Chengdong Ma

Japanese apricot (Prunus mume Sieb. et Zucc.) is a dicotyledonous plant from the Rosaceae family that originated in China. Functional genomic studies in Japanese apricot are essential to elucidate the molecular mechanisms underlying key agronomic traits and to accelerate crop improvement. However, the lack of an efficient genetic transformation system has hindered gene function analysis and impeded molecular breeding efforts. Agrobacterium rhizogenes-mediated transformation has emerged as a robust tool for functional gene validation and studying root-specific processes across diverse plant species, due to its simple protocol and rapid turnaround time. Notably, Agrobacterium-mediated transformation remains notoriously recalcitrant in Rosaceae species, particularly in Japanese apricot. Through screening of ten Japanese apricot varieties, we identified ‘Muguamei’ (MGM) as the optimal cultivar for tissue culture. Using its genotype, we established an Agrobacterium rhizogenes-mediated transformation system for Japanese apricot via an in vitro approach. The binary vector incorporated the RUBY reporter for visual selection and eYGFPuv for fluorescent validation of transformation events. Furthermore, CRISPR/Cas9-mediated knockout of PmPDS in ‘Muguamei’ calli generated albino phenotypes, confirming successful genome editing. Through optimization of antibiotics, the study achieved an 80% explant survival rate using Woody Plant Medium (WPM) supplemented with 6-BA (0.5 mg/L) and TDZ (0.05 mg/L). For in vitro micropropagation, we found that ‘Muguamei’ exhibited optimal shoot growth in the presence of 6-BA (0.06 mg/L) and TDZ (0.1 mg/L), and up to 8 bud proliferation lines could be reached under 4.0 mg/L 6-BA. During the rooting of micro shoots, ½MS medium performed better and reached the optimum root length (35.70 ± 4.56 mm) and number (6.00 ± 1.00) under IAA (0.5 mg/L) and IBA (0.4 mg/L). Leaf explants were cultured on WPM supplemented with TDZ (4.0 mg/L) and NAA (0.2 mg/L). 50 mg/L kanamycin concentrations were the suitable screening concentration.

ALMT (aluminum-activated malate transporter) proteins play crucial roles in the transport of organic acids and have significant implications for plant stress responses and development. While extensively studied in some plants, the characteristics and functional divergence of the ALMT gene family have not yet been thoroughly explored in Prunus mume and some other Rosaceae species. In this study, we systematically analyzed the ALMT gene family across nine Rosaceae species to explore their evolutionary relationships, structural characteristics, and functional roles. A total of 138 ALMT genes were identified and categorized into four groups based on a phylogenetic analysis. The motif analysis confirmed the accuracy of the phylogenetic grouping. The collinearity analysis indicated that whole-genome duplication events were the primary drivers of ALMT gene expansion in these species. Furthermore, the cis-acting element analysis revealed diverse regulatory elements associated with environmental responses, including abscisic acid, light, and jasmonic acid. The gene expression correlation analysis showed that PmALMT1 is primarily associated with malic acid accumulation, whereas PmALMT8 is related to citric acid accumulation. Further transient expression in Nicotiana benthamiana validated the above conclusion. This comprehensive analysis provides valuable insights into the evolution, function, and regulation of the ALMT gene family in Rosaceae species.

The formation of multi-pistil flowers reduces the yield and quality in Japanese apricot (Prunus mume). However, the molecular mechanism underlying the formation of multi-pistil flowers remains unknown. In the current study, overexpression of PmKNAT2/6-a, a class I KNOTTED1-like homeobox (KNOX) member, in Arabidopsis (Arabidopsis thaliana) resulted in a multi-pistil phenotype. Analysis of the upstream regulators of PmKNAT2/6-a showed that AGAMOUS-like 24 (PmAGL24) could directly bind to the PmKNAT2/6-a promoter and regulate its expression. PmAGL24 also interacted with Like Heterochromatin Protein 1 (PmLHP1) to recruit lysine trimethylation at position 27 on histone H3 (H3K27me3) to regulate PmKNAT2/6-a expression, which is indirectly involved in multiple pistils formation in Japanese apricot flowers. Our study reveals that the PmAGL24 transcription factor, an upstream regulator of PmKNAT2/6-a, regulates PmKNAT2/6-a expression via direct and indirect pathways and is involved in the formation of multiple pistils in Japanese apricot.

The Knotted1-like Homeobox gene is crucial for plant morphological development and growth. Physicochemical characteristics, phylogenetic relationships, chromosomal localization, cis-acting elements, and tissue-specific expression patterns of the 11 PmKNOX genes found in the Japanese apricot genome in this study were examined. Proteins of 11 PmKNOX were soluble proteins with isoelectric points between 4.29 and 6.53, molecular masses between 15.732 and 44.011 kDa, and amino acid counts between 140 and 430. The identified PmKNOX gene family was split into three subfamilies by jointly constructing the phylogenetic tree of KNOX proteins in Japanese apricot and Arabidopsis thaliana. Combined outcomes of the analyzed conserved motifs and gene structures of the 11 PmKNOX genes from the same subfamily displayed comparable gene structure and motif patterns. The 11 PmKNOX members were distributed across six chromosomes, while two sets of PmKNOX genes were found to be collinear. Analysis of the 2000 bp promoter upstream of the coding region of the PmKNOX gene revealed that most PmKNOX genes might be involved in the physiological metabolism, growth and development processes of plants. The PmKNOX gene expression profile revealed that these genes were expressed at varying levels in different tissues, and most of them were linked to the meristems of leaf and flower buds, suggesting that PmKNOX may be involved in plants’ apical meristems. In Arabidopsis thaliana, functional validation of PmKNAT2a and PmKNAT2b revealed that these two genes might be involved in regulating leaf and stem development. In addition to laying the groundwork for future research on the function of these genes, understanding the evolutionary relationships between members of the PmKNOX gene family provides opportunities for future breeding in Japanese apricots.

NF-Y transcription factor consists of three subsets, A, B, and C, which play various roles during biological processes in plants, particularly in growth and development, stress response, and microorganism interactions. The current study was exhaustively conducted on the NF-Y gene family in Prunus mume (Japanese apricot). We identified 27 PmNF-Y genes that were distributed on eight chromosomes of the Japanese apricot genome, and which were divided into three subgroups according to their phylogenetic relationship. Protein replication analysis showed that the Ka/Ks ratio of one pair of tandem repeats and two pairs of segmental repeats was less than 1. Cis-acting elements upstream of PmNF-Y genes were divided into four distinct categories: light response, growth metabolism, stress, and hormones, among which the numbers of hormone and light-responsive cis-acting elements were higher. RNA-seq analysis revealed that most of the genes were expressed with slight variation in various tissues. Interestingly, some genes were expressed differently during dormancy. During dormancy with exogenous hormones and low temperature, the qRT-PCR results showed that these differentially expressed genes had specific expression responses under GA₄, ABA, MeJA, and low-temperature treatments. Therefore, these findings could provide a novel theoretical foundation for future research into the function and molecular regulation mechanism of the NF-Y gene family in the Prunus species.

Self-incompatibility (SI) is a common strategy to avoid inbreeding and, consequently, keep genetic diversity within a species. In its mechanism, pollen rejection happens in the style when the single multiallelic locus (SFB in prunus species) of the haploid pollen matches one of the S-alleles existing in the diploid pistil. The SFB gene for the pollen S gene has been identified in many Prunus species. However, Japanese apricot is a species with a typical gametophytic self-incompatibility (GSI), and its SFB alleles available are limited, although they are required for studying GSI. Therefore, we used an AS-PCR amplification method, sequencing, and the pair primers SFB-C1F and Pm-Vb designed based on the conserved region of the Prunus SFB gene to identify SFB genotypes of 48 Japanese apricot (P. mume) accessions. Eleven novel SFB alleles were isolated from these accessions and shared typical structural features with SFB alleles from other Prunus species. These novel SFB alleles were uniquely expressed in pollen. Hence, we concluded that these 11 PmSFB were pollen S determinants of P. mume. This current study offers the novel SFB genes of the P. mume S locus, which could be a useful potential resource for studies on pollen SI mechanisms.

Japanese apricot (Prunus mume Sieb. et Zucc.) is among the most valued fruits and flowering plants in eastern Asia. However, few comparative studies have been conducted with respect to its agro-morphological and pomological traits, chloroplast (cp) genome sequences and plastid diversity. Therefore, a comparative study was, conducted to investigate the divergence and geographic distribution of ten Japanese apricot accessions from three Chinese provinces (Zhejiang, Jiangsu and Sichuan). Phenotypic characteristics of the evaluated accessions, such as leaf length, tip leaf length, flower diameter, anther number, fruit weight, longitudinal height, transversal height, lateral height, fruit stone weight, stone longitudinal height, stone transversal height, stone lateral heigh, titratable acid content and total soluble solids, varied significantly (p < 0.05) among the ten investigated accessions. On the other hand, most of the investigated accessions were statistically similar within the same province. Comparing the Cp genomes of P. mume accessions with those of the genus Prunus revealed a similarity in structure and composition with slight differences. “Bayes empirical Bayes” (BEB) analysis in Prunus species, including P. mume, revealed BEB in rps16, rps3, rpoC1(4*), rpl32, rpl16, rbcL, psbF, petB, ndhF, clpP and ccsA genes. The BEB value of the rpoC1 gene is higher than 0.95, indicating that it is potentially under positive selection. Interestingly, the accessions from the same province of origin had the same number of forward repeat sequences. Furthermore, all accessions from Zhejiang province had the same number of simple sequence repeats. Similarly, nucleotide deletion/insertion of the ycf1 sequence and the results of phylogenetic trees revealed that accessions were mainly clustered according to their province of origin. Our comparative study of agronomical traits, chloroplast composition, structure, nucleotide variability of cp genome and phylogeography in Japanese apricot accessions provides valuable information on their diversity and geographic distribution.

Japanese apricot (Prunus mume) is remarkably valuable for its high ornamental and economic importance due to its distinctive features. Low temperature is a serious environmental constraint for this species, restricting its cultivation and dispersal in the north of China. To address this issue, breeding requires an understanding of the molecular mechanisms underlying responses to cold stress. We examined the leaf physiological and transcriptome profile by RNA sequencing in ‘Bungo’ scion cultivar grafted onto Prunus mume (cold-sensitive) and Prunus armeniaca (cold-tolerant) rootstocks at 4 °C for 0, 6, and 24 h. Our results revealed that the increased MDA concentration in the leaves of P. mume cultivar (cold-sensitive) suggests that cold stress might cause oxidative damage and increased sensitivity. Moreover, the cold-tolerant cultivar (P. armeniaca) considerably enhances the enzyme activities (i.e., SOD, POD, and CAT), as well as osmo-protectants (soluble sugars and proline) compared with sensitive cultivar, which helps plants to withstand oxidative damage caused by cold stress. Additionally, differentially expressed genes were shown to be enriched in plant hormone signal transduction, ribosome, MAPK signaling, and circadian rhythm pathway. After 24 h of cold stress, genes related to PYL4, histidine kinase 1, SAUR36, bHLH130, bHLH123, TIFY 6B-like, WRKY 40, WRKY 57, and 60S acidic ribosomal protein P1 were differentially expressed, implying that these DEGs involved in multiple pathways are involved in cold tolerance in Japanese apricot. This study improved our current understanding of the mechanism of cold tolerance in Japanese apricot, and the findings could be utilized for other related fruit species.