RNA-Seq-based analysis of transcriptomic signatures elicited by mutations conferring salt tolerance in Cucurbita pepo

• Pairwise comparisons of transcriptomes of wild-type and salt-tolerant mutants reveal the molecular mechanisms behind salt tolerance in squash. • Brassinosteroid, ABA, ethylene, and JA biosynthesis genes like CpBZR1, CpTCH4–1, CpTCH4–2, CpCYP707A1, CpACO1–2 , and CpLOX3 were key regulators of salt tolerance in Cucurbita . • Salt-tolerance-associated DEGs were significantly enriched in plant hormone, MAPK signalling, carotenoid biosynthesis and starch/sucrose metabolism pathways. • Squash response to salt stress involves regulation of several transcription factors of the ERF, C3H, MYB, HSF, NAC, knotted and WRKY families, along with several Lncr loci. Salinity is a major determinant of plant growth and crop productivity, resulting in significant economic losses in agriculture. Improving salinity tolerance in plant breeding programs requires not only donor tolerant genotypes but also a thorough knowledge of the genes controlling the trait. Taking advantage of two recently identified salinity-tolerant EMS mutants of squash ( sal-1 and sal-2 ), this study aimed to analyse whether these two sources of salt tolerance are associated with similar transcriptomic changes in leaves. RNA sequencing revealed that the two mutants have a very distinct transcriptomic response to salt stress compared to the WT, with 154 and 1068 salt-tolerance-associated differentially expressed genes (DEGs) in sal-1 and sal-2 , respectively. GO and KEGG enrichment analyses revealed the importance of several phytohormone biosynthesis, signalling and transport genes ( CpAUX22B/22D, CpSAUR32–2, CpARR5/12, CpAHK2/3, CpBZR1, CpTCH4, CpNCED1, CpCYP707A1, CpPP2C, CpSnRK1/2, CpLOX2 and CpACX ) in the salt tolerance response. MAPK genes ( CpMPK3 and CpMEKK1 ) and the Ca²⁺ signalling network ( CpCPK26/28/34, CpCML31/36/48, CpPBP1, CpCBL1 and CpRBOHD ) were also specifically activated in salt-tolerant mutants, indicating their contribution to salt tolerance. Genes for antioxidant enzymes (PP2, POD, CAT, PRX, GST and GRX) and cell wall metabolism were also up-regulated in salt-tolerant mutants, reducing oxidative stress and maintaining the integrity of membranes and other cellular structures. Genes for ion transporters were significantly up-regulated in response to salt stress in sal-2 , probably involved in maintaining ion homeostasis. Several genes encoding transcription factors of the ERF, C3H, Dof, HD-ZIP, MYB, HSF, NAC, knotted and WRKY families, as well as long non-coding RNA, were also found to positively or negatively regulate salt stress tolerance in the sal-1 and sal-2 mutants. Overall, the results highlight the complexity of the molecular response involved in salt stress tolerance in C. pepo and prioritise further investigation of specific genes that contribute to the resilience of crops under saline conditions.