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The orange subfamily (Aurantioideae) contains several Citrusspecies cultivated worldwide, such as sweet orange and lemon. The origin of Citrusspecies has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrusgenus. We found substantial variations in the sequence and expression of the PH4gene in Citrusrelative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

Carotenoid biosynthesis is closely associated with abscisic acid (ABA) during the ripening process of non-climacteric fruits, but the regulatory mechanism that links ABA signaling to carotenoid metabolism remains largely unclear. Here, we identified two master regulators of ABA-mediated citrus fruit coloration, CsERF110 and CsERF53, which activate the expression of carotenoid metabolism genes (CsGGPPS, CsPSY, CsPDS, CsCRTISO, CsLCYB2, CsLCYE, CsHYD, CsZEP, and CsNCED2) to facilitate carotenoid accumulation. Further investigations showed that CsERF110 not only activates the expression of CsERF53by binding to its promoter but also interacts with CsERF53 to form the transcriptional regulatory module CsERF110–CsERF53. We also discovered a positive feedback regulatory loop between the ABA signal and carotenoid metabolism regulated by the transcriptional regulatory module CsERF110–CsERF53. Our results reveal that the CsERF110–CsERF53 module responds to ABA signaling, thereby orchestrating citrus fruit coloration. Considering the importance of carotenoid content for citrus and many other carotenoid-rich crops, the revelation of molecular mechanisms that underlie ABA-mediated carotenoid biosynthesis in plants will facilitate the development of transgenic/gene-editing approaches, further contributing to improving the quality of citrus and other carotenoid-rich crops.

Somatic variations are a major source of genetic diversification in asexual plants, and underpin clonal evolution and the breeding of asexual crops. Sweet orange is a model species for studying somatic variation because it reproduces asexually through apomixis and is propagated asexually through grafting. To dissect the genomic basis of somatic variation, we de novo assembled a reference genome of sweet orange with an average of three gaps per chromosome and a N50 contig of 24.2 Mb, as well as six diploid genomes of somatic mutants of sweet oranges. We then sequenced 114 somatic mutants with an average genome coverage of 41×. Categorization of the somatic variations yielded insights into the single-nucleotide somatic mutations, structural variations and transposable element (TE) transpositions. We detected 877 TE insertions, and found TE insertions in the transporter or its regulatory genes associated with variation in fruit acidity. Comparative genomic analysis of sweet oranges from three diversity centres supported a dispersal from South China to the Mediterranean region and to the Americas. This study provides a global view on the somatic variations, the diversification and dispersal history of sweet orange and a set of candidate genes that will be useful for improving fruit taste and flavour.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene β-cyclase (LCYB) to β-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene β-cyclase (CsLCYb2a) produced “golden” transgenic tomato fruits with 9.3-fold increased levels of β-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of β-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYBgenes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYBgenes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and inbreeding in flowering plants. The most widespread SI system utilizes Sribonucleases (S-RNases) and S-locus F-boxes (SLFs) as Sdeterminants. In citrus, SI is ancestral, and Citrus maxima(pummelo) is self-incompatible, while Citrus reticulata(mandarin) and its hybrids are self-compatible (SC). Here, we identify nine highly polymorphic pistil-specific, developmentally expressed S-RNases from pummelo that segregate with Shaplotypes in a gametophytic manner and cluster with authentic S-RNases. We provide evidence that these S-RNases function as the female Sdeterminants in citrus. Moreover, we show that each S-RNase is linked to approximately nine SLFs. In an analysis of 117 citrus SLFand SFL-like (SLFL) genes, we reveal that they cluster into 12 types and that the S-RNases and intra-haplotypic SLFand SLFLgenes co-evolved. Our data support the notion that citrus have a Slocus comprising a S-RNaseand several SLFs that fit the non-self-recognition model. We identify a predominant single nucleotide mutation, Sm-RNase, in SC citrus, which provides a ‘natural’ loss of function. We show that SI–SC transitions due to the Sm-RNase initially arose in mandarin, spreading to its hybrids and became fixed. Identification of an evolutionarily distant new genus utilizing the S-RNase-based SI system, >100 million years separated from the nearest S-RNase family, is a milestone for evolutionary comparative studies.

Although revisiting the discoveries and implications of genetic variations using phased genomics is critical, such efforts are still lacking. Somatic mutations represent a crucial source of genetic diversity for breeding and are especially remarkable in heterozygous perennial and asexual crops. In this study, we focused on a diploid sweet orange (Citrus sinensis) and constructed a haplotype-resolved genome using high fidelity (HiFi) reads, which revealed 10.6% new sequences. Based on the phased genome, we elucidate significant genetic admixtures and haplotype differences. We developed a somatic detection strategy that reveals hidden somatic mutations overlooked in a single reference genome. We generated a phased somatic variation map by combining high-depth whole-genome sequencing (WGS) data from 87 sweet orange somatic varieties. Notably, we found twice as many somatic mutations relative to a single reference genome. Using these hidden somatic mutations, we separated sweet oranges into seven major clades and provide insight into unprecedented genetic mosaicism and strong positive selection. Furthermore, these phased genomics data indicate that genomic heterozygous variations contribute to allele-specific expression during fruit development. By integrating allelic expression differences and somatic mutations, we identified a somatic mutation that induces increases in fruit size. Applications of phased genomics will lead to powerful approaches for discovering genetic variations and uncovering their effects in highly heterozygous plants. Our data provide insight into the hidden somatic mutation landscape in the sweet orange genome, which will facilitate citrus breeding.

Citrus fruits are widely consumed worldwide in juices or as fresh and provide a broad range of phytonutrients that are important for human health. Here, a citrus multi-omics resource is presented: comprehensive metabolic profiling of various citrus species was performed and metabolic profiles were compared among species, with a focus on the phenylpropanoid metabolic pathway. A metabolite-based genome-wide association analysis (mGWAS) of 154 pummelo accessions was performed using factored spectrally transformed linear mixed models (FaST-LMM) and efficient mixed-model association eXpedited (EMMAX), and the genetic and biochemical basis of metabolomic variation was comprehensively analysed. A metabolite-single nucleotide polymorphism-gene (metabolite-SNP-gene) interaction network was constructed for pummelo, and many candidate loci controlling the synthesis and regulation of bioactive compounds were identified; among these loci, three BAHD malonyltransferases were involved in the malonylation of flavonoid glycosides. Further investigation revealed that an R2R3-MYB transcription factor CgMYB1 positively controls the metabolism of phenylpropanoid molecules, particularly the flavonoid derivatives. This study provides valuable data resources on the metabolic regulatory networks of bioactive components in citrus, in addition to demonstrating an efficient method for metabolic pathway dissection and providing targets for future breeding work with the aim of improving nutritional value.

Carotenoids in plant foods provide health benefits by functioning as provitamin A. One of the vital provitamin A carotenoids, β-cryptoxanthin, is typically plentiful in citrus fruit. However, little is known about the genetic basis of β-cryptoxanthin accumulation in citrus. Here, we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrusand determine the taxonomic profile of β-cryptoxanthin. We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citruscultivars to reveal the genetic basis of β-cryptoxanthin accumulation through a genome-wide association study. We identified a causal gene, CitCYP97B, which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined. We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the β-ring of β-cryptoxanthin in a heterologous expression system. In plantaexperiments provided further evidence that CitCYP97Bnegatively regulates β-cryptoxanthin content. Using the sequenced Citrusaccessions, we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B, thereby altering β-cryptoxanthin accumulation in fruit. Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrustypes during citrus evolution. Overall, these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of β-cryptoxanthin-biofortified products.

Carotenoids are important antioxidant components in the human diet. To develop carotenoid-rich agricultural products by genetic intervention, understanding the genetic basis of carotenoids variation is essential. In this study, we constructed a high-density integrated genetic map with 3817 molecular markers using specific locus amplified fragment (SLAF) sequencing from a C. reticulata× P. trifoliataF1pseudotestcross population. A total of 17 significant quantitative trait loci (QTLs) distributed on Chromosomes (Chr) 2, 3, 5, 6, and 9 were detected to determine the carotenoid variation in the population. In particular, three QTL colocalizations for multiple carotenoid constituents were observed on Chr 2, 3, and 9, one of which was located on Chr2:34,654,608–35430715 accounted for 20.1–25.4% of the variation of luteoxanthin, auroxanthin, lutein, violaxanthin, and total carotenoid content. Overall, this study provides a genetic foundation for marker-assisted selection (MAS) breeding of nutritionally enhanced citrus fruit.

Elaioplasts of citrus peel are colorless plastids which accumulate significant amounts of terpenes. However, other functions of elaioplasts have not been fully characterized to date. Here, a LC–MS/MS shotgun technology was applied to identify the proteins from elaioplasts that were highly purified from young fruit peel of kumquat. A total of 655 putative plastid proteins were identified from elaioplasts according to sequence homology in silicoand manual curation. Based on functional classification via Mapman, ~50% of the identified proteins fall into six categories, including protein metabolism, transport, and lipid metabolism. Of note, elaioplasts contained ATP synthase and ADP, ATP carrier proteins at high abundance, indicating important roles for ATP generation and transport in elaioplast biogenesis. Additionally, a comparison of proteins between citrus chromoplast and elaioplast proteomes suggest a high level of functional conservation. However, some distinctive protein profiles were also observed in both types of plastids notably for isoprene biosynthesis in elaioplasts, and carotenoid metabolism in chromoplasts. In conclusion, this comprehensive proteomic study provides new insights into the major metabolic pathways and unique characteristics of elaioplasts and chromoplasts in citrus fruit. A study of the proteins in bodies called elaioplasts in citrus fruit cells will help to clarify elaioplast formation and functions. Plant cells contain several types of membrane-bound bodies called plastids, with elaioplasts being an incompletely characterized type. They are known to store oils in the cells, especially molecules called terpenes that influence the fragrances and flavors of citrus fruits. Researchers in China, led by Yunliu Zeng at Huazhong Agricultural University, analyzed the protein molecules in elaioplasts of kumquat peel to investigate possible roles for elaioplasts other than oil storage. They identified more than 600 proteins and assigned likely functions to many. The results suggest the proteins are involved in chemical energy transactions and the manufacture of various categories of biomolecules. This will guide research to fully characterize the activities and significance of elaioplasts.

Naturally, resistant crop germplasms are important resources for managing the issues of agricultural product safety and environment deterioration. We found a spontaneous mutant of ‘Newhall’ navel orange (Citrus sinensisOsbeck) (MT) with broad-spectrum protections against fungal pathogens in the orchard, postharvest-storage, and artificial inoculation conditions. To understand the defense mechanism of MT fruit, we constructed a genome-scale metabolic network that integrated metabolome and transcriptome datasets. The coordinated transcriptomic and metabolic data were enriched in two sub-networks, showing the decrease in very long chain fatty acid (by 41.53%) and cuticular wax synthesis (by 81.34%), and increase in the synthesis of jasmonic acid (JA) (by 95.23%) and JA-induced metabolites such as 5-dimethylnobietin (by 28.37%) in MT. Furthermore, cytological and biochemical analyses confirmed that the response to fungal infection in MT was independent of wax deficiency and was correlated with the levels of jasmonates, and the expression of plant defensin gene PDF1.2. Results of exogenous application of MeJA and JA inhibitors such as propyl gallate proved that JA-mediated defense contributes to the strong tolerance against pathogens in MT. Our results indicated that jasmonate biosynthesis and signaling are stimulated by the fatty acid redirection of MT, and participate in the tolerance of pathogenic fungi. Elevated hormone signaling underpins fungal protection in a naturally occurring variety of orange found in China. Yunjiang Cheng from Huazhong Agricultural 
University in Wuhan, China, and coworkers analyzed all the genes expressed and metabolites produced by a mutant variety of ‘Newhall’ navel orange (Citrus sinensisOsbeck). The researchers found that, compared to wild-type oranges, the fungus-resistant mutant showed a decrease in both fatty acid synthesis and surface wax production, as well as an increase in synthesis of jasmonic acid, a plant hormone involved in anti-microbial defenses. The mutant seems to redirect production of fatty acids to make more jasmonic acid—and it’s this hormone that mediates the plant’s strong tolerance to fungal pathogens. This information could help agronomists breed other varieties of pest-resistant oranges that require less chemical fungicide under field and storage conditions.

Carotenoids directly influence citrus fruit color and nutritional value, which is critical to consumer acceptance. Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality. Despite the well-established carotenoid biosynthetic pathways, the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood. Our previous studies have reported that the Myc-type basic helix–loop–helix (bHLH) transcription factor (TF) regulates citrus proanthocyanidin biosynthesis. Transgenic analyses further showed that overexpression of CsTT8could significantly promote carotenoid accumulation in transgenic citrus calli, but its regulatory mechanism is still unclear. In the present study, we found that overexpression of CsTT8enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR, CsHDS, CsHDR, CsPDS, CsLCYE, CsZEP, and CsNCED2, which was accompanied by changes in the contents of abscisic acid and gibberellin. The in vitroand in vivoassays indicated that CsTT8 directly bound to the promoters of CsDXR, CsHDS, and CsHDR, the key metabolic enzymes of the methylerythritol 4-phosphate (MEP) pathway, thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes. In addition, CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes, CsPDS, CsLCYE, CsZEP, and CsNCED2, directly promoting carotenoid biosynthesis. This study reveals a novel network of carotenoid metabolism regulated by CsTT8. Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize anthocyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins. We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus. Different Ruby2alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Fullencodes an anthocyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Shortwas identified in purple pummelo and encodes an anthocyanin repressor. CgRuby2Shorthas lost the ability to activate anthocyanin biosynthesis. However, it retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory complex. Further investigation in different citrus species indicated that the Ruby2–Ruby1cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2–Ruby1cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.

Review on citrus reproduction. Citrus is one of the most important and widely grown fruit crops. It possesses several special reproductive characteristics, such as nucellar embryony and self-incompatibility. The special phenomenon of nucellar embryony in citrus, also known as the polyembryony, is a kind of sporophytic apomixis. During the past decade, the emergence of novel technologies and the construction of multiple citrus reference genomes have facilitated rapid advances to our understanding of nucellar embryony. Indeed, several research teams have preliminarily determined the genetic basis of citrus apomixis. On the other hand, the phenomenon of self-incompatibility that promotes genetic diversity by rejecting self-pollen and accepting non-self-pollen is difficult to study in citrus because the long juvenile period of citrus presents challenges to identifying candidate genes that control this phenomenon. In this review, we focus on advances to our understanding of reproduction in citrus from the last decade and discuss priorities for the coming decade.

The emergence of apomixis—the transition from sexual to asexual reproduction—is a prominent feature of modern citrus. Here we de novo sequenced and comprehensively studied the genomes of four representative citrus species. Additionally, we sequenced 100 accessions of primitive, wild and cultivated citrus. Comparative population analysis suggested that genomic regions harboring energy- and reproduction-associated genes are probably under selection in cultivated citrus. We also narrowed the genetic locus responsible for citrus polyembryony, a form of apomixis, to an 80-kb region containing 11 candidate genes. One of these, CitRWP, is expressed at higher levels in ovules of polyembryonic cultivars. We found a miniature inverted-repeat transposable element insertion in the promoter region of CitRWP that cosegregated with polyembryony. This study provides new insights into citrus apomixis and constitutes a promising resource for the mining of agriculturally important genes.

As an essential horticultural crop, Citrushas carotenoid diversity, which affects its aesthetic and nutritional values. ß,ß-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for ß,ß-xanthophyll synthesis. Previous studies have focused on the hydroxylation of BCH1, but the role of its paralogous gene in citrus, BCH2, remains largely unknown. In this study, we revealed the ß-hydroxylation activity of citrus BCH2 (CsBCH2) for the first time through the functional complementation assay using Escherichia coli, although CsBCH2 exhibited a lower activity in hydroxylating ß-carotene into ß-cryptoxanthin than citrus BCH1 (CsBCH1). Our results showed that overexpression of CsBCH2in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression. This study revealed the distinct expression patterns and functional characteristics of two paralogous genes, CsBCH1and CsBCH2, and illustrated the backup compensatory role of CsBCH2for CsBCH1in citrus xanthophyll biosynthesis. The independent function of CsBCH2and its cooperative function with CsBCH1inß-cryptoxanthin biosynthesis suggested the potential of CsBCH2to be employed for expanding the synthetic biology toolkit in carotenoid engineering.

Citrus sinensisis the most cultivated and economically valuable Citrusspecies in the world, whose genome has been assembled by three generation sequencing. However, chromosome recognition remains a problem due to the small size of chromosomes, and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome. We employed fluorescence in situhybridization (FISH) with nine chromosome painting probes, thirty oligo pools and eight repetitive sequences to visualize 18 chromosomes. Then we developed an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3 (CMA) staining. By this approach, we constructed a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions, which enable us to visualize significant differences between homologous chromosomes. Based on the map, we located several highly repetitive sequences on chromosomes, and estimated sizes and copy numbers of each site. In particular, we discovered the translocation regions of chromosomes 4 and 9 in C. sinensis'Valencia'. The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.

Apomixis, or asexual seed formation, is prevalent in Citrinaevia a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in Citrinae, a chromosome-level genome of the Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinaeaccessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinaediversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella, we found that apomixis of some varieties originated via introgression. In apomictic Fortunella, the locus associated with apomixis contains the FhRWPgene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWPand CitRWPpromoters from apomictic Citrusand Fortunella, due to either two or three miniature inverted transposon element (MITE) insertions. A transcription factor, FhARID, encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties, which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinaeand has potential ramifications for citrus breeding.

ABSTRACTTo illustrate the mechanism of lycopene accumulation in red-fleshed Cara Cara navel orange (Citrus sinensis Osbeck), the genes encoding citrus lycopene β-cyclases (CLCYbs) were compared among citrus accessions. Protein sequence alignment showed that CLCYbs could be classified into two types, CLCYb1s and CLCYb2s, based on the polymorphisms in the 4thand the 50thamino acids. Polymerase chain reaction—Restriction fragment length polymorphisms analysis (PCR-RFLP) further indicated that all studied eighteen accessions of citrus and its relatives (including four with lycopene accumulated flesh) could be classified into three groups. Pummelo (Citrus grandis), two grapefruits (C. paradisi), trifoliata orange (Pocirus trifoliata), two citranges (C. sinensis x P. trifoliata) and kumquat (Fortunella crassifolia) are in Group-1, which are homozygous for CLCYb1s. Genomes in Group-2 and 3 are heterozygous for CLCYb1s and CLCYb2s. However, Group-2 includes two navel oranges and two sour oranges (C. aurantium), in which close amounts of Cslcybls and Cslcyb2s were amplified. Whereas Group-3 includes two lemons (C. limon), two mandarins (C. reticulata) and Calamondin, in which unequal and unstable levels of two variants were amplified. Southern blot analysis implied that CLCYbs had at least two copies in Cara Cara navel orange and other three tested genotypes. The data presented here indicated the irrelevancy of the two variants in their genomes to the accumulation of lycopene in the citrus fruits.

Field observations indicate that boron Btoxicity symptoms may occur in citrus plants from inappropriate foliar spraying or overfertilizing with B especially under lowrainfall conditions, where B can accumulate to levels that become toxic to plant growth. Previous work has indicated that different rootstocks can greatly influence the scions tolerance to B toxicity, however, little is known about the response of different citrus scion–rootstock combinations to excessB conditions. In the present study, we investigated the effects of excess B on plant growth, gas exchange, B concentration, and distribution of four scion–rootstock combinations, Newhall and Skaggs Bonanza navel orange Citrus sinensisOsb. scions grafted on Carrizo citrange C. sinensisL. Osb. × Poncirus trifoliataL. Raf. and Trifoliate orange Poncirus trifoliataL. Raf. rootstocks. Oneyearold plants of the four scion–rootstock combinations were grown for 183 d in sand–perlite 1 : 1, vv medium under greenhouse conditions. The plants were irrigated with halfstrength Hoaglands nutrient solution containing two B concentrations, 0.25 control and 2.50 excess B mg L–1. It was found that, apart from the combination of Newhall grafted on Carrizo citrange, the dry weights in various parts of the other three combinations were reduced by the excessB treatment. Furthermore, the plants of Skaggs Bonanza grafted on Carrizo citrange showed the highest growth reduction amongst the four scion–rootstock combinations. In most cases, the greater reductions in dry weight were found in roots as compared to the other plant parts under excessB conditions, indicating that roots were more sensitive to B toxicity than the other tissues. In the case of Newhall plants grafted on Carrizo citrange, the entire plant growth was increased by excessB treatment. Boron concentrations in all plants parts increased significantly by increasing the B supply in the nutrient solution. Leaves were the dominant sites of B accumulation and showed the greatest increase in B concentration compared to the other plant parts, as B concentration in the nutrient solution increased. Our results indicate that the combination of Newhall grafted on Carrizo citrange was more tolerant to B toxicity, while the combination of Skaggs Bonanza grafted on Carrizo citrange was relatively more sensitive to B toxicity, in comparison with the other scion–rootstock combinations. However, Newhall plants contained more B in leaves and in roots than Skaggs Bonanza plants when they were both grafted on Carrizo citrange, indicating that the mechanism underlying such great differential growth responses of the two scion–rootstock combinations to B toxicity may not be associated with B exclusion from roots or reduced translocation of B to shoots. Furthermore, B distribution in different plant parts implied that the mechanism was also unlikely related to altered distributions of accumulated B in plant tissues. However, inherent ability to tolerate excessive B concentration in plant tissues may be involved in B tolerance.

Apomixis is the phenomenon of clonal reproduction by seed. As apomixis can produce clonal progeny with exactly the same genotype as the maternal plant, it has an important application in genotype fixation and accelerating agricultural breeding strategies. The introduction of apomixis to major crops would bring many benefits to agriculture, including permanent fixation of superior genotypes and simplifying the procedures of hybrid seed production, as well as purification and rejuvenation of crops propagated vegetatively. Although apomixis naturally occurs in more than 400 plant species, it is rare among the major crops. Currently, with better understanding of apomixis, some achievements have been made in synthetic apomixis. However, due to prevailing limitations, there is still a long way to go to achieve large-scale application of apomixis to crop breeding. Here, we compare the developmental features of apomixis and sexual plant reproduction and review the recent identification of apomixis genes, transposons, epigenetic regulation, and genetic events leading to apomixis. We also summarize the possible strategies and potential genes for engineering apomixis into crop plants.

Iron-deficiency chlorosis is a common nutritional disorder in crops grown on alkaline or calcareous soils. Although the acclimation mechanism to iron deficiency has been investigated, the genetic regulation of iron acquisition is still unclear. Here, by comparing the iron uptake process between the iron-poor-soil-tolerant citrus species Zhique (ZQ) and the iron-poor-soil-sensitive citrus species trifoliate orange (TO), we discovered that enhanced root H +efflux is crucial for the tolerance to iron deficiency in ZQ. The H+efflux is mainly regulated by a plasma membrane-localized H+-ATPase, HA6, the expression of which is upregulated in plants grown in soil with low iron content, and significantly higher in the roots of ZQ than TO. Overexpression of the HA6gene in the Arabidopsis thaliana aha2mutant, defective in iron uptake, recovered the wild-type phenotype. In parallel, overexpression of the HA6gene in TO significantly increased iron content of plants. Moreover, an iron deficiency-induced transcription factor, MYB308, was revealed to bind the promoter and activate the expression of HA6in ZQ in yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Overexpression of MYB308in ZQ roots significantly increased the expression level of the HA6gene. However, MYB308 cannot bind or activate the HA6promoter in TO due to the sequence variation of the corresponding MYB308 binding motif. Taking these results together, we propose that the MYB308 could activate HA6to promote root H+efflux and iron uptake, and that the distinctive MYB308-HA6transcriptional module may be, at least in part, responsible for the iron deficiency tolerance in citrus.

Somaclonal variation arising from tissue culture may provide a valuable resource for the selection of new germplasm, but may not preserve true-to-type characteristics, which is a major concern for germplasm conservation or genome editing. The genomic changes associated with dedifferentiation and somaclonal variation during long-term in vitroculture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitroand induced via somatic embryogenesis. We compared four sweet orange callus lines after 30 years of constant tissue culture with newly induced calli by comprehensively determining the single-nucleotide polymorphisms, copy number variations, transposable element insertions, methylomic and transcriptomic changes. We identified a burst of variation during early dedifferentiation, including a retrotransposon outbreak, followed by a variation purge during long-term in vitroculture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30 years of in vitroculture. We also analyzed the effects of somaclonal variation on transcriptional reprogramming, and indicated subgenome dominance was evident in the tetraploid callus. We identified a retrotransposon insertion and DNA modification alternations in the potential regeneration-related gene CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16. This study provides the foundation to harness in vitrovariation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.

In citrus, 1,6-rhamnosytransferase (1,6RhaT) and 1,2-rhamnosytransferase (1,2RhaT) catalyze flavanone-7-O-glucosides to form nonbitter flavanone rutinosides (FRs) and bitter flavanone neohesperidosides (FNs), respectively. As revealed in this study of fruit peels from 36 citrus accessions, FRs varied from undetectable levels in pummelo and kumquat to being the dominant flavonoids in sweet orange and loose-skin mandarins. Furthermore, a previously annotated full-length 1,6RhaT-likegene was identified as another 1,6RhaT-encoding gene by in vitroexperiments. In total, 28 alleles of full-length 1,6RhaTswere isolated and classified into A, B and C types with only type A alleles encoding a functional protein. Coincidently, only the accessions that contained FRs harbored type A alleles, as was further verified in two F1 hybrid populations. Moreover, the inferior substrate conversion efficiency of 1,6RhaTs in comparison with that of 1,2RhaT in vitromight partly explain the lower proportions of FRs to total flavanone disaccharides in citrus hybrids harboring both functional rhamnosyltransferases. Our findings provide a better understanding of FR content variations among citrus and are meaningful for a mechanistic illustration of citrus flavonoid metabolism and fruit quality improvement practices.

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in ‘Guiyou No. 1’ pummelo (Citrus maxima), which is a spontaneous mutant of ‘Shatian’ pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of ‘Guiyou No. 1’. Segregation ratios of the Sgenotype in F1progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNasemRNA contribute to the loss of SI in ‘Guiyou No. 1’. Furthermore, we report a phased assembly of the ‘Guiyou No. 1’ pummelo genome and obtained two complete and well-annotated Shaplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNasepromoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in ‘Guiyou No. 1’ by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNasegenes.

Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifoliaSwingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.

Abstract Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.

Tetraploid plants were obtained in pomegranate (Punica granatumL. var. `Nana') by colchicine treatment of shoots propagated in vitro. Shoots cultured on MS medium supplemented with 10 mg l−1colchicine, 1.0 mg l−1BA and 0.1 mg l−1NAA for 30 days produced tetraploids at a high frequency of 20%. No tetraploids were detected by treating the shoots in 5000 mg l−1colchicine for 114 h. Shoots treated by 5000 mg l−1colchicine for 96 h produced three morphological mutants with narrow leaves, which were later confirmed as mixoploids that separated into diploids and tetraploids after further subculture. In vitrotetraploid plants had shorter roots, wider and shorter leaves than the diploid ones. Tetraploid pomegranate plants grew and flowered normally in pots, but possessed flowers with increased diameter and decreased length compared to diploids. The number of pollen grains per anther was higher in tetraploids, but the viability of pollen decreased significantly.

Abstract: Embryogenic protoplasts of Dancy tangerine (Citrus reticulata Blanco) were X-ray irradiated at three doses and electrofused with iodoacetic acid-treated embryogenic protoplasts of Page tangelo (C. reticulata Blanco C. paradisi Macf.). Shoots could regenerate only from the fusion combination with the lowest irradiation dose, but were recalcitrant to rooting. In vitro grafting was applied to obtain complete plants. Chromosome examination showed that the plants contained mainly diploid and aneuploid cells, together with few tetraploid cells, indicating that they were mixoploids. Random amplified polymorphic DNA analyses with 10-mer arbitrary primers confirmed the plants as true somatic hybrids. This is the first report on regeneration of mixoploid hybrid plants via protoplast asymmetric fusion in Citrus. Negative effects of ionizing irradiation on regeneration of embryoids and plantlets and possible agronomic interest of the mixoploid plants are also discussed.

Abstract: X-ray irradiated embryogenic protoplasts of Microcitrus papuana Swing. were electrically fused with iodoacetic acid-treated embryogenic protoplasts of Newhall navel orange [Citrus sinensis (L.) Osb.]. Seven cell lines were established by low-melting agarose embedding culture of fusion-treated protoplasts. Cytological examination of 4 cell lines showed that each cell line consisted of many aneuploid (45.10%, 38.98%, 32.69% and 34.85%, respectively) and diploid cells (52.94%, 59.33%, 63.46% and 62.12%. respectively), whereas only a few tetraploid cells (1.96%, 1.69%, 3.85% and 3.03%, respectively) were detected. Analyses of random amplified polymorphic DNA with four 10-mer primers confirmed the hybrid characteristics of the cell lines, which in combination with chromosome counting proved that the cell lines were asymmetric hybrids.

Apocarotenoids contribute to fruit color and aroma, which are critical quality and marketability attributes. Previously, we reported that the red peels of citrus fruits, which are characterized by higher expression levels of a carotenoid cleavage dioxygenase4b(CitCCD4b) gene, accumulate higher levels of β-citraurin and β-citraurinene than yellow peels. Here, we identified and quantified 12 apocarotenoids, either volatile or nonvolatile, in citrus peel using liquid chromatography–mass spectrometry (LC–MS). Our results show that red peels contain also dramatically higher amounts of β-apo-8′-carotenal, crocetin dialdehyde known from saffron, β-citraurol, β-cyclocitral, and 3-OH-β-cyclocitral and up to about 17-fold higher levels of 3-OH-β-cyclocitral glucoside (picrocrocin isomer). The content of these apocarotenoids was also significantly increased in different CitCCD4b-overexpressing transgenic callus lines, compared with corresponding controls. Transient expression of CitCCD4bin Nicotiana benthamianaleaves resulted in a striking increase in the 3-OH-β-cyclocitral level and the accumulation of picrocrocin. Thus, our work reinforces the specific function of CitCCD4b in producing C10apocarotenoid volatiles and C30pigments in citrus peel and uncovers its involvement in the biosynthesis of picrocrocin, C20dialdehyde, and C30alcohol apocarotenoids, suggesting the potential of this enzyme in metabolic engineering of apocarotenoids and their derivatives.

Both cuticle and membrane lipids play essential roles in quality maintenance and disease resistance in fresh fruits. Many reports have indicated the modification of alternative branch pathways in epicuticular wax mutants; however, the specific alterations concerning lipids have not been clarified thus far. Here, we conducted a comprehensive, time-resolved lipidomic, and transcriptomic analysis on the “Newhall” navel orange (WT) and its glossy mutant (MT) “Gannan No. 1”. The results revealed severely suppressed wax formation accompanied by significantly elevated production of 36-carbon plastid lipids with increasing fruit maturation in MT. Transcriptomics analysis further identified a series of key functional enzymes and transcription factors putatively involved in the biosynthesis pathways of wax and membrane lipids. Moreover, the high accumulation of jasmonic acid (JA) in MT was possibly due to the need to maintain plastid lipid homeostasis, as the expression levels of two significantly upregulated lipases (CsDAD1 and CsDALL2) were positively correlated with plastid lipids and characterized to hydrolyze plastid lipids to increase the JA content. Our results will provide new insights into the molecular mechanisms underlying the natural variation of plant lipids to lay a foundation for the quality improvement of citrus fruit.

This corrects the article DOI: 10.1038/hortres.2016.17

It has been observed that the consumption of litchi often causes symptoms characterized by itching or sore throat, gum swelling, oral cavity ulcers and even fever and inflammation, which significantly impair the quality of life of a large population. Using the RAW264.7 cell line, a step-by-step strategy was used to screen for the components in litchi fruits that elicited adverse reactions. The adverse reaction fractions were identified by mass spectrometry and analyzed using the SMART program, and a sequence alignment of the homologous proteins was performed. MTT tests were used to determine the cytotoxicity of a litchi protein extract in RAW264.7 macrophages, and real-time PCR was applied to analyze the expression of inflammatory genes in the RAW264.7 cells treated with lipopolysaccharide or the litchi protein extract. The results showed that the litchi water-soluble protein extract could increase the production of the pro-inflammatory mediators IL-1β, iNOS and COX-2, and the anti-inflammatory mediator HO-1 in the RAW264.7 cell line. The 14-3-3-like proteins GF14 lambda, GF14 omega and GF14 upsilon were likely the candidate proteins that caused the adverse effects.