Jiangli Shi

Expansins are crucial cell wall-loosening proteins that play a vital role in various plant developmental processes, including fruit ripening and softening. However, a comprehensive genome-wide analysis of the expansin family in apple (Malus × domestica) and the specific functions of its members in postharvest fruit ripening remain to be explored. In this study, we identified 51 expansin genes in the apple genome and classified them into four subfamilies (EXPA, EXPB, EXLA, and EXLB). Cis-element analysis of the promoters of apple expansin genes showed that these promoters are rich in various hormone-responsive elements, including abscisic acid (ABA)-responsive elements (ABREs) and ethylene-responsive elements (EREs), suggesting potential hormonal regulation of expansin genes. Expression profiling identified six ripening-associated expansin genes. Among them, MdEXPA5, MdEXPA17, and MdEXPA23 were positively regulated by both ethylene and ABA, while being suppressed by the ethylene action inhibitor 1-MCP. Further functional characterization demonstrated that transient overexpression of MdEXPA17 accelerated fruit softening, skin yellowing, ethylene production, and increased total soluble solid (TSS) content. Conversely, silencing of MdEXPA17 significantly delayed these ripening processes. Our study provides a systematic overview of the apple expansin gene family and supports a role for MdEXPA17 in promoting postharvest fruit ripening and softening. These findings offer valuable insights into the molecular mechanisms of apple fruit ripening and provide potential targets for genetic improvement of fruit quality and shelf life.

Plant seed number depends on ovule number initiated within the carpels, and it serves as a primary factor shaping fruit yield. Pomegranate trees exhibit bisexual flowers and functional male flowers. Pomegranate have anatropous ovules which are bitegmic and crassinucellate. Bisexual flowers possess the fertile pistil, while functional male flowers have abnormally developed ovules, a small ovary with few chambers, and a short style. The formation of functional male flowers is due to abnormal and stagnant development of ovule integument. Ovule number directly determines the yield of pomegranate seeds. Recent studies have highlighted the molecular mechanisms through which ovule-related genes regulate pomegranate ovule development. Pomegranate PgCRC and PgINO genes positively regulate the increase in the number of ovules, and PgBEL1 to synergistically regulate seed development. PgAGL11 (the SEEDSTICK orthologous gene) promotes ovule development in transgenic Arabidopsis. PgSEP protein can bridge interactions among PgBEL1, PgSTK and PgAG, which regulate ovule development. At the level of post-transcriptional regulation, PgmiRNA167, PgmiRNA164 and PgmiRNA160 are differentially expressed during pomegranate flower development, and PgmiR166a interacts with its target genes to affect ovule development. This review summarizes the key regulators of ovule development and their molecular pathways, integrating these interactions into a model that describes pomegranate ovule development.

Single–nucleus RNA sequencing (snRNA–seq) provides a powerful tool to profile cell–type–specific transcriptional programs in woody fruit trees. Yet its application in Malus domestica has been limited by heterogeneous reference genomes and the lack of validated marker genes. Here, we first established a comprehensive set of marker genes corresponding to 29 plant cell types and identified their orthologs in apple. Using these resources, we systematically evaluated six publicly available apple genome assemblies as references for snRNA–seq analysis. Our results demonstrate that reference genome selection has a major impact on mapping performance and downstream cell type annotation, with the GDDH13 assembly consistently producing the highest transcript counts per nucleus and the greatest number of identifiable transcriptional clusters (15 clusters representing 11 distinct cell types across 35,557 high–quality nuclei). To achieve robust cell type annotation, we integrated classical marker gene–based annotation with two cross–species computational frameworks—Orthologous Marker Gene Groups (OMGs) and XSpeciesSpanner. These three approaches enabled confident identification of 11 major cell types in apple seedlings and provided cross-validation for ambiguous or previously unannotated clusters. Pseudotime analysis reconstructed the developmental trajectories of both procambial and stomatal lineages, revealing their progressive transcriptional transitions and enabling the delineation of key molecular programs underlying vascular differentiation and early guard-cell formation. Together, this work provides a reference–quality single–nucleus transcriptomic atlas for apple and underlined the critical impact of genome choice and multi–method cell-type annotation on single–cell studies in non–model crops.

Ascorbate oxidases (AAOs) are key regulators of extracellular redox homeostasis and plant stress responses, but their roles in grapevine defense remain unclear. Here, we performed a genome-wide analysis and characterization of the AAO gene family in grapevine Vitis amurensis, identifying 10 VaAAO genes that are unevenly distributed across six chromosomes, with notable clustering on chromosome 7. Promoter analysis revealed multiple phytohormone- and stress-responsive cis-elements (e.g., ARE, STRE, and TCA-element) and transcription factor binding sites (e.g., MYC/MYB, and WRKY), suggesting involvement in redox- and stress-related signaling pathways. Analysis of previously published transcriptomic data under Botrytis cinerea infection identified VaAAO7 as a key pathogen-responsive gene. VaAAO7 was rapidly induced by H₂O₂, and its transient ectopic overexpression in susceptible V. vinifera ‘Red Globe’ leaves significantly reduced lesion development. Together, these results demonstrate that VaAAO7 functions as a positive regulator of B. cinerea resistance and highlight its potential for genetic engineering to enhance systemic defense and develop disease-resistant grapevine cultivars.

Laccases are the key enzymes responsible for plant lignin biosynthesis and responses to environment stress. However, the roles of LAC genes in plant disease resistance are still largely unknown, especially in grapevine, one of the most important horticultural crops in the world. Its quality and yield are very vulnerable to gray mold disease caused by Botrytis cinerea. In total, 30 VvLAC genes were identified and found to be unevenly distributed on seven chromosomes; they were classified into seven groups based on phylogenetic analysis according to the criteria applied in Arabidopsis thaliana. Collinearity and synteny analyses identified some orthologous gene pairs in Vitis vinifera and a few paralogous gene pairs among grape and peach. The VvLAC gene family has diverse gene structures and a highly conserved motif composition. The prominent presence of the MYB cis-elements in each VvLAC promoter highlighted MYB transcriptional factors as the main regulators of VvLAC genes. Furthermore, twenty-five VvLAC genes with functional redundancy are probably implicated in grape lignin biosynthesis. The expression patterns of the LAC genes in grape leaves of Chinese wild V. amurensis ‘Shuangyou’ (SY), a germplasm highly resistant to B. cinerea, were investigated through transcriptomic data and qRT-PCR verification. Combined with the phylogenetic analysis, with AtLACs participating in lignin metabolism, and the cis-element analysis, VaLAC14, VaLAC19, VaLAC24 and VaLAC30 were identified as key candidate genes for lignin biosynthesis in the grape response to B. cinerea. This study supplies a comprehensive understanding of the classification, evolution, structure and responses of the grape LAC genes against B. cinerea. It also provides valuable genetic resources for functional characterization towards enhancing grapevine disease resistance.

The interaction between miRNAs (microRNAs) and target genes plays an important role in plant pistil development. MiRNAs related to pistils were explored in pomegranate. The differentially expressed miRNAs were screened at different developmental stages of pomegranate pistils, and their target differentially expressed mRNAs were further identified to clarify the regulatory effect of miRNAs on pistil development. In our study, 61 conserved miRNAs were identified in 30 families, including miR395, miR394, miR393, miR161, miR162, and miR168. Among them, miR156, miR157, miR159, miR160, miR164, miR165, miR166, miR167, miR169, and miR172 were involved in the development of flower organs. Eight miRNAs were randomly selected and verified for qRT-PCR analysis. The result analysis indicated that miR160, miR164, and miR172 might be positive factors in the regulation of pomegranate pistil development. MiR156 and miR166 might be involved in regulation of pomegranate pistil development as negative factors.

Pomegranate fruit production and consumption are restricted by appropriate postharvest handling practices. 1–MCP (1–methylcyclopropene) is a natural preservative of fruits and vegetables; however, its effects on the storage of different pomegranate varieties have not been extensively investigated. Herein, the effects of 1.0 μL L⁻¹ 1–MCP on postharvest pomegranate fruit of three soft-seed ‘Mollar’, ‘Malisi’, and ‘Tunisan soft seed’ and two semi-soft-seed ‘Moyuruanzi’ and ‘Dongyan’ were investigated over 90 d (days) under low-temperature storage at 4 ± 0.5 °C with a relative humidity of 85–90%. Several indexes of exterior and interior quality were recorded, the sensory quality was evaluated, and the respiration and ethylene production were also determined. The results showed that peel browning was generally more severe in the soft-seed varieties than in the semi-soft-seed varieties. Significantly lighter peel browning presented in the three soft-seed fruits from 45 d after the 1–MCP treatment, with 35%, 19%, and 28% less than those controls at 90 d, correspondingly. However, 1–MCP only significantly decreased peel browning in the semi soft-seed fruits at 60 days. A prominent decrease in weight loss was recorded in all five varieties, with ‘Malisi’ showing the largest and ‘Dongyan’ the smallest difference between the 1–MCP and control treatments. Through the results of color, physiological, and chemical changes, as well as sensory properties, better color and total acceptance were found with higher titratable acids and vitamin C but with decreased anthocyanins in most fruits treated with 1–MCP. In contrast to the control, remarkable suppression of ethylene production peaks in all whole fruits and periodical increase in respiration rates in the soft-seed whole fruits were activated at 30–60 d after storage by the 1–MCP treatment, roughly when peel browning occurred and began increasing. Overall, our findings provided a crucial foundation for extending the application of 1–MCP in postharvest preservation of pomegranates.

Laccase (LAC) is the key enzyme responsible for lignin biosynthesis. Here, 57 PgLACs from pomegranate were identified and distributed on eight chromosomes and one unplaced scaffold. They were divided into six groups containing three typical Cu-oxidase domains. Totally, 51 cis-acting elements in the promoter region of PgLACs are involved in response to ABA, GA, light, stress, etc., indicating diverse functions of PgLACs. The expression profiles of 13 PgLACs during the seed development stage showed that most PgLACs expressed at a higher level earlier than at the later seed development stage in two pomegranate cultivars except PgLAC4. Also, PgLAC1/6/7/16 expressed at a significantly higher level in soft-seed ‘Tunisia’; on the contrary, PgLAC37 and PgLAC50 with a significantly higher expression in hard-seed ‘Taishanhong’. Combined with their distinguishing cis-acting elements, it was concluded that PgLAC1/6/7 may respond to GA via TATC-box and GARE-motif, and PgLAC16 repressed the promotor activity of embryo mid-maturation genes via RY-element so as to contribute to softer seed formation, whereas PgLAC37/50 may participate in seed formation and accelerate seed maturity via ABRE and G-box elements. Collectively, the dramatic gene expressions of PgLAC1/6/7/16/37/50 will provide valuable information to explore the formation of soft- and hard-seed in pomegranate.