Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non-photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty-four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG-localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi-feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops.

Geographic range size, water temperature, and extrinsic threats predict the extinction risk in global cetaceans.

Despite the fact that cetaceans provide significant ecological contributions to the health and stability of aquatic ecosystems, many are highly endangered with nearly one-third of species assessed as threatened with extinction. Nevertheless, to date, few studies have explicitly examined the patterns and processes of extinction risk and threats for this taxon, and even less between the two subclades (Mysticeti and Odontoceti). To fill this gap, we compiled a dataset of six intrinsic traits (active region, geographic range size, body weight, diving depth, school size, and reproductive cycle), six environmental factors relating to sea surface temperature and chlorophyll concentration, and two human-related threat indices that are commonly recognized for cetaceans. We then employed phylogenetic generalized least squares models and model selection to identify the key predictors of extinction risk in all cetaceans, as well as in the two subclades. We found that geographic range size, sea surface temperature, and human threat index were the most important predictors of extinction risk in all cetaceans and in odontocetes. Interestingly, maximum body weight was positively associated with the extinction risk in mysticetes, but negatively related to that for odontocetes. By linking seven major threat types to extinction risk, we further revealed that fisheries bycatch was the most common threat, yet the impacts of certain threats could be overestimated when considering all species rather than just threatened ones. Overall, we suggest that conservation efforts should focus on small-ranged cetaceans and species living in warmer waters or under strong anthropogenic pressures. Moreover, further studies should consider the threatened status of species when superimposing risk maps and quantifying risk severity. Finally, we emphasize that mysticetes and odontocetes should be conserved with different strategies, because their extinction risk patterns and major threat types are considerably different. For instance, large-bodied mysticetes and small-ranged odontocetes require special conservation priority.

Do functional and phylogenetic nestedness follow the same mechanisms as taxonomic nestedness? Evidence from amphibians in the largest archipelago of China.

Nested subset pattern (nestedness) has been raised to explain the distribution of species on islands and habitat fragments for over 60 years. However, previous studies on nestedness focused on species richness and composition and overlooked the role of species traits and phylogeny in generating and explaining nestedness. To address this gap, we sampled amphibians on 37 land-bridge islands in the largest archipelago of China, the Zhoushan Archipelago, to explore nestedness as well as the underlying causal processes through three facets of diversity, that is, taxonomic, functional and phylogenetic diversity. The taxonomic nestedness was measured through organizing the species incidence matrix to achieve a maximum value, while the functional and phylogenetic nestedness were quantified by incorporating the similarity of species in terms of their ecological traits and phylogeny. We also obtained six island characteristics and seven species traits as predictors of nestedness. Amphibian metacommunities were significantly nested in these three facets of diversity. When relating different predictors to nestedness, island area, habitat diversity and species traits were highly correlated with taxonomic nestedness. Moreover, island area and habitat diversity significantly influenced functional and phylogenetic nestedness. Therefore, the results support the selective extinction and habitat nestedness hypotheses. Interestingly, although we did not observe significant influences of island isolation on taxonomic nestedness, functional and phylogenetic diversities were significantly higher than expected when matrices were ordered by increasing distance to mainland. The result suggests that there are more functionally and phylogenetically diverse species on less-isolated islands, reflecting a selective colonization process overlooked by the traditional analysis of taxonomic nestedness. Although the three facets of nestedness and underlying processes were largely congruent, we detected the distance-related functional and phylogenetic nestedness for amphibian assemblages. Therefore, we highlight that a framework that simultaneously considers taxonomic, functional and phylogenetic nestedness can contribute to a complementary understanding of nestedness processes. In addition, it also improves our ability to conserve insular biodiversity from different perspectives.

CHH methylation of genes associated with fatty acid and jasmonate biosynthesis contributes to cold tolerance in autotetraploids of Poncirus trifoliata.

Polyploids have elevated stress tolerance, but the underlying mechanisms remain largely elusive. In this study, we showed that naturally occurring tetraploid plants of trifoliate orange (Poncirus trifoliata (L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors. Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes. Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress. CHH methylation in the promoters was associated with up-regulation of related genes, whereas CG, CHG, and CHH methylation in the 3'-regions was relevant to gene down-regulation. Of note, genes involved in unsaturated fatty acids (UFAs) and jasmonate (JA) biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated, consistent with greater accumulation of UFAs and JA when exposed to the cold stress. Collectively, our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels, and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid. These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.

Red light-induced kumquat fruit coloration is attributable to increased carotenoid metabolism regulated by FcrNAC22.

Carotenoids play vital roles in the coloration of plant tissues and organs, particularly fruits; however, the regulation of carotenoid metabolism in fruits during ripening is largely unknown. Here, we show that red light promotes fruit coloration by inducing accelerated degreening and carotenoid accumulation in kumquat fruits. Transcriptome profiling revealed that a NAC (NAM/ATAF/CUC2) family transcription factor, FcrNAC22, is specifically induced in red light-irradiated fruits. FcrNAC22 localizes to the nucleus, and its gene expression is up-regulated as fruits change color. Results from dual luciferase, yeast one-hybrid assays and electrophoretic mobility shift assays indicate that FcrNAC22 directly binds to, and activates the promoters of three genes encoding key enzymes in the carotenoid metabolic pathway. Moreover, FcrNAC22 overexpression in citrus and tomato fruits as well as in citrus callus enhances expression of most carotenoid biosynthetic genes, accelerates plastid conversion into chromoplasts, and promotes color change. Knock down of FcrNAC22 expression in transiently transformed citrus fruits attenuates fruit coloration induced by red light. Taken together, our results demonstrate that FcrNAC22 is an important transcription factor that mediates red light-induced fruit coloration via up-regulation of carotenoid metabolism.

Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata.

Tetraploids have been reported to exhibit increased stress tolerance, but the underlying molecular and physiological mechanisms remain poorly understood. In this study, autotetraploid plants were identified by screening natural seedlings of trifoliate orange (Poncirus trifoliata). The tetraploids exhibited different morphology and displayed significantly enhanced drought and dehydration tolerance in comparison with the diploid progenitor. Transcriptome analysis indicated that a number of stress-responsive genes and pathways were differentially influenced and enriched in the tetraploids, in particular those coding for enzymes related to antioxidant process and sugar metabolism. Transcript levels and activities of antioxidant enzymes (peroxidase and superoxide dismutase) and sucrose-hydrolysing enzyme (vacuolar invertase) were increased in the tetraploids upon exposure to the drought, concomitant with greater levels of glucose but lower level of reactive oxygen species (ROS). These data indicate that the tetraploids might undergo extensive transcriptome reprogramming of genes involved in ROS scavenging and sugar metabolism, which contributes, synergistically or independently, to the enhanced stress tolerance of the tetraploid. Our results reveal that the tetraploids take priority over the diploid for stress tolerance by maintaining a more robust system of ROS detoxification and osmotic adjustment via elevating antioxidant capacity and sugar accumulation in comparison with the diploid counterpart.

A global synthesis of the small-island effect in habitat islands.

Habitat loss and fragmentation are considered to be the leading drivers of biodiversity loss. The small-island effect (SIE) can be used to predict species extinctions resulting from habitat loss and has important implications for species conservation. However, to date, no study has explicitly evaluated the prevalence of SIEs in habitat islands. Here, we compiled 90 global datasets to systematically investigate the prevalence and underlying factors determining the ubiquity of SIEs in habitat island systems. Among the 90 datasets, SIEs were unambiguously detected in 36 cases. We found significant effects of habitat island types and taxon groups on the threshold area of SIEs. The number of islands, area range, species range, island type and taxon group were key variables that determined the prevalence of SIEs. Our study demonstrates that SIEs occur in 40% of cases and thus are common in habitat islands. We conclude that conservation biologists and applied ecologists should consider the prevalence of SIEs when making management strategies in fragmented landscapes.

Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus.

S-RNase-based self-incompatibility is found in Solanaceae, Rosaceae, and Scrophulariaceae, and is the most widespread mechanism that prevents self-fertilization in plants. Although 'Shatian' pummelo (Citrus grandis), a traditional cultivated variety, possesses the self-incompatible trait, the role of S-RNases in the self-incompatibility of 'Shatian' pummelo is poorly understood. To identify genes associated with self-incompatibility in citrus, we identified 16 genes encoding homologs of ribonucleases in the genomes of sweet orange (Citrus sinensis) and clementine mandarin (Citrus clementine). We preliminarily distinguished S-RNases from S-like RNases with a phylogenetic analysis that classified these homologs into three groups, which is consistent with the previous reports. Expression analysis provided evidence that CsRNS1 and CsRNS6 are S-like RNase genes. The expression level of CsRNS1 was increased during fruit development. The expression of CsRNS6 was increased during the formation of embryogenic callus. In contrast, we found that CsRNS3 possessed several common characteristics of the pistil determinant of self-incompatibility: it has an alkaline isoelectric point (pI), harbors only one intron, and is specifically expressed in style. We obtained a cDNA encoding CgRNS3 from 'Shatian' pummelo and found that it is homolog to CsRNS3 and that CgRNS3 exhibited the same expression pattern as CsRNS3. In an in vitro culture system, the CgRNS3 protein significantly inhibited the growth of self-pollen tubes from 'Shatian' pummelo, but after a heat treatment, this protein did not significantly inhibit the elongation of self- or non-self-pollen tubes. In conclusion, an S-RNase gene, CgRNS3, was obtained by searching the genomes of sweet orange and clementine for genes exhibiting sequence similarity to ribonucleases followed by expression analyses. Using this approach, we identified a protein that significantly inhibited the growth of self-pollen tubes, which is the defining property of an S-RNase.

Selective extinction and habitat nestedness are the main drivers of lizard nestedness in the Zhoushan Archipelago.

Nested subset pattern (nestedness) is an important part of the theoretical framework of island biogeography and community ecology. However, most previous studies often used nestedness metrics or randomization algorithms that are vulnerable to type I error. In this study, we investigated the nestedness of lizard assemblages on 37 islands in the Zhoushan Archipelago, China. We used the line-transect method to survey species occurrence, abundance, and habitat types of lizards on 37 islands during 2 breeding seasons in 2021 and 2022. We applied the nested metric WNODF and the conservative rc null model to control for type I error and quantify the significance of nestedness. Spearman rank correlations were used to evaluate the role of 4 habitat variables (island area, 2 isolation indices, and habitat diversity) and 4 ecological traits (body size, geographic range size, clutch size, and minimum area requirement) in generating nestedness. The results of WNODF analyses showed that lizard assemblages were significantly nested. The habitat-by-site matrix estimated by the program NODF was also significantly nested, supporting the habitat nestedness hypothesis. The nestedness of lizard assemblages were significantly correlated with island area, habitat diversity, clutch size, and minimum area requirement. Overall, our results suggest that selective extinction and habitat nestedness were the main drivers of lizard nestedness in our system. In contrast, the nestedness of lizard assemblages was not due to passive sampling or selective colonization. To maximize the number of species preserved, our results indicate that we should protect both large islands with diverse habitats and species with large area requirement and clutch size.

Characteristics of Rhizosphere Microbiome, Soil Chemical Properties, and Plant Biomass and Nutrients in Citrus reticulata cv. Shatangju Exposed to Increasing Soil Cu Levels.

The prolonged utilization of copper (Cu)-containing fungicides results in Cu accumulation and affects soil ecological health. Thus, a pot experiment was conducted using Citrus reticulata cv. Shatangju with five Cu levels (38, 108, 178, 318, and 388 mg kg-1) to evaluate the impacts of the soil microbial processes, chemistry properties, and citrus growth. These results revealed that, with the soil Cu levels increased, the soil total Cu (TCu), available Cu (ACu), organic matter (SOM), available potassium (AK), and pH increased while the soil available phosphorus (AP) and alkali-hydrolyzable nitrogen (AN) decreased. Moreover, the soil extracellular enzyme activities related to C and P metabolism decreased while the enzymes related to N metabolism increased, and the expression of soil genes involved in C, N, and P cycling was regulated. Moreover, it was observed that tolerant microorganisms (e.g., p_Proteobacteria, p_Actinobacteria, g_Lysobacter, g_Sphingobium, f_Aspergillaceae, and g_Penicillium) were enriched but sensitive taxa (p_Myxococcota) were suppressed in the citrus rhizosphere. The citrus biomass was mainly positively correlated with soil AN and AP; plant N and P were mainly positively correlated with soil AP, AN, and acid phosphatase (ACP); and plant K was mainly negatively related with soil ß-glucosidase (ßG) and positively related with the soil fungal Shannon index. The dominant bacterial taxa p_Actinobacteriota presented positively correlated with the plant biomass and plant N, P, and K and was negatively correlated with plant Cu. The dominant fungal taxa p_Ascomycota was positively related to plant Cu but negatively with the plant biomass and plant N, P, and K. Notably, arbuscular mycorrhizal fungi (p_Glomeromycota) were positively related with plant P below soil Cu 108 mg kg-1, and pathogenic fungi (p_Mortierellomycota) was negatively correlated with plant K above soil Cu 178 mg kg-1. These findings provided a new perspective on soil microbes and chemistry properties and the healthy development of the citrus industry at increasing soil Cu levels.

Molecular regulation of oil gland development and biosynthesis of essential oils in Citrus spp.

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis-regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

Fe-TiO2-x/TiO2 S-scheme homojunction for efficient photocatalytic CO2 reduction.

CO2-to-high value-added chemicals via a photocatalytic route is of interest but strangled by the low efficiency. Herein, a novel Fe-TiO2-x/TiO2 S-scheme homojunction was designed and constructed by using a facile surface modification approach whereby oxygen vacancy (OV) and Fe introducing on the TiO2 nanorod surface. The as-synthesized Fe-TiO2-x/TiO2 S-scheme homojunction exhibits positive properties on promoting photocatalytic CO2 reduction: i) the nanorod structure provides numerous active sites and a radical charge transfer path; ii) the doped Fe and OV not only synergistically enhance light utilization but also promote CO2 adsorption; iii) the Fe-TiO2-x/TiO2 S-scheme homojunction benefits photoexcited charge separation and retains stronger redox capacity. Thanks to those good characters, the Fe-TiO2-x/TiO2 homojunction exhibits superior CO2 reduction performances with optimized CO/CH4 generation rates of 122/22 µmol g-1h-1 which exceed those of pure TiO2 by more than 9.4/7.3 folds and most currently reported catalytic systems. This manuscript develops a facile and universal approach to synthesize well-defined homojunction and may inspire the construction of other more high-efficiency photocatalysts toward CO2 reduction and beyond.

Fruit Characteristics of Citrus Trees Grown under Different Soil Cu Levels.

The effects of the increased soil copper (Cu) on fruit quality due to the overuse of Cu agents have been a hot social issue. Seven representative citrus orchards in Guangxi province, China, were investigated to explore the fruit quality characteristics under different soil Cu levels and the relationship between soil-tree Cu and fruit quality. These results showed that pericarp color a value, titratable acid (TA), and vitamin C (Vc) were higher by 90.0, 166.6, and 22.4% in high Cu orchards and by 50.5, 204.2, and 55.3% in excess Cu orchards, compared with optimum Cu orchards. However, the ratio of total soluble solids (TSS)/TA was lower by 68.7% in high Cu orchards and by 61.6% in excess Cu orchards. With the increase of soil Cu concentrations, pericarp color a value and Vc were improved, TA with a trend of rising first then falling, and TSS/TA with a trend of falling first then rising were recorded. As fruit Cu increased, pericarp color a value and TSS reduced and as leaf Cu increased, TSS/TA decreased while Vc was improved. Moreover, a rise in soil Cu enhanced leaf Cu accumulation, and a rise in leaf Cu improved fruit Cu accumulation. Fruit Cu accumulation reduced fruit quality by direct effects, leaf Cu improved fruit quality by direct and indirect effects. Soil Cu affected fruit quality by indirect effects by regulating leaf Cu and fruit Cu. Therefore, reasonable regulation and control of soil Cu concentrations can effectively increase pericarp color, sugar, and acid accumulation in citrus fruit.

Ectopic expression of MdSPDS1 in sweet orange (Citrus sinensis Osbeck) reduces canker susceptibility: involvement of H2O2 production and transcriptional alteration.

BACKGROUND: Enormous work has shown that polyamines are involved in a variety of physiological processes, but information is scarce on the potential of modifying disease response through genetic transformation of a polyamine biosynthetic gene. RESULTS: In the present work, an apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange (Citrus sinensis Osbeck 'Anliucheng') via Agrobacterium-mediated transformation of embryogenic calluses. Two transgenic lines (TG4 and TG9) varied in the transgene expression and cellular endogenous polyamine contents. Pinprick inoculation demonstrated that the transgenic lines were less susceptible to Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker, than the wild type plants (WT). In addition, our data showed that upon Xac attack TG9 had significantly higher free spermine (Spm) and polyamine oxidase (PAO) activity when compared with the WT, concurrent with an apparent hypersensitive response and the accumulation of more H2O2. Pretreatment of TG9 leaves with guazatine acetate, an inhibitor of PAO, repressed PAO activity and reduced H2O2 accumulation, leading to more conspicuous disease symptoms than the controls when both were challenged with Xac. Moreover, mRNA levels of most of the defense-related genes involved in synthesis of pathogenesis-related protein and jasmonic acid were upregulated in TG9 than in the WT regardless of Xac infection. CONCLUSION: Our results demonstrated that overexpression of the MdSPDS1 gene prominently lowered the sensitivity of the transgenic plants to canker. This may be, at least partially, correlated with the generation of more H2O2 due to increased production of polyamines and enhanced PAO-mediated catabolism, triggering hypersensitive response or activation of defense-related genes.

Nestedness and underlying processes of bird assemblages in Nanjing urban parks.

Nestedness is an important pattern frequently reported for species assemblages on islands or fragmented systems. However, to date, there are few studies that comprehensively investigated faunal nestedness and underlying processes in urbanized landscapes. In this study, we examined the nestedness of bird assemblages and its underlying causal mechanisms in 37 urban parks in Nanjing, China. We used the line-transect method to survey birds from April 2019 to January 2020. We used the Weighted Nestedness metric based on Overlap and Decreasing Fill (WNODF) to estimate the nestedness of bird assemblages. We applied spearman partial correlation test to examine the relationships between nestedness ranks of sites and park characteristics (area, isolation, anthropogenic noise, number of habitat types, and building index), as well as between nestedness ranks of species and their ecological traits (body size, geographic range size, clutch size, minimum area requirement, dispersal ratio, and habitat specificity). We found that bird assemblages in urban parks were significantly nested. Park area, habitat diversity, building index, habitat specificity, and minimum area requirement of birds were significantly correlated with nestedness. Therefore, the nestedness of bird assemblages was caused by selective extinction, habitat nestedness, and urbanization. However, the nestedness of bird assemblages did not result from passive sampling, selective colonization, or human disturbance. Overall, to maximize the number of species preserved in our system, conservation priority should be given to parks with large area, rich habitat diversity, and less building index. From a species perspective, we should focus on species with large area requirement and high habitat specificity for their effective conservation.

Illuminating the cells: transient transformation of citrus to study gene functions and organelle activities related to fruit quality.

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 crassifolia Swingle) 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.

Downregulated expression of S2-RNase attenuates self-incompatibility in "Guiyou No. 1" pummelo.

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 S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA 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 S haplotypes. 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-RNase promoter. 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-RNase genes.

Evolution of self-compatibility by a mutant Sm-RNase in citrus.

Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and inbreeding in flowering plants. The most widespread SI system utilizes S ribonucleases (S-RNases) and S-locus F-boxes (SLFs) as S determinants. 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 S haplotypes in a gametophytic manner and cluster with authentic S-RNases. We provide evidence that these S-RNases function as the female S determinants in citrus. Moreover, we show that each S-RNase is linked to approximately nine SLFs. In an analysis of 117 citrus SLF and SFL-like (SLFL) genes, we reveal that they cluster into 12 types and that the S-RNases and intra-haplotypic SLF and SLFL genes co-evolved. Our data support the notion that citrus have a S locus comprising a S-RNase and 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.

A comprehensive proteomic analysis of elaioplasts from citrus fruits reveals insights into elaioplast biogenesis and function.

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 silico and 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.

Genome of Wild Mandarin and Domestication History of Mandarin.

Mandarin (Citrus reticulata) is one of the most important citrus crops worldwide. Its domestication is believed to have occurred in South China, which has been one of the centers of mandarin cultivation for four millennia. We collected natural wild populations of mandarin around the Nanling region and cultivated landraces in the vicinity. We found that the citric acid level was dramatically reduced in cultivated mandarins. To understand genetic basis of mandarin domestication, we de novo assembled a draft genome of wild mandarin and analyzed a set of 104 citrus genomes. We found that the Mangshan mandarin is a primitive type and that two independent domestication events have occurred, resulting in two groups of cultivated mandarins (MD1 and MD2) in the North and South Nanling Mountains, respectively. Two bottlenecks and two expansions of effective population size were identified for the MD1 group of cultivated mandarins. However, in the MD2 group there was a long and continuous decrease in the population size. MD1 and MD2 mandarins showed different patterns of interspecific introgression from cultivated pummelo species. We identified a region of high divergence in an aconitate hydratase (ACO) gene involved in the regulation of citrate content, which was possibly under selection during the domestication of mandarin. This study provides concrete genetic evidence for the geographical origin of extant wild mandarin populations and sheds light on the domestication and evolutionary history of mandarin.