Abscisic acid induces PpeKIL1 to terminate fruit growth and promote fruit abortion in peach (Prunus persica).

Abnormal pollination from chance events or hybridization between species leads to unusual embryo development, resulting in fruit abortion. To elucidate the mechanism underlying fruit abortion, we conducted a comprehensive analysis of the transcriptome and hormone profiles in aborting fruits (AF) derived from an interspecific cross between the peach cultivar 'Huangjinmi 3' and the Prunus mume cultivar 'Jiangmei', as well as in normal-seeded fruits (NF) resulting from an intraspecific cross of 'Huangjinmi 3' with the 'Manyuanhong' peach cultivars. Growth of AF was inhibited during the exponential growth phase, with up-regulation of oxidative stress related genes and down-regulation of DNA replication and cell cycle genes. Accumulation of the tissue growth-related hormones auxin and cytokinin was reduced in AF, while levels of the growth inhibiting hormone abscisic acid (ABA) were higher compared to NF. The increased ABA concentration aligned with down-regulation of the ABA catabolism gene CYP707A2, which encodes abscisic acid 8'-hydroxylase. Correlation analysis showed ABA could explain the maximum proportion of differently expressed genes between NF and AF. We also showed that expression of KIRA1-LIKE1 (PpeKIL1), a peach ortholog of the Arabidopsis KIRA1 gene, was up-regulated in AF. PpeKIL1 promotes senescence or delays normal growth in tobacco and Arabidopsis, and its promoter activity increases with exogenous ABA treatment. Our study demonstrates a candidate mechanism where ABA induces expression of PpeKIL1, which further blocks normal fruit growth and triggers fruit abscission.

Discovery of a key gene associated with fruit maturity date and analysis of its regulatory pathway in peach.

Fruit maturity is an important agronomic trait of fruit crops. Although in previous studies, several molecular markers are developed for the trait, the knowledge about its candidate genes is particularly limited. In this study, a total of 357 peach accessions were re-sequenced to obtain 949,638 SNPs. Combing with 3-year fruit maturity dates, a genome-wide association analysis was performed, and 5, 8, and 9 association loci were identified. To screen the candidate genes for those year-stable loci on chromosomes 4 and 5, two maturity date mutants were used for transcriptome sequencing. Gene expression analysis indicated that Prupe.4G186800 and Prupe.4G187100 on chromosome 4 were essential to fruit ripening in peaches. However, the expression analysis of different tissues showed that the first gene has no tissue-specific character, but transgenic studies showed that the latter is more likely to be a key candidate gene than the first for the maturity date in peach. The yeast two-hybrid assay showed that the proteins encoded by the two genes interacted and then regulated fruit ripening. Moreover, the previously identified 9 bp insertion in Prupe.4G186800 may affect their interaction ability. This research is of great significance for understanding the molecular mechanism of peach fruit ripening and developing practical molecular markers in a breeding program.

The Peach (Prunus persica) CBL and CIPK Family Genes: Protein Interaction Profiling and Expression Analysis in Response to Various Abiotic Stresses.

The plant calcineurin B-like protein-CBL interacting protein kinase (CBL-CIPK) signaling pathway is a Ca2+-related signaling pathway that responds strongly to both biological and abiotic environmental stimuli. This study identified eight CBL and eighteen CIPK genes from peach for the first time. Their basic properties and gene structure were analyzed, and the CBL and CIPK members from Arabidopsis and apple were combined to study their evolutionary relationships. Using RT-qPCR and RNA-seq data, we detected the expression patterns of PprCBLs and PprCIPKs in different tissues and fruit development stages of peach. Among them, the expression levels of PprCBL1 and PprCIPK18 were stable in various tissues and stages. The expression patterns of other members showed specificity between cultivars and developmental stages. By treating shoots with drought and salt stress simulated using PEG6000 and NaCl, it was found that PprCIPK3, PprCIPK6, PprCIPK15 and PprCIPK16 were strongly responsive to salt stress, and PprCIPK3, PprCIPK4, PprCIPK10, PprCIPK14, PprCIPK15, PprCIPK16 and PprCIPK18 were sensitive to drought stress. Three genes, PprCIPK3, PprCIPK15 and PprCIPK16, were sensitive to both salt and drought stress. We cloned four PprCBL and several PprCIPK genes and detected their interaction by yeast two-hybrid assay (Y2H). The results of Y2H show not only the evolutionary conservation of the interaction network of CBL-CIPK but also the specificity among different species. In conclusion, CBL and CIPK genes are important in peach and play an important role in the response to various abiotic stresses.

Genome-wide identification and characterization of long noncoding RNAs during peach (Prunus persica) fruit development and ripening.

LncRNAs represent a class of RNA transcripts of more than 200 nucleotides (nt) in length without discernible protein-coding potential. The expression levels of lncRNAs are significantly affected by stress or developmental cues. Recent studies have shown that lncRNAs participate in fruit development and ripening processes in tomato and strawberry; however, in other fleshy fruits, the association between lncRNAs and fruit ripening remains largely elusive. Here, we constructed 9 ssRNA-Seq libraries from three different peach (Prunus persica) fruit developmental stages comprising the first and second exponential stages and the fruit-ripening stage. In total, 1500 confident lncRNAs from 887 loci were obtained according to the bioinformatics analysis. The lncRNAs identified in peach fruits showed distinct characteristics compared with protein-coding mRNAs, including lower expression levels, lower complexity of alternative splicing, shorter isoforms and smaller numbers of exons. Expression analysis identified 575 differentially expressed lncRNAs (DELs) classified into 6 clusters, among which members of Clusters 1, 2, 4 and 5 were putatively associated with fruit development and ripening processes. Quantitative real-time PCR revealed that the DELs indeed had stage-specific expression patterns in peach fruits. GO and KEGG enrichment analysis revealed that DELs might be associated with fruit-ripening-related physiological and metabolic changes, such as flavonoid biosynthesis, fruit texture softening, chlorophyll breakdown and aroma compound accumulation. Finally, the similarity analysis of lncRNAs within different plant species indicated the low sequence conservation of lncRNAs. Our study reports a large number of fruit-expressed lncRNAs and identifies fruit development phase-specific expressed lncRNA members, which highlights their potential functions in fruit development and ripening processes and lays the foundations for future functional research.

Genome-Wide Analysis of the Gene Structure, Expression and Protein Interactions of the Peach (Prunus persica) TIFY Gene Family.

The TIFY family is a plant-specific gene family involved in regulating many plant processes, such as development and growth, defense and stress responses, fertility and reproduction, and the biosynthesis of secondary metabolites. The v2.0 peach (Prunus persica) genome, which has an improved chromosome-scale assembly and contiguity, has recently been released, but a genome-wide investigation of the peach TIFY family is lacking. In this study, 16 TIFY family genes from the peach genome were identified according to the peach reference genome sequence information and further validated by cloning sequencing. The synteny, phylogenetics, location, structure, and conserved domains and motifs of these genes were analyzed, and finally, the peach TIFY family was characterized into 9 JAZ, 1 TIFY, 1 PPD and 5 ZML subfamily members. Expression profiles of peach JAZ, PPD, and ZML genes in various organs and fruit developmental stages were analyzed, and they showed limited effects with fruit ripening cues. Four TIFY members were significantly affected at the mRNA level by exogenous treatment with MeJA in the peach epicarp, and among them, PpJAZ1, PpJAZ4 and PpJAZ5 were significantly correlated with fruit epicarp pigmentation. In addition, the TIFY family member protein interaction networks established by the yeast two-hybrid (Y2H) assay not only showed similar JAZ-MYC2 and JAZ homo- and heterodimer patterns as those found in Arabidopsis but also extended the JAZ dimer network to ZML-ZML and JAZ-ZML interactions. The PpJAZ3-PpZML4 interaction found in this study suggests the potential formation of the ZML-JAZ-MYC complex in the JA-signaling pathway, which may extend our knowledge of this gene family's functions in diverse biological processes.

Diversity of metabolite accumulation patterns in inner and outer seed coats of pomegranate: exploring their relationship with genetic mechanisms of seed coat development.

The expanded outer seed coat and the rigid inner seed coat of pomegranate seeds, both affect the sensory qualities of the fruit and its acceptability to consumers. Pomegranate seeds are also an appealing model for the study of seed coat differentiation and development. We conducted nontarget metabolic profiling to detect metabolites that contribute to the morphological differentiation of the seed coats along with transcriptomic profiling to unravel the genetic mechanisms underlying this process. Comparisons of metabolites in the lignin biosynthetic pathway accumulating in seed coat layers at different developmental stages revealed that monolignols, including coniferyl alcohol and sinapyl alcohol, greatly accumulated in inner seed coats and monolignol glucosides greatly accumulated in outer seed coats. Strong expression of genes involved in monolignol biosynthesis and transport might explain the spatial patterns of biosynthesis and accumulation of these metabolites. Hemicellulose constituents and flavonoids in particular accumulated in the inner seed coat, and candidate genes that might be involved in their accumulation were also identified. Genes encoding transcription factors regulating monolignol, cellulose, and hemicellulose metabolism were chosen by coexpression analysis. These results provide insights into metabolic factors influencing seed coat differentiation and a reference for studying seed coat developmental biology and pomegranate genetic improvement.

Characterization of the plastome of Camellia pingguoensis (Theaceae), an endangered and endemic yellow camellia species in China.

Camellia pingguoensis D. Fang is a shrub which is found on limestone of karst forests in Guangxi, China. In this study, we characterized the whole plastid genome of C. pingguoensis using Illumina paired-end sequencing reads. The plastome is 156,621 bp in length, containing two copies of inverted repeat (IR) regions (26,046 bp), a large-single copy (LSC) region (86,289 bp), and a small-single copy (SSC) region (18,240 bp). A total of 114 unique genes in the genome has 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The phylogenetic result indicates C. pingguoensis is closely related to C. nitidissima C. W. Chi.

The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis.

Pomegranate (Punica granatum L.) is a perennial fruit crop grown since ancient times that has been planted worldwide and is known for its functional metabolites, particularly punicalagins. We have sequenced and assembled the pomegranate genome with 328 Mb anchored into nine pseudo-chromosomes and annotated 29 229 gene models. A Myrtales lineage-specific whole-genome duplication event was detected that occurred in the common ancestor before the divergence of pomegranate and Eucalyptus. Repetitive sequences accounted for 46.1% of the assembled genome. We found that the integument development gene INNER NO OUTER (INO) was under positive selection and potentially contributed to the development of the fleshy outer layer of the seed coat, an edible part of pomegranate fruit. The genes encoding the enzymes for synthesis and degradation of lignin, hemicelluloses and cellulose were also differentially expressed between soft- and hard-seeded varieties, reflecting differences in their accumulation in cultivars differing in seed hardness. Candidate genes for punicalagin biosynthesis were identified and their expression patterns indicated that gallic acid synthesis in tissues could follow different biochemical pathways. The genome sequence of pomegranate provides a valuable resource for the dissection of many biological and biochemical traits and also provides important insights for the acceleration of breeding. Elucidation of the biochemical pathway(s) involved in punicalagin biosynthesis could assist breeding efforts to increase production of this bioactive compound.

Iron deficiency stress can induce MxNRAMP1 protein endocytosis in M. xiaojinensis.

BACKGROUND: Iron deficiency is one of the most common nutritional disorders in plants, especially in fruit trees grown in calcareous soil. Iron deficiency stress can induce a series of adaptive responses in plants, the cellular and molecular mechanisms of which remain unclear. NRAMPs (natural resistance-associated macrophage proteins) play an important role in divalent metal ion transportation. RESULTS: In this study, we cloned MxNRAMP1, an NRAMP family gene from a highly iron-efficient apple genotype, Malus xiaojinensis. Further research showed that iron deficiency stress could induce MxNRAMP1 expression in roots and leaves. A protoplast transient expression system and immune electron microscopy localization techniques were used to prove that MxNRAMP1 mainly exists in the plasma membrane and vesicles. Interestingly, iron deficiency stress could induce the MxNRAMP protein to transport iron ions to specific organelles (lysosome and chloroplast) through vesicle endocytosis. Stable transgenic tobacco showed that MxNRAMP1 over-expression could promote iron absorption and accumulation in plants, and increase the plant's resistance against iron deficiency stress. CONCLUSIONS: These results showed that, in M. xiaojinensis, MxNRAMP1 not only plays an important role in iron absorption and transportation, it can also produce adaptive responses against iron deficiency through endocytosis.

Molecular cloning, polyclonal antibody preparation, and characterization of a functional iron-related transcription factor IRO2 from Malus xiaojinensis.

Transcription factors play important roles in plant growth and responses to environmental stresses. In this study, a novel basic helix-loop-helix iron-related transcription factor, IRO2, containing a 762-bp open reading frame and encoding 253 amino acids, was cloned from the iron-efficient genotype of Malus xiaojinensis. Localization analyses in onion showed that the MxIRO2 protein was targeted to the nucleus and activation studies in yeast indicated MxIRO2-BD had weak transcriptional activation activity. Prokaryotic expression of MxIRO2 in Escherichia coli BL21 (DE3) pLysS cells resulted in high expression levels of the protein when induced with isopropyl-ß-d-thiogalactoside. The fusion protein was purified using Ni-NTA His-bind resin, and the purified MxIRO2-His fusion protein was used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was purified by precipitation with 50% saturated ammonium sulfate and DEAE Sephadex A-50 chromatography to obtain the immunoglobulin G fraction. The expression of MxIRO2 in roots and leaves of M. xiaojinensis seedlings under iron deficiency was determined. The results indicated that MxIRO2 was induced in both roots and leaves under iron deficiency. In these experimental conditions, the transcription and translation levels first increased and then decreased under iron deficiency. This work offers an important basis for further investigating the mechanisms of fruit tree adaptation to iron deficiency.