The structure of two CONSTANS-LIKE1 genes in potato and its wild relatives

Day length controls development in many plants. In Arabidopsis thaliana, the CONSTANS (CO) gene has been firmly established as a key component in the photoperiodic pathway of floral transition; less is known about CONSTANS-LIKE1 (COL1) orthologues of this gene in Arabidopsis and several other species. The CONSTANS protein comprises two B-box-type zinc fingers, CCT domain, and a variable middle region (MR) which corresponds to exon 2 in the COL1 genes of Solanum species. Solanum COL1 proteins are over 85% identical within the genus and about 50% similar to Arabidopsis CO. Comparative COL1 analysis in several cultivated and wild Solanum species discerned two gene variants, which differed in the structures of exon 2 and introns 1 and 2. In exon 2, two variants were primarily discerned by the numbers of AAC/AAT and CAA/CAG repeats coding for polyasparagine and polyglutamine tracts in MR; therefore two variants were dubbed short and long COL1 genes (sCOL1 and lCOL1). However, intron 1 in lCOL1 was shorter than in sCOL1 due to three indels, whereas intron 2 in available COL1 sequences was represented by three different variants. The temporal profiles of sCOL1 and lCOL1 expression in tuberosum potato dramatically differed under short and long day, and the level of sCOL1 expression exceeded that of lSOL1 by an order of magnitude. Both sCOL1 and lCOL1 were found in each Solanum genome under study and in each individual plant, and the ratio of their copy numbers was not related to plant ploidy and photoperiodic response. Evidently the evolution of two COL1 genes preceded Solanum speciation, and the day-length response of diverse Solanum genotypes does not stem from the primary COL1 structure.     

Identification of LOV KELCH PROTEIN2 (LKP2)-interacting factors that can recruit LKP2 to nuclear bodies

LOV KELCH PROTEIN2 (LKP2) is an F-box protein that has been postulated to function centrally, or near to the circadian clock oscillator. As a first step to determine which proteins act as substrates of LKP2, yeast two-hybrid screening was performed using LKP2 as bait, and two interaction factors, Di19 and COL1, were isolated. The transiently expressed Di19-GUS fusion protein was localized in the nucleus of Arabidopsis petiole cells. COL1 and other CO/COL family proteins could also interact with LKP1/ZTL, LKP2 or FKF1. The LKP2-binding site in CO or COL1 was near the center of each protein. The CCT motif in CO or COL1 was not sufficient for interaction with LKP2. LKP2 recognized CO with F-box and kelch repeat-containing regions, while it recognized COL1 with an LOV domain. When LKP2 was fused with cyan fluorescent proein (CFP) and transiently expressed in onion epidermal cells, CFP-LKP2 signals were localized in the nucleus and cytosol. Both yellow fluorescent protein (YFP)-CO and YFP-COL1 were located in the nucleus, forming nuclear bodies when they were transiently expressed. However, co-expression of CFP-LKP2 with YFP fused to either CO or COL1 resulted in the recruitment of CFP-LKP2 in nuclear bodies. Furthermore, the CFP-LKP2 and YFP-CO signals co-localized with signals for pU2B'-mRFP, which is a marker for Cajal bodies. These results suggest the possibility that LKP2 functions with CO/COL family proteins in the nuclear bodies.     

The CCAAT binding factor can mediate interactions between CONSTANS-like proteins and DNA

CONSTANS-Like (COL) proteins are plant-specific nuclear regulators of gene expression but do not contain a known DNA-binding motif. We tested whether a common DNA-binding protein can deliver these proteins to specific cis-acting elements. We screened for proteins that interact with two members of a subgroup of COL proteins. These COL proteins were Tomato COL1 (TCOL1), which does not seem to be involved in the control of flowering time, and the Arabidopsis thaliana CONSTANS (AtCO) protein which mediates photoperiodic induction of flowering. We show that the C-terminal plant-specific CCT (CO, CO-like, TIMING OF CAB EXPRESSION 1) domain of both proteins binds the trimeric CCAAT binding factor (CBF) via its HAP5/NF-YC component. Chromatin immunoprecipitation demonstrated that TCOL is recruited to the CCAAT motifs of the yeast CYC1 and HEM1 promoters by HAP5. In Arabidopsis, each of the three CBF components is encoded by several different genes that are highly transcribed. Under warm long days, high levels of expression of a tomato HAP5 (THAP5a) gene can reduce the flowering time of Arabidopsis. A mutation in the CCT domain of TCOL1 disrupts the interaction with THAP5 and the analogous mutation in AtCO impairs its function and delays flowering. CBFs are therefore likely to recruit COL proteins to their DNA target motifs in planta.     

CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis

The CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain is found in 45 Arabidopsis thaliana proteins involved in processes such as photoperiodic flowering, light signaling, and regulation of circadian rhythms. We show that this domain exhibits similarities to yeast HEME ACTIVATOR PROTEIN2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters. Moreover, we demonstrate that CONSTANS (CO), which promotes Arabidopsis flowering, interacts with At HAP3 and At HAP5 in yeast, in vitro, and in planta. Mutations in CO that delay flowering affect residues highly conserved between CCT and the DNA binding domain of HAP2. Taken together, these data suggest that CO might replace At HAP2 in the HAP complex to form a trimeric CO/At HAP3/At HAP5 complex. Flowering was delayed by overexpression of At HAP2 or At HAP3 throughout the plant or in phloem companion cells, where CO is expressed. This phenotype was correlated with reduced abundance of FLOWERING LOCUS T (FT) mRNA and no change in CO mRNA levels. At HAP2 or At HAP3 overexpression may therefore impair formation of a CO/At HAP3/At HAP5 complex leading to reduced expression of FT. During plant evolution, the number of genes encoding HAP proteins was greatly amplified, and these proteins may have acquired novel functions, such as mediating the effect of CCT domain proteins on gene expression.     

Overexpression of the mango MiCO gene delayed flowering time in transgenic Arabidopsis

Plant Cell, Tissue and Organ Culture (PCTOC) - CONSTANS (CO)/CONSTANS-like (COL) genes play an important role in the photoperiodic flowering pathway. However, the functional roles of the CO/COL...     

Over-expression of <Emphasis Type="Italic">CONSTANS-LIKE 5</Emphasis> can induce flowering in short-day grown <Emphasis Type="Italic">Arabidopsis</Emphasis>

To ensure that the initiation of flowering occurs at the correct time of year, plants need to integrate a diverse range of external and internal signals. In Arabidopsis, the photoperiodic flowering pathway is controlled by a set of regulators that include CONSTANS (CO). In addition, Arabidopsis plants also have a family of genes with homologies to CO known as CO-LIKE (COL) about which relatively little is known. In this paper, we describe the regulation and interactions of a novel member of the family, COL5. The expression of COL5 is under circadian and diurnal regulation, but COL5 itself does not appear to affect circadian rhythms. COL5, like CO, is regulated by GIGANTEA. Furthermore, COL5 is expressed in the vascular tissue. Using COL5 over-expressing lines we show that, under short days, constitutive expression of COL5 affects flowering time and the expression of the floral integrator genes, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO 1. Constitutive expression of COL5 partially suppresses the late flowering phenotype of the co-mutant plants. However, plants with loss of COL5 function do not show altered flowering. Taken together, our results suggest that COL5 has COL activity, but may either not have a role in regulating flowering in wild-type plants or may act redundantly with other flowering regulators. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana

CONSTANS (CO) is an important floral regulator in the photoperiod pathway, integrating the circadian clock and light signal into a control for flowering time. It is known that CO promotes flowering in Arabidopsis under long-day conditions. CONSTANS-LIKE 9 (COL9) is a member of the CONSTANS-LIKE gene family, encoding a nuclear protein. The expression of COL9 is regulated by the circadian clock in the photoperiod pathway and is detected in various organs. Unexpectedly, overexpression of COL9 in transgenic Arabidopsis resulted in delayed flowering, while co-suppression lines and a transferred DNA (T-DNA) knockout line showed earlier flowering under long-day conditions. Overexpression of COL9 did not enhance the late-flowering phenotype in a co mutant background. Double overexpressors produced by overexpression of CO in COL9 transgenic lines showed an early flowering phenotype similar to single CO overexpressors. The pattern of oscillation of a number of circadian-associated genes remained unchanged in the COL9 transgenic lines. Compared with wild-type plants, the abundance of CO and FLOWERING LOCUS T (FT) mRNA was reduced in the COL9 overexpression lines. Our results indicate that COL9 is involved in regulation of flowering time by repressing the expression of CO, concomitantly reducing the expression of FT and delaying floral transition.     

Molecular cloning and expression analysis of a CONSTANS homologue,PnCOL1, from Pharbitis nil

The Arabidopsis CONSTANS (CO) gene is a key regulator of the long day (LD)-dependent flowering pathway and two CO homologous genes COL1 and COL2 are involved in the regulation of the circadian rhythm. In order to understand the role of CO and COL in short-day plants, a CO homologue, PnCOL1, was isolated and characterized from Japanese morning glory (Pharbitis nil). The deduced PnCOL1 protein of 386 amino acid residues contained two putative zinc finger motifs at the N-terminal region and a conserved CCT domain at the C-terminal region. The deduced amino acid sequence of PnCOL1 was 34% identical to that of PnCO, but 32%, 34%, and 34% identical to those of CO, COL1, and COL2, respectively. Expression of PnCOL1 was barely detected in the cotyledons of plants grown under continuous light (CL), but highly expressed in the cotyledons of plants grown under SD. Expression of PnCOL1 showed a pattern of circadian rhythm as well as daily oscillation. The overexpression of PnCOL1 by a 35S promoter did not overcome the late-flowering phenotype of Arabidopsis co mutants. The results provided in this study suggest that PnCOL1 may have a role in the circadian rhythm in Pharbitis nil.