. moschata diverged. The subgenomes have largely maintained the chromosome structures of their diploid progenitors. Such long-term karyotype stability after polyploidization has not been commonly observed in plant polyploids. The two subgenomes have retained similar numbers of genes, and neither subgenome is globally dominant in gene expression. Allele-specific expression analysis in the C. maxima ×C. moschata interspecific F1 hybrid and their two parents indicates the predominance of trans-regulatory effects underlying expression divergence of the parents, and detects transgressive gene expression changes in the hybrid correlated with heterosis in important agronomic traits. Our study provides insights into polyploid genome evolution and valuable resources for genetic improvement of cucurbit crops.

The Or Gene Enhances Carotenoid Accumulation and Stability During Post-Harvest Storage of Potato Tubers

Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that expressed the cauliflower Orange （Or） gene. We found that the Or transgene not only promoted retention of 13-carotene level, but also continuously stimulated its accumulation during 5 months of cold storage. In contrast, no increased levels of carotenoids were observed in the tubers of vector-only controls or a yellow- flesh variety during the same period of storage. The increased carotenoid accumulation was found to be associated with the formation of lipoprotein-carotenoid sequestering structures, as well as with the enhanced abundance of phytoene synthase, a key enzyme in the carotenoid biosynthetic pathway. Furthermore, the provitamin A carotenoids stored were shown to be stable during simulated digestion and accessible for uptake by human intestinal absorptive cells. Proteomic analysis identified three major functional groups of proteins （i.e. heat shock proteins, glutathione-S-transferases, and carbohydrate metabolic proteins） that are potentially important in the Or-regulated carotenoid accumulation. Our results show that regulation of carotenoid sequestration capacity is an important mechanism by which carotenoid stability is regulated. Our findings suggest that induction of a proper sink structure formation in staple crops may provide the crops with a unique ability to promote and/or stabilize provitamin A accumulation during plant growth and post-harvest storage.

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.

Identification of a novel pathway involving a GATA transcription factor in yeast and possibly in plant Zn uptake and homeostasis

To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Noccaea caerulescens, was screened for its ability to restore growth under Zn limiting conditions in the yeast mutant zaplA. ZAP1 is a transcription factor that activates the Zn dependent transcription of yeast genes involved in Zn uptake, including ZRT1, the yeast high affinity Zn transporter. From this screen two members of the E2F family of transcription factors were found to activate ZRT1 expression in a Zn independent manner. The activation of ZRTI by the plant E2F proteins involves E2F- mediated activation of a yeast GATA transcription factor which in turn activates ZRT1 expression. A ZRT1 promoter region necessary for activation by E2F and GATA proteins is upstream of two zinc responsive elements previously shown to bind ZAP1 in ZRT1. This activation may not involve direct binding of E2F to the ZRT1 promoter. The expression of E2F genes in yeast does not replace function of ZAP1; instead it appears to activate a novel GATA regulatory pathway involved in Zn uptake and homeostasis that is not Zn responsive.

Plant Genome Editing Database (PGED): A Call for Submission of Information about Genome-Edited Plant Mutants

Recent advances in genome editing technologies, particularly CRISPR/Cas, enable the alteration of DNA sequences to produce deletions, insertions, and substitutions in genes (Jaganathan et al., 2018), as well as large or entire chromosome deletions in the genomes of plants and animals (Zhou et al., 2014;Adikusuma et al., 2017).