Emails: 320383@studenti.unimore.it, cristian.forestan@unibo.it, pasquale.devita@crea.gov.it, salvatore.esposito@crea.gov.it, nicola.pecchioni@crea.gov.it

The CCT (CO, COL, and TOC1) domain-containing gene family functions as a pivotal regulator in fundamental biological processes, encompassing the intricate regulation of flowering, the synchronization of circadian rhythms, and defense against abiotic stresses. In particular, the CCT gene family includes two key players, VERNALIZATION2 (VRN2) and PHOTOPERIOD1 (PPD1), indispensable for governing the timing of flowering in response to environmental cues.

However, to date, no comprehensive genome-wide analysis of the CCT gene family in durum wheat has been conducted. Hence, using the recent high-quality Svevo reference, we identified 81 TtCCT candidate genes unevenly distributed across 13 chromosomes, except chromosome 3A, lacking any candidate. Phylogenetic analysis delineated four distinct subfamilies, namely, COL (34), CMF (29), PRR (10), and ZIM (8).

The exon-intron distribution and protein motifs demonstrated remarkably similar compositions within the same subfamily. Twelve TtCCTs, all belonging to the CMF subfamily arose from tandem duplications, while thirty-two TtCCTs underwent whole-genome duplications (WGD)/segmental events, leading to three duplicated gene pairs within the CMF subfamily, ten in COL, two in ZIM and one in PRR.

Despite the number of duplicated TtCCTs, the Ka/Ks ratio indicated that they were under purifying selection, confirming the important role of this gene family. Furthermore, the promoter survey highlighted cis-regulatory elements (CREs) involved in different mechanisms including phytohormone signaling, plant development, and abiotic/biotic stress responses, suggesting that TtCCTs might be involved in various plant processes.

Using RNAseq data from 30 samples representing different tissues and developmental stages, two clusters (I and II) were identified. Cluster I peaked during the spike development and pistil morphogenesis, whereas Cluster II was induced during (early) seed development. Interestingly, among the putative TtCCT targets predicted through the gene regulatory network (GRN) approach, two common genes interconnecting clusters I and II were identified as potential switchers, probably enabling the activation of Cluster II through the targets regulated by Cluster I.

This underscores the fine-tuning regulation of durum wheat development stages, where TtCCT genes might be involved. Our findings offer an in-depth insight into the functions of CCT genes in durum wheat, hypothesize their molecular mechanisms, and establish a foundation for further functional characterization of CCT genes in durum wheat.