Alternative splicing of the PECTINESTERASE gene encoding a cell wall-degrading enzyme affects postharvest softening in grape

The firmness of table grape berries is a crucial quality parameter. Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive. To enhance our comprehension of the underlying molecular factors, we initially identified differentially expressed genes(DEGs) by comparing the transcriptomes of folic acid(FA)-treated and water-treated(CK) berries at different time points. We then analyzed the sequences to detect alternatively spliced(AS) genes associated with postharvest softening. A total of 2,559 DEGs were identified and categorized into four subclusters based on their expression patterns, with subcluster-4 genes exhibiting higher expression in the CK group compared with the FA treatment group. There were 1,045 AS-associated genes specific to FA-treated berries and 1,042 in the CK-treated berries, respectively. Gene Ontology(GO) annotation indicated that the AS-associated genes in CK-treated berries were predominantly enriched in cell wall metabolic processes,particularly cell wall degradation processes. Through a comparison between treatment-associated AS genes and subcluster-4 DEGs, we identified eight genes, including Pectinesterase 2(VvPE2, Vitvi15g00704), which encodes a cell wall-degrading enzyme and was predicted to undergo an A3SS event. The reverse transcription polymerase chain reaction further confirmed the presence of a truncated transcript variant of VvPE2 in the FA-treated berries.Our study provides a comprehensive analysis of AS events in postharvest grape berries using transcriptome sequencing and underscores the pivotal role of VvPE2 during the postharvest storage of grape berries.

Identification,characterization,and verification of miR399 target gene in grape

The microRNA miR399 plays an important role in phosphorus signal transduction pathways in plants.Previously,miR399 was shown to be closely associated with berry ripening in grape(Vitis vinifera).The objective of the present study was to elucidate the evolutionary characteristics of the miR399 gene family in grape and to verify the cleavage effect on the target genes.Grape miR399s were identified by miRNA sequencing and retrieval from the miRBase database.The mature sequences and precursor sequences were subjected to phylogenetic analysis to reconstruct evolutionary trees,as well as secondary structure analysis of the precursor sequence,and prediction of target genes.The cisacting elements in the miR399 promoter were predicted and the cleavage effect of grape miR399b on its target genes was verified.The grape miR399 family comprised nine precursor sequences and nine mature sequences.The precursor sequences formed a typical and stable stem—loop structure.The minimum folding free energy ranged from-55.70 kcal·mol^(-1)to-37.40 kcal·mol^(-1).Multiple sequence alignment revealed that the miR399 family was highly conserved.The grape miR399 family was phylogenetically closely related to peach,apple,and citrus miR399s.Grape miR399s were predicted to target inorganic phosphate transporter 1—3,phospholipase D delta-like,and beta-glucuronosyltransferase.The cleavage effect of grape miR399b on the target genes was verified by means of a dual-luciferase assay and 5’RLM-RACE.Histochemical GUS staining showed that the promoter activity of miR399b was promoted by GA3treatment.

The PyPIF5-PymiR156a-PySPL9-PyMYB114/ MYB10 module regulates light-induced anthocyanin biosynthesis in red pear

Some cultivars of pear(Pyrus L.)show attractive red fruit skin due to anthocyanin accumulation.This pigmentation can be affected by environmental conditions,especially light.To explore the light-induced regulation network for anthocyanin biosynthesis and fruit coloration in pear,small RNA libraries and mRNA libraries from fruit skins of‘Yunhongyihao’pear were constructed to compare the difference between bagging and debagging treatments.Analysis of RNA-seq of fruit skins with limited light(bagged)and exposed to light(debagged),showed that PyPIF5 was down-regulated after bag removal.PymiR156a was also differentially expressed between bagged and debagged fruit skins.We found that PyPIF5 negatively regulated PymiR156a expression in bagged fruits by directly binding to the G-box motif in its promoter.In addition,PymiR156a overexpression promoted anthocyanin accumulation in both pear skin and apple calli.We confirmed that PymiR156a mediated the cleavage of PySPL9,and that the target PySPL9 protein could form heterodimers with two key anthocyanin regulators(PyMYB114/PyMYB10).We proposed a new module of PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10.When the bagged fruits were re-exposed to light,PyPIF5 was down-regulated and its inhibitory effect on PymiR156a was weakened,which leads to degradation of the target PySPL,thus eliminating the blocking effect of PySPL on the formation of the regulatory MYB complexes.Ultimately,this promotes anthocyanin biosynthesis in pear skin.