Introduction: Proso millet ( Panicum miliaceum L.) is an important short-day cereal crop whose heading date is highly sensitive to photoperiod, a key factor influencing its yield and geographical adaptation. The CCT (CONSTANS, CONSTANS-LIKE and TOC1) gene family plays a crucial role in regulating plant photoperiod pathway and flowering time. Methods: In this study, a comprehensive genome-wide identification, characterization and expression analysis were performed of the CCT gene family in proso millet. CCT family members were identified, and their phylogenetic relationships, gene structures, conserved motifs, and duplication events were analyzed. Expression patterns were examined using RNA-seq data from leaves and shoot apical meristems under different photoperiod conditions, and further analyzed by qRT‑PCR across proso millet germplasms with varying heading dates. Results: A total of 74 CCT genes ( PmCCT1–PmCCT74 ) were identified and found to be unevenly distributed across the 18 chromosomes. Phylogenetic analysis classified them into four major subfamilies: CONSTANS (COL)-like, CCT MOTIF FAMILY (CMF)-like, pseudo-response regulator (PRR)-like, and TIFY-like. Expression profiling revealed that 17 PmCCT genes exhibited differential expression under long-day (LD) and short-day (SD) conditions, with elevated transcript levels in both leaves and spikelets. Subsequent qRT-PCR analysis across proso millet germplasms with varying heading dates further identified ten marker PmCCT genes that were strongly induced during the heading transition under SD treatment, specifically in the photoperiod-sensitive accession. Their dynamic expression patterns closely correlated with heading time variation. Discussion: This study provides a systematic understanding of the potential functions of PmCCT genes in photoperiod-mediated regulation of heading date in proso millet. The identified ten candidate marker genes highlight their role in photoperiod-dependent regulation, offering valuable molecular targets and a theoretical foundation for molecular breeding aimed at improving geographical adaptations of proso millet accessions.

Background: Proso millet ( Panicum miliaceum L.) is a drought-tolerant cereal crop cultivated in arid and semi-arid regions. VQ proteins, a class of plant-specific proteins characterized by a conserved VQ motif (FxxxVQxxTG), are known to play critical roles in plant responses to abiotic stress. To elucidate the genetic basis of drought tolerance in proso millet, a genome-wide identification and characterization of the VQ gene family was undertaken. Methods: This study involved the identification of all VQ family members from the proso millet genome, followed by comprehensive analyses including chromosomal localization, phylogenetic relationships, gene structure, conserved motifs, collinearity, and promoter cis -acting elements. Expression profiling was conducted using transcriptomics and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) to investigate potential gene functions. Additionally, the drought tolerance of various germplasm materials was systematically assessed. Results: A total of 70 VQ genes ( PmVQ1 – PmVQ70 ) were identified and classified into four distinct subfamilies. Based on the screening of root-preferential and drought-responsive candidate genes, combined with phenotypic and qRT-PCR analyses of drought-tolerant and drought-sensitive materials, four key candidate genes were identified. qRT-PCR analysis revealed that the four genes exhibited differential expression patterns between drought-tolerant and drought-sensitive materials, suggesting their potential roles as core regulators in proso millet’s drought response, particularly in root-specific regulatory pathways under drought stress. This study provides a systematic analysis of the VQ gene family in proso millet and offers valuable genetic resources for elucidating drought tolerance mechanisms and advancing molecular breeding.

Leaf senescence is a pivotal developmental program in plants, precisely regulated by diverse signals including salicylic acid (SA). The Arabidopsis VQ protein AtVQ25 has been previously characterized as a positive regulator of SA-mediated leaf senescence, functioning through interaction with AtWRKY53 to relieve the transcriptional self-repression of AtWRKY53 at its own promoter. However, the upstream regulatory mechanisms governing AtVQ25 itself remain elusive. In this study, a yeast library screening was performed, and the mitogen-activated protein kinases AtMPK1 and AtMPK2 were identified as interacting partners of AtVQ25. The direct physical interaction was validated by yeast two-hybrid (Y2H), luciferase complementation imaging (LCI), pull-down, and co-immunoprecipitation assays (Co-IP). Furthermore, AtVQ25 was shown to be directly phosphorylated by AtMPK1/AtMPK2, which enhanced its protein stability and retarded its degradation, thereby positively modulating leaf senescence progression. Genetic analyses revealed that the function of AtVQ25 in SA-mediated leaf senescence depends on the functional integrity of AtMPK1/AtMPK2. Collectively, these findings establish AtMPK1/AtMPK2 as upstream interactors of AtVQ25 that coordinate SA-mediated leaf senescence through phosphorylation-dependent enhancement of AtVQ25 protein stability, providing novel insights into the upstream regulatory circuitry of VQ proteins.