Peculiarity of transcriptional and H3K27me3 dynamics during peach bud dormancy

Bud dormancy facilitates the survival of meristems under harsh environmental conditions.To elucidate how molecular responses to chilling accumulation controlling dormancy in peach buds,chromatin immunoprecipitation sequencing to identify the H3K27me3 modifications and RNA sequencing of two peach cultivars with pronounced differences in chilling requirement were carried out,the results showed that genes associated with abscisic acid and gibberellic acid signal pathways play key roles in dormancy regulation.The results demonstrated that peach flower bud differentiation occurred continuously in both cultivars during chilling accumulation,which was correlated with the transcript abundance of key genes involved in phytohormone metabolism and flower bud development under adverse conditions.The more increased strength in high chillingrequirement cultivar along with the chilling accumulation at the genome-wide level.The function of the dormancy-associated MADS-box gene PpDAM6 was identified,which is involved in leaf bud break in peach and flower development in transgenic Nicotiana tabacum(NC89).In addition,PpDAM6 was positively regulated by PpCBF,and the genes of putative dormancy-related and associated with metabolic pathways were proposed.Taken together,these results constituted a theoretical basis for elucidating the regulation of peach bud dormancy transition.

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

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 keymetabolic 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.

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 expressedmiRNAs 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 correlatedwith 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.

DR5 is a Suitable System for Studying the Auxin Response in the Poncirus trifoliata-Xanthomonas axonopodis pv.citri Interaction

The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this research.The auxin response maxima could be observed in the apex of the root tip,primary phloem of the tender stem,and the margin of the young leaves according to the activity of theβ-glucuronidase(GUS)reporter gene triggered by the auxin responsive DR5 promoter.Auxin responses in the apex of the root tips increased when treated with synthetic auxin 1-naphthylacetic acid(NAA),but decreased when treated with the auxin polar transportation inhibitor 2,3,5-triiodobenzoic acid(TIBA).These results indicated that the DR5 reporter system worked in P.trifoliata for auxin distribution and response observation.Trifoliate orange is highly susceptible to citrus canker disease.Auxin accumulation was observed visually in the invasion sites of the detached leaves inoculated with Xanthomonas axonopodis pv.citri(Xac)by GUS staining;the upregulated expression of the YFP,GH3.1,GH3.9,and SAUR genes assessed by quantitative real-time PCR(qRT-PCR)also identified auxin accumulation in the inoculated tissues following Xac infection.Overall,these findings indicated that the plantlets of P.trifoliata engineered with the auxin reporter gene provided a promising system for studying auxin responses during Xac infection.

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.

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.

UNREDUCED MEGAGAMETOPHYTE FORMATION VIA SECOND DIVISION RESTITUTION CONTRIBUTES TO TETRAPLOID PRODUCTION IN INTERPLOIDY CROSSES WITH/ORAH/MANDARIN(CITRUS RETICULATA)

Seedless fruits are desirable in the citrus fresh fruit market.Triploid production via diploid x tetraploid interploidy crosses is thought to be the most efficient and widely-used strategy for the breeding of seedless citrus.Although'Orah'mandarin has desirable organoleptic qualities,seeds in the fruits weaken its market competitiveness.To produce new seedless cultivars that are similar to'Orah'mandarin,we performed three 2x x 4x crosses using'Orah'mandarin as the seed parent to regenerate triploid plantlets.A total of 182 triploid and 36 tetraploid plantlets were obtained.By analyzing their genetic origins using nine novel single nucleotide polymorphism(SNP)markers,all of the triploids and tetraploids derived from these three crosses were proven to be hybrids.Also,we demonstrated that 2n megagametophyte formation in'Orah'mandarin result in tetraploid production in these three interploidy crosses.These tetraploid plantlets were genotyped using eight pericentromeric SNP markers and nine centromere distal SNP markers.Based on the genotypes of the 2n megagametophytes,the parental heterozygosity rates in 16 SNP loci and all 2n megagametophytes were less than 50%,indicating that second division restitution was the mechanism underlying 2n megagametophyte formation at both the population and individual levels.These triploid hybrids enrich the germplasm available for seedless breeding.Moreover,the tetraploid hybrids are valuable as parents for ploidy breeding for the production of seedless citrus fruits.