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Abstract The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. “Helfensteiner” (cross of cv. “Pinot noir” and “Schiava grossa”) instead of a single “Pinot noir”. These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome. 

Summary Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green‐to‐mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood.We investigated the function of the transcriptional regulatorVviNAC33by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP‐seq) approach combined with transcriptomic data.We demonstrated that VviNAC33 induces leaf de‐greening, inhibits organ growth and directly activates the expression ofSTAY‐GREEN PROTEIN 1(SGR1), which is involved in Chl and photosystem degradation, andAUTOPHAGY 8f(ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibitsAUXIN EFFLUX FACILITATOR PIN1,RopGEF1andATP SYNTHASE GAMMA CHAIN 1T(ATPC1), which are involved in photosystem II integrity and activity.Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative‐to‐mature phase transition.