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.

The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.).

Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA-mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism-related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid-driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5-CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5-CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5-CsbZIP44 precisely modulates ABA signal-mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops.

Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.).

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 CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli, but its regulatory mechanism is still unclear. In the present study, we found that overexpression of CsTT8 enhances 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 vitro and in vivo assays 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.

Constructing a NiMnS electrode with a Mn-rich surface for hydrogen production in anion exchange membrane water electrolyzers.

Efficient alkaline hydrogen evolution electrodes must be used for hydrogen production in anion exchange membrane water electrolyzers (AEMWEs). Therefore, we fabricated a NiMnS catalyst with a Mn-rich surface, which was self-supported on Ti paper through one-step electrodeposition. Mn doping endowed the catalyst with a unique hollow morphology and lattice-distorted structure. Consequently, the NiMnS/Ti electrode exhibited a large number of exposed electrochemical surfaces and effective active sites and a high hydrogen evolution reaction (HER) activity. Specifically, in half-cell measurements, the NiMnS/Ti electrode exhibited an overpotential of 65 mV at 10 mA cm-2, which was lower than that of NiS/Ti (102 mV) and indicated its superior HER activity. When the proposed cathode was applied in an AEMWE single cell, the device achieved a high current density of up to 0.9 A cm-2 at a cell potential of 2.0 V.

Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus.

Cytoplasmic male sterility (CMS) has long been used to produce seedless fruits in perennial woody crops like citrus. A male-sterile somatic cybrid citrus (G1 + HBP) was generated by protoplast fusion between a CMS callus parent 'Guoqing No. 1' Satsuma mandarin (Citrus unshiu, G1) and a fertile mesophyll parent Hirado Buntan pummelo (Citrus grandis, HBP). To uncover the male-sterile mechanism of G1 + HBP, we compared the transcriptome profiles of stamen organ and cell types at five stages between G1 + HBP and HBP, including the initial stamen primordia, enlarged stamen primordia, pollen mother cells, tetrads, and microspores captured by laser microdissection. The stamen organ and cell types showed distinct gene expression profiles. A majority of genes involved in stamen development were differentially expressed, especially CgAP3.2, which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1 + HBP compared with HBP. Jasmonic acid- and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia, and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1 + HBP. In contrast, the content of auxin biosynthesis metabolites was lower in G1 + HBP. The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia, meiocytes, and microspores, indicating the dysfunction of mitochondria in stamen organ and cell types of G1 + HBP. Taken together, the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development, and thus lead to male sterility in the citrus cybrid.

Genomic conservation of crop wild relatives: A case study of citrus.

Conservation of crop wild relatives is critical for plant breeding and food security. The lack of clarity on the genetic factors that lead to endangered status or extinction create difficulties when attempting to develop concrete recommendations for conserving a citrus wild relative: the wild relatives of crops. Here, we evaluate the conservation of wild kumquat (Fortunella hindsii) using genomic, geographical, environmental, and phenotypic data, and forward simulations. Genome resequencing data from 73 accessions from the Fortunella genus were combined to investigate population structure, demography, inbreeding, introgression, and genetic load. Population structure was correlated with reproductive type (i.e., sexual and apomictic) and with a significant differentiation within the sexually reproducing population. The effective population size for one of the sexually reproducing subpopulations has recently declined to ~1,000, resulting in high levels of inbreeding. In particular, we found that 58% of the ecological niche overlapped between wild and cultivated populations and that there was extensive introgression into wild samples from cultivated populations. Interestingly, the introgression pattern and accumulation of genetic load may be influenced by the type of reproduction. In wild apomictic samples, the introgressed regions were primarily heterozygous, and genome-wide deleterious variants were hidden in the heterozygous state. In contrast, wild sexually reproducing samples carried a higher recessive deleterious burden. Furthermore, we also found that sexually reproducing samples were self-incompatible, which prevented the reduction of genetic diversity by selfing. Our population genomic analyses provide specific recommendations for distinct reproductive types and monitoring during conservation. This study highlights the genomic landscape of a wild relative of citrus and provides recommendations for the conservation of crop wild relatives.

miR171-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 regulate somatic embryogenesis in citrus.

Somatic embryogenesis (SE) is a key regeneration pathway in various biotechnology approaches to crop improvement, especially for economically important perennial woody crops like citrus. However, maintenance of SE capability has long been a challenge and becomes a bottleneck in biotechnology-facilitated plant improvement. In the embryogenic callus (EC) of citrus, we identified 2 csi-miR171c-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 (CsSCL2/3), which exert positive feedback regulation on csi-miR171c expression. Suppression of CsSCL2 expression by RNA interference (RNAi) enhanced SE in citrus callus. A thioredoxin superfamily protein CsClot was identified as an interactive protein of CsSCL2/3. Overexpression of CsClot disturbed reactive oxygen species (ROS) homeostasis in EC and enhanced SE. Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq identified 660 genes directly suppressed by CsSCL2 that were enriched in biological processes including development-related processes, auxin signaling pathway, and cell wall organization. CsSCL2/3 bound to the promoters of regeneration-related genes, such as WUSCHEL-RELATED HOMEOBOX 2 (CsWOX2), CsWOX13, and Lateral Organ Boundaries Domain 40 (LBD40), and repressed their expression. Overall, CsSCL2/3 modulate ROS homeostasis through the interactive protein CsClot and directly suppress the expression of regeneration-related genes, thus regulating SE in citrus. We uncovered a regulatory pathway of miR171c-targeted CsSCL2/3 in SE, which shed light on the mechanism of SE and regeneration capability maintenance in citrus.

MADS-box protein PpDAM6 regulates chilling requirement-mediated dormancy and bud break in peach.

Bud dormancy is crucial for winter survival and is characterized by the inability of the bud meristem to respond to growth-promotive signals before the chilling requirement (CR) is met. However, our understanding of the genetic mechanism regulating CR and bud dormancy remains limited. This study identified PpDAM6 (DORMANCY-ASSOCIATED MADS-box) as a key gene for CR using a genome-wide association study analysis based on structural variations in 345 peach (Prunus persica (L.) Batsch) accessions. The function of PpDAM6 in CR regulation was demonstrated by transiently silencing the gene in peach buds and stably overexpressing the gene in transgenic apple (Malus × domestica) plants. The results showed an evolutionarily conserved function of PpDAM6 in regulating bud dormancy release, followed by vegetative growth and flowering, in peach and apple. The 30-bp deletion in the PpDAM6 promoter was substantially associated with reducing PpDAM6 expression in low-CR accessions. A PCR marker based on the 30-bp indel was developed to distinguish peach plants with non-low and low CR. Modification of the H3K27me3 marker at the PpDAM6 locus showed no apparent change across the dormancy process in low- and non-low- CR cultivars. Additionally, H3K27me3 modification occurred earlier in low-CR cultivars on a genome-wide scale. PpDAM6 could mediate cell-cell communication by inducing the expression of the downstream genes PpNCED1 (9-cis-epoxycarotenoid dioxygenase 1), encoding a key enzyme for ABA biosynthesis, and CALS (CALLOSE SYNTHASE), encoding callose synthase. We shed light on a gene regulatory network formed by PpDAM6-containing complexes that mediate CR underlying dormancy and bud break in peach. A better understanding of the genetic basis for natural variations of CR can help breeders develop cultivars with different CR for growing in different geographical regions.

Genome-wide identification of the C2H2-Zinc finger gene family and functional validation of CsZFP7 in citrus nucellar embryogenesis.

KEY MESSAGE: Genome-wide identification of C2H2-ZF gene family in the poly- and mono-embryonic citrus species and validation of the positive role of CsZFP7 in sporophytic apomixis. The C2H2 zinc finger (C2H2-ZF) gene family is involved in plant vegetative and reproductive development. Although a large number of C2H2 zinc-finger proteins (C2H2-ZFPs) have been well characterized in some horticultural plants, little is known about the C2H2-ZFPs and their function in citrus. In this work, we performed a genome-wide sequence analysis and identified 97 and 101 putative C2H2-ZF gene family members in the genomes of sweet orange (C. sinensis, poly-embryonic) and pummelo (C. grandis, mono-embryonic), respectively. Phylogenetic analysis categorized citrus C2H2-ZF gene family into four clades, and their possible functions were inferred. According to the numerous regulatory elements on promoter, citrus C2H2-ZFPs can be divided into five different regulatory function types that indicate functional differentiation. RNA-seq data revealed 20 differentially expressed C2H2-ZF genes between poly-embryonic and mono-embryonic ovules at two stages of citrus nucellar embryogenesis, among them CsZFP52 specifically expressed in mono-embryonic pummelo ovules, while CsZFP7, 37, 44, 45, 67 and 68 specifically expressed in poly-embryonic sweet orange ovules. RT-qPCR further validated that CsZFP7 specifically expressed at higher levels in poly-embryonic ovules, and down-regulation of CsZFP7 in the poly-embryonic mini citrus (Fortunella hindsii) increased rate of mono-embryonic seeds compared with the wild type, indicating the regulatory potential of CsZFP7 in nucellar embryogenesis of citrus. This work provided a comprehensive analysis of C2H2-ZF gene family in citrus, including genome organization and gene structure, phylogenetic relationships, gene duplications, possible cis-elements on promoter regions and expression profiles, especially in the poly- and mono-embryogenic ovules, and suggested that CsZFP7 is involved in nucellar embryogenesis.

Overexpression of miR390b promotes stem elongation and height growth in Populus.

MicroRNA390 (miR390) is involved in plant growth and development by down-regulating the expression of the downstream genes trans-acting short interfering RNA3 (TAS3) and AUXIN RESPONSE FACTORs (ARFs). There is a scarcity of research on the involvement of the miR390-TAS3-ARFs pathway in the stem development of Populus. Here, differentially expressed miRNAs during poplar stem development were screened by small RNA sequencing analysis, and a novel function of miR390b in stem development was revealed. Overexpression of miR390b (OE-miR390b) resulted in a large increase in the number of xylem fiber cells and a slight decrease in the cell length at the longitudinal axis. Overall increases in stem elongation and plant height were observed in the OE-miR390b plants. According to transcriptome sequencing results and transient co-expression analysis, TAS3.1 and TAS3.2 were identified as the target genes of miR390 in poplar and were negatively regulated by miR390 in the apex. The transcription levels of ARF3.2 and ARF4 were significantly repressed in OE-miR390b plants and strongly negatively correlated with the number of xylem fiber cells along the longitudinal axis. These findings indicate that the conserved miR390-TAS3-ARFs pathway in poplar is involved in stem elongation and plant height growth.

Structural variation and parallel evolution of apomixis in citrus during domestication and diversification.

Apomixis, or asexual seed formation, is prevalent in Citrinae via 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 Citrinae accessions. 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 Citrinae diversification. 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 FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus and 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 Citrinae and has potential ramifications for citrus breeding.

Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification.

Although interactions between the cytoplasmic and nuclear genomes occurred during diversification of many plants, the evolutionary conflicts due to cytonuclear interactions are poorly understood in crop breeding. Here, we constructed a pan-mitogenome and identified chimeric open reading frames (ORFs) generated by extensive structural variations (SVs). Meanwhile, short reads from 184 accessions of citrus species were combined to construct three variation maps for the nuclear, mitochondrial, and chloroplast genomes. The population genomic data showed discordant topologies between the cytoplasmic and nuclear genomes because of differences in mutation rates and levels of heteroplasmy from paternal leakage. An analysis of species-specific SVs indicated that mitochondrial heteroplasmy was common and that chloroplast heteroplasmy was undetectable. Interestingly, we found a prominent divergence in the mitogenomes and the highest genetic load in the, which may provide the basis for cytoplasmic male sterility (CMS) and thus influence the reshuffling of the cytoplasmic and nuclear genomes during hybridization. Using cytoplasmic replacement experiments, we identified a type of species-specific CMS in mandarin related to two chimeric mitochondrial genes. Our analyses indicate that cytoplasmic genomes from mandarin have rarely been maintained in hybrids and that paternal leakage produced very low levels of mitochondrial heteroplasmy in mandarin. A genome-wide association study (GWAS) provided evidence for three nuclear genes that encode pentatricopeptide repeat (PPR) proteins contributing to the cytonuclear interactions in the Citrus genus. Our study demonstrates the occurrence of evolutionary conflicts between cytoplasmic and nuclear genomes in citrus and has important implications for genetics and breeding.

Acetylome reprograming participates in the establishment of fruit metabolism during polyploidization in citrus.

Polyploidization leads to novel phenotypes and is a major force in evolution. However, the relationship between the evolution of new traits and variations in the post-translational modifications (PTM) of proteins during polyploidization has not been studied. Acetylation of lysine residues is a common protein PTM that plays a critical regulatory role in central metabolism. To test whether changes in metabolism in citrus fruit is associated with the reprogramming of lysine acetylation (Kac) in non-histone proteins during allotetraploidization, we performed a global acetylome analysis of fruits from a synthetic allotetraploid citrus and its diploid parents. A total of 4,175 Kac sites were identified on 1,640 proteins involved in a wide range of fruit traits. In the allotetraploid, parental dominance (i.e. resemblance to one of the two parents) in specific fruit traits, such as fruit acidity and flavonol metabolism, was highly associated with parental Kac level dominance in pertinent enzymes. This association is due to Kac-mediated regulation of enzyme activity. Moreover, protein Kac probably contributes to the discordance between the transcriptomic and proteomic variations during allotetraploidization. The acetylome reprogramming can be partially explained by the expression pattern of several lysine deacetylases (KDACs). Overexpression of silent information regulator 2 (CgSRT2) and histone deacetylase 8 (CgHDA8) diverted metabolic flux from primary metabolism to secondary metabolism and partially restored a metabolic status to the allotetraploid, which expressed attenuated levels of CgSRT2 and CgHDA8. Additionally, KDAC inhibitor treatment greatly altered metabolism in citrus fruit. Collectively, these findings reveal the important role of acetylome reprogramming in trait evolution during polyploidization.

miR156 regulates somatic embryogenesis by modulating starch accumulation in citrus.

Somatic embryogenesis (SE) is a major regeneration approach for in vitro cultured tissues of plants, including citrus. However, SE capability is difficult to maintain, and recalcitrance to SE has become a major obstacle to plant biotechnology. We previously reported that miR156-SPL modules regulate SE in citrus callus. However, the downstream regulatory pathway of the miR156-SPL module in SE remains unclear. In this study, we found that transcription factors CsAGL15 and CsFUS3 bind to the CsMIR156A promoter and activate its expression. Suppression of csi-miR156a function leads to up-regulation of four target genes, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (CsSPL) genes, and reduction of SE efficiency. In the short tandem target mimic (STTM)-miR156a overexpression callus (MIM156), the number of amyloplasts and starch content were significantly reduced, and genes involved in starch synthesis and transport were down-regulated. csi-miR172d was down-regulated, whereas the target genes, CsTOE1.1 and CsTOE1.2, which inhibit the expression of starch biosynthesis genes, were up-regulated. In our working model, CsAGL15 and CsFUS3 activate csi-miR156a, which represses CsSPLs and further regulates csi-miR172d and CsTOEs, thus altering starch accumulation in callus cells and regulating SE in citrus. This study elucidates the pathway of miR156-SPLs and miR172-TOEs-mediated regulation of SE, and provides new insights into enhancing SE capability in citrus.

miR171 modulates induction of somatic embryogenesis in citrus callus.

KEY MESSAGE: Overexpression of miR171 restored SE competence in the recalcitrant citrus callus, and inhibition of miR171 function weakened SE competence in the strongly embryogenic citrus callus. Somatic embryogenesis (SE) is an important way of in vitro regeneration for plants. For perennial woody crops such as citrus, embryogenic callus is usually induced from unfertilized aborted ovules and widely used in biotechnology aided breeding. However, SE capacity always declines in callus during subculture, which makes regeneration difficult and hinders the application of biotechnology. We previously found that miR171 may be a regulator of SE in citrus, based on the abundant expression of csi-miR171c in the embryogenic callus and during SE of citrus. Here, we report that miR171 promotes SE and is required for SE in citrus. Overexpression of miR171 restored SE competence in the recalcitrant callus of 'Guoqing No.1' Satsuma mandarin (G1), whereas inhibition of miR171 function by Short Tandem Target Mimic (STTM) weakened SE competence in the strongly embryogenic callus of 'Valencia' sweet orange (V). The comparative transcriptomic analysis in miR171 overexpressed callus line (OE) and the wild type callus (WT) indicated that overexpression of miR171 decreased the expression level of its SCARECROW-LIKE (CsSCL) targets, and activated stress response related biological processes and metabolic processes that are required for cell differentiation. However, CsSCLs were up-regulated in the OE callus during SE induction process, which activated the cell division and developmental processes that are required for embryogenesis progress. Our results validate the function of miR171 in regulation of SE and reveal the biological responses provoked by miR171 in citrus that may promote SE.

Low-Crystalline AuCuIn Catalyst for Gaseous CO2 Electrolyzer.

Despite its importance for the establishment of a carbon-neutral society, the electrochemical reduction of CO2  to value-added products has not been commercialized yet because of its sluggish kinetics and low selectivity. The present work reports the fabrication of a low-crystalline trimetallic (AuCuIn) CO2  electroreduction catalyst and demonstrates its high performance in a gaseous CO2  electrolyzer. The high Faradaic efficiency (FE) of CO formation observed at a low overpotential in a half-cell test is ascribed to the controlled crystallinity and composition of this catalyst as well as to its faster charge transfer, downshifted d-band center, and low oxophilicity. The gaseous CO2  electrolyzer with the optimal catalyst as the cathode exhibits superior cell performance with a high CO FE and production rate, outperforming state-of-the-art analogs. Thus, the obtained results pave the way to the commercialization of CO2  electrolyzers and promote the establishment of a greener society.

Variation burst during dedifferentiation and increased CHH-type DNA methylation after 30 years of in vitro culture of sweet orange.

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 vitro culture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitro and 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 vitro culture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30 years of in vitro culture. 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 vitro variation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.

Genomic and transcriptomic analyses of Citrus sinensis varieties provide insights into Valencia orange fruit mastication trait formation.

Valencia orange (Citrus sinensis Osbeck) (VO) is a type of late-ripening sweet orange whose ripening occurs 4 to 5 months later than that of the mid-ripening common sweet orange (CO). Notably, the mastication trait of VO fruit is inferior to that of CO fruit. To date, how inferior pulp mastication trait forms in VO has not been determined. In this study, 13 VO varieties and 12 CO varieties were subjected to whole-genome resequencing. A total of 2.98 million SNPs were identified from 25 varieties, and a SNP molecular marker was developed to distinguish VO and CO. Moreover, 144 and 141 genes identified by selective sweep analysis were selected during VO and CO evolution, respectively. Based on gene functional enrichment analysis, most of the selected VO genes were related to the stress response and lignin biosynthesis. Simultaneously, we comparatively analyzed the transcriptome profiles of peel and pulp tissues among three VO varieties and three CO varieties, and the results demonstrated differences in lignin biosynthesis between VO and CO fruits. Furthermore, coexpression network analysis was performed to identify hub genes of lignin-related and variety-specific networks, which included CsERF74, CsNAC25, CsHSFB3, CsSPL4/13, etc. Overall, this study provides important insights into the mastication trait formation of Valencia orange fruit.

Transcriptomes and DNA methylomes in apomictic cells delineate nucellar embryogenesis initiation in citrus.

Citrus nucellar poly-embryony (NPE) is a mode of sporophytic apomixis that asexual embryos formed in the seed through adventitious embryogenesis from the somatic nucellar cells. NPE allows clonal propagation of rootstocks, but it impedes citrus cross breeding. To understand the cellular processes involved in NPE initiation, we profiled the transcriptomes and DNA methylomes in laser microdissection captured citrus apomictic cells. In apomictic cells, ribosome biogenesis and protein degradation were activated, whereas auxin polar transport was repressed. Reactive oxygen species (ROS) accumulated in the poly-embryonic ovules, and response to oxidative stress was provoked. The global DNA methylation level, especially that of CHH context, was decreased, whereas the methylation level of the NPE-controlling key gene CitRWP was increased. A C2H2 domain-containing transcription factor gene and CitRWP co-expressed specifically in apomictic cells may coordinate to initiate NPE. The activated embryogenic development and callose deposition processes indicated embryogenic fate of nucellar embryo initial (NEI) cells. In our working model for citrus NPE initiation, DNA hyper-methylation may activate transcription of CitRWP, which increases C2H2 expression and ROS accumulation, triggers epigenetic regulation and regulates cell fate transition and NEI cell identity in the apomictic cells.

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.