The CONSTANS/FLOWERING LOCUS T (CO/FT) regulon plays a central role in the control of flowering time in photoperiod-sensitive plants. Flowering time in wild cotton (Gossypium spp.) has strict photoperiod sensitivity, but domesticated cotton is day-neutral. Information on the molecular characterization of the CO and CO-like (COL) genes in cotton is very limited. In this study, we identified 42 COL homologs (GhCOLs) in the G. hirsutum genome, and many of them were previously unreported. We studied their chromosome distribution, phylogenetic relationships, and structures of genes and proteins. Our results showed that GhCOLs were classified into three groups, and 14 COLs in group I showed conserved structure when compared with other plants. Two homoeologous pairs, GhCOL1-A and GhCOL1-D in Group I, showed the highest sequence similarity to Arabidopsis thaliana CO and rice CO homologous gene Heading date1 (Hd1). Tissue-specific expression showed that 42 GhCOL genes may function as tissue-specific regulators in different cells or organs. We cloned and sequenced the 14 GhCOL genes in Group I related to flowering induction to study their diurnal expression pattern, and found that their expression showed distinct circadian regulation. Most of them peaked at dawn and decreased rapidly to their minima at dusk, then started to accumulate until following dawn under long- or short-day conditions. Transgenic study in the Arabidopsis co-2 mutant demonstrated that GhCOL1-A and GhCOL1-D fully rescued the late-flowering phenotype, whereas GhCOL3-A, GhCOL3-D, GhCOL7-A, and GhCOL7-D partially rescued the late-flowering phenotype, and the other five homoeologous pairs in Group I did not promote flowering. These results indicate that GhCOL1-A and GhCOL1-D were potential flowering inducers, and are candidate genes for research in flowering regulation in cotton.

Plant phosphatidylethanolamine-binding protein (PEBP) is comprised of three clades: FLOWERING LOCUS T (FT), TERMINAL FLOWER1 (TFL1) and MOTHER OF FT AND TFL1 (MFT). FT/TFL1-like clades regulate identities of the determinate and indeterminate meristems, and ultimately affect flowering time and plant architecture. MFT is generally considered to be the ancestor of FT/TFL1, but its function is not well understood. Here, two MFT homoeologous gene pairs in Gossypium hirsutum , GhMFT1-A/D and GhMFT2-A/D , were identified by genome-wide identification of MFT -like genes. Detailed expression analysis revealed that GhMFT1 and GhMFT2 homoeologous genes were predominately expressed in ovules, and their expression increased remarkably during ovule development but decreased quickly during seed germination. Expressions of GhMFT1 and GhMFT2 homoeologous genes in germinating seeds were upregulated in response to abscisic acid (ABA), and their expressions also responded to gibberellin (GA). In addition, ectopic overexpression of GhMFT1 and GhMFT2 in Arabidopsis inhibited seed germination at the early stage. Gene transcription analysis showed that ABA metabolism genes ABA-INSENSITIVE3 ( ABI3 ) and ABI5 , GA signal transduction pathway genes REPRESSOR OF ga1-3 ( RGA ) and RGA-LIKE2 ( RGL2 ) were all upregulated in the 35S : GhMFT1 and 35S : GhMFT2 transgenic Arabidopsis seeds. GhMFT1 and GhMFT2 localize in the cytoplasm and nucleus, and both interact with a cotton bZIP transcription factor GhFD, suggesting that both of GhMFT1, 2 have similar intracellular regulation mechanisms. Taken together, the results suggest that GhMFT1 and GhMFT2 may act redundantly and differentially in the regulation of seed germination.

Flowering transition is a crucial development process in cotton (Gossypium hirsutum L.), and the flowering time is closely correlated with the timing of FLOWERING LOCUS T (FT) expression. However, the mechanism underlying the coordination of various cis-regulatory elements in the FT promoter of cotton has not been determined. In this study, a 5.9-kb promoter of FT was identified from cotton. A bioinformatics analysis showed that multiple insertion–deletion sites existed in the 5.9-kb promoter. Different expression levels of a reporter gene, and the induction by sequential deletions in GhFT promoter, demonstrated that 1.8-kb of the GhFT promoter was stronger than 4.2-, 4.8-, and 5.9-kb promoter fragments. The binding sites of the CONSTANS (CO) and NUCLEAR FACTOR Y transcription factors were located within the 1.0-kb sequence upstream of the FT transcription start site. A large number of repeat segments were identified in proximal promoter regions (−1.1 to −1.4 kb). A complementation analysis of deletion constructs between 1.0 and 1.8 kb of G. hirsutum, Gossypium arboretum, and Gossypium raimondii FT promoters revealed that the 1.0-kb fragment significantly rescued the late-flowering phenotype of the Arabidopsis FT loss-of-function mutant ft-10, whereas the 1.8-kb promoter only slightly rescued the late-flowering phenotype. Furthermore, the conserved CORE motif in the cotton FT promoter is an atypical TGTG(N2-3)ATG, but the number of arbitrary bases between TGTG and ATG is uncertain. Thus, the proximal FT promoter region might play an important role affecting the activity levels of FT promoters in cotton flowering.

Background In plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF gene family has been performed in cotton, and their functions in flowering are largely unknown. Results In this study, 17, 17, 31, and 17 GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering. Conclusions Here, 82 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.

In shoot apex cells of rice, a hexameric florigen activation complex (FAC), comprising FLOWERING LOCUS T (FT), 14-3-3 and the bZIP transcription factor FD, activates downstream target genes and regulates several developmental transitions, including flowering. The allotetraploid cotton (Gossypium hirsutum L.) contains only one FT locus in both of the A- and D-subgenomes. However, there is limited information regarding cotton FACs. Here, we identified a 14-3-3 protein that interacts strongly with GhFT in the cytoplasm and nuclei, and five FD homoeologous gene pairs were characterized. In vivo, all five GhFD proteins interacted with Gh14-3-3 and GhFT in the nucleus. GhFT, 14-3-3, and all the GhFDs interacted in the nucleus as well, suggesting that they formed a ternary complex. Virus-induced silencing of GhFD1, -2, and -4 in cotton delayed flowering and inhibited the expression of floral meristem identity genes. Silencing GhFD3 strongly decreased lateral root formation, suggesting a function in lateral root development. GhFD overexpression in Arabidopsis and transcriptional activation assays suggested that FACs containing GhFD1 and GhFD2 function mainly in promoting flowering with partial functional redundancy. Moreover, GhFD3 was specifically expressed in lateral root meristems and dominantly activated the transcription of AUXIN RESPONSE FACTOR genes, such as ARF19. Thus, the diverse functions of FACs may depend on the recruited GhFD. Creating targeted genetic mutations in the florigen system using CRISPR-Cas genome editing may fine-tune flowering and improve plant architecture.