Apple (Malus × domestica Borkh.) is one of the most cultivated fruit crops in China. Apple trees frequently encounter waterlogging stress, mainly due to excess rainfall, soil compaction, or poor soil drainage, results in yellowing leaves and declined fruit quality and yield in some regions. However, the mechanism underlying the response to waterlogging has not been well elucidated. Therefore, we performed a physiological and transcriptomic analysis to examine the differential responses of two apple rootstocks (waterlogging-tolerant M. hupehensis and waterlogging-sensitive M. toringoides) to waterlogging stress. The results showed that M. toringoides displayed more severe leaf chlorosis during the waterlogging treatment than M. hupehensis. Compared with M. hupehensis, the more severe leaf chlorosis induced by waterlogging stress in M. toringoides was highly correlated with increased electrolyte leakage and superoxide radicals, hydrogen peroxide accumulation, and increased stomata closure. Interestingly, M. toringoides also conveyed a higher ethylene production under waterlogging stress. Furthermore, RNA-seq revealed that a total of 13,913 common differentially expressed genes (DEGs) were differentially regulated between M. hupehensis and M. toringoides under waterlogging stress, especially those DEGs involved in the biosynthesis of flavonoids and hormone signaling. This suggests a possible link of flavonoids and hormone signaling to waterlogging tolerance. Taken together, our data provide the targeted genes for further investigation of the functions, as well as for future molecular breeding of waterlogging-tolerant apple rootstocks.

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.

Drought significantly impairs the growth, yield, and quality of apple (Malus domestica Borkh.). Nuclear factor-Y (NF-Y) transcription factors have well-documented functions in regulating stress tolerance in various plants. However, the specific functions and molecular mechanisms of NF-Ys in conferring drought tolerance in apples remain largely unexplored. Here, we identified and characterized a NF-Y transcription factor, MhNF-YA3-like, from Malus hupehensis, the expression of which is upregulated by drought stress and abscisic acid (ABA). Overexpressing MhNF-YA3-like enhanced tolerance to drought stress, while silencing MhNF-YA3-like increased susceptibility to drought stress. Further research demonstrated that MhNF-YA3-like directly binds to the promoter of MhAAO3 and activates its transcription. Notably, the interaction between MhNF-YA3-like and MhMSI4-like further amplified the regulatory effect of MhNF-YA3-like on MhAAO3, promoting the synthesis of ABA, which consequently improved drought stress tolerance. This study provides insights into how the MhNF-YA3-like-MhMSI4-like module finely tunes apple drought tolerance.

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.

Drought stress severely impacts the growth, yield and quality of apple (Malus domestica). Abscisic acid (ABA) and basic helix–loop–helix (bHLH) transcription factors play crucial roles in regulating the drought response in many plants, but the potential interactions between bHLH and ABA in response to drought in apple still need to be discovered. Herein, we identified a bHLH transcription factor, ORG2 (OBP3-responsive gene 2), from M. hupehensis, and the expression of which is induced by drought and ABA. Apple plants that overexpressed MhORG2 were more sensitive to drought stress, while silencing MhORG2 caused the opposite phenotype. Specifically, we found that MhORG2 could directly bind to the DRE element in the MhAAO3 promoter and repress its expression, thereby ultimately reducing drought tolerance. Furthermore, MhORG2 represses the expression of antioxidant enzyme genes (MhSOD, MhAPX1 and MhCAT), leading to the accumulation of reactive oxygen species (ROS) and consequently reducing the drought tolerance of apple plants. Our findings uncover a novel mechanism by which MhORG2 negatively regulates drought tolerance in apple plants, offering a potential target for the development of drought-tolerant crops via biotechnological approaches.

Drought is a major environmental stress that severely affects plant growth and poses a serious threat to crop productivity. Abscisic acid (ABA) is a key plant hormone that plays a critical role in regulating plant responses to drought stress. However, the regulatory pathways controlling ABA for drought tolerance in apple (Malus domestica Borkh) remain largely unclear. This study identified an NF-Y transcription factor, MhNF-YC6, from the drought-tolerant wild apple species (Malus hupehensis). Functional analysis revealed that MhNF-YC6 is a positive regulator in apple drought tolerance. Further research demonstrated that MhNF-YC6 directly binds to the promoters of the ABA biosynthesis genes MhNCED3 and MhAAO3, and activates their transcription. Silencing of MhNCED3 and MhAAO3 resulted in significantly reduced ABA contents and drought tolerance. Furthermore, CONSTANS-like 4 (COL4) is identified as an MhNF-YC6-interacting protein that acts as a positive regulator of drought tolerance in apple. Notably, we discovered that the interaction between MhNF-YC6 and MhCOL4 substantially enhances the regulatory effect of MhNF-YC6 on MhNCED3 and MhAAO3, thereby promoting ABA accumulation, inducing stomatal closure, and ultimately improving drought tolerance. Our findings offer valuable insights into the transcriptional regulation of ABA biosynthesis and provide promising targets for the molecular breeding of drought-tolerant crops.