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Even in other eukaryotes, only a few families of transcription factors with multiple distinct DNA-binding domains are known. These include POU-domain proteins 22 and Pax proteins 23 , both of which have been studied extensively with respect to how the multiple DNA binding structures contribute to the specificity and affinity of DNA-binding 24 , Thus, these proteins contain three HTHs and have been shown to recognize different types of target sites using multiple combinations of their HTHs We demonstrate here that the two DNA-binding domains of RAV1 can separately recognize each of two motifs that constitute a bipartite binding sequence and together cooperatively enhance its DNA-binding affinity and specificity.
Expression of recombinant proteins For the expression of recombinant proteins, the JM Escherichia coli host strain was transformed with the plasmids described above. Purification of the recombinant proteins by affinity chromatography using glutathione-Sepharose Pharmacia or nickel ion resin Novagen was performed by the procedures recommended by the manufacturers.
The concentration of each protein solution was determined by the method of Bradford Probes for other experiments were synthetic double-stranded oligonucleotides with sequences listed in each figure. These pBluescript plasmid clones were sequenced. The inserts of these plasmid clones were cut out with XhoI or XbaI, labeled by the fill-in reaction, and then digested with either XbaI or XhoI, which was not used for the first digestion. Amino acid residues identical to those of RAV1 are indicated by dots ….
The N-terminal and C-terminal conserved sequence blocks are indicated by lines. Amino acid residue numbers of the regions compared are indicated in parentheses. The search revealed the presence of at least two types of Arabidopsis cDNAs potentially encoding amino acid sequences significantly similar to the B3 domains of maize VP1 and its homologues from other plant species.
Among 13 and 15 positive clones obtained by hybridization screening with P5T and 97E4T7, respectively, those with the longest cDNA inserts were chosen for sequence analyses. In Figure 1D , domain organizations of AP2 -like or B3 -like domain-containing proteins are summarized.
The photograph of ethidium bromide-stained agarose gel before RNA transfer is shown at the bottom. However, the expression levels were relatively high in rosette leaves and roots, and very low in flowers. Oligonucleotides recovered after the fourth and sixth rounds of selection were PCR-amplified and cloned into a plasmid vector.
DNA sequences were analyzed for 68 independent clones 19 from the fourth round and 49 from the sixth round among those whose binding to GRAV1-ent were confirmed Fig. In summary, RAV1 binds specifically to bipartite sequences comprising two unrelated motifs, separated by various spacings, and in different relative orientations.
Among the analyzed 68 sequences, there were three sequences that did not follow this rule. The interference signal was more marked with the G corresponding to the fourth base C of the CAC3C4TG motif than that corresponding to the third base C in every case, regardless of whether the motif was present in the forward sequences , and or reverse orientation sequence No apparent interferences resulted from methylation of any other Gs in the four sequences.
However these results do not exclude the possibility that preformed dimers or other oligomers of GRAV1-ent or HRAV1-ent in solution are highly stable and cannot exchange their subunits even after a prolonged incubation. No such complexes with an intermediate mobility were observed when GST tag was partially cleaved off by digestion with factor Xa Fig.
A Sixty-eight nucleotide sequences selected after the fourth or sixth round of BSSAs are classified according to the type and combination of sequence motifs. The number of sequences in each category is indicated in parentheses.
The numbers of sequences with the indicated bases at each position are shown. Bases corresponding to the linker sequences, indicated with lower case letters, are excluded from counting. All other mutants, which lacked either a part of or entire region of the B3 or AP2-like domain did not form detectable levels of DNA-protein complex.
However, it exhibited a stronger and clearer binding when different target sequences were used Fig. These results suggest that both AP2 and B3-like domains of RAV1 possess an autonomous DNA-binding activity, although the binding efficiency or affinity for a target sequence is greatly increased when the two domains are combined.
GRAV1-ent ent bound equally well to the tested sequences except for sequences and In contrast, the autonomous DNA-binding activities of mutant proteins containing either the AP2 or B3-like domain alone exhibited a strong dependence on the target sequences Fig. GRAV1-dN dN containing the B3-like domain alone bound strongly to sequences , and , compared with other sequences.
This binding preference appeared to correlate with the degrees of deviation in the CACCTG motif in each sequence from the consensus Fig. Sequence contained no apparent CACCTG motif, and the binding to this sequence was almost at the background level. Methylation interference experiments were conducted using sequences , , and Fig.
Results with only the bottom strand or both strands , and are shown. Bands corresponding to G residues that strongly and less strongly interfered with protein binding when methylated are indicated by closed and open circles, respectively. Results are summarized at the bottom. The digestion reactions were terminated by the addition of phenylmethanesulfonyl fluoride, and the digests were used for EMSAs.
Near-complete digestion was obtained with 0. Note that the mobility of the complex with RAV1 without the GST tag is approximately the same as that of the HRAV1-ent complex because the molecular masses of the two proteins are very similar to each other. GRAV1-dC3 hardly bound sequences , , and or only weakly with different efficiencies sequences , and to the sequences containing a single copy of the CAACA motif, although the efficiency of the binding to sequence , which exhibited the strongest binding among them, was very low compared with that to sequence Such differences could not simply be accounted for by the deviation from the consensus sequence of the CAACA motif Fig.
A hidden motif, which behaves like a second CAACA motif as in sequence , might be present in some of these sequences and affect the binding to different degrees. Contacts of each DNA-binding domain with each sequence motif were further confirmed by methylation interference experiments. Methylation of the G in this top strand sequence also interfered with the binding. A Schematic illustrations of the GST fusion proteins used in the experiments.
Filled and hatched boxes indicate the AP2 and B3-like domains, respectively. However, CO is already expressed in the phloem early in development Takada and Goto, , and changes in CO expression levels do not seem to account for the increase in FT accumulation for inducing flowering Castillejo and Pelaz, Consequently, something else that accounts for this late FT accumulation must exist. In the photoperiod pathway, FT promotes flowering in response to LD. Single loss-of-function alleles of TEM1, tem1—1, and TEM2, tem2—2, cause a slight early flowering phenotype in LD, and a double tem1—1 tem2—2 mutant shows enhanced early flowering compared with the single mutants under LD conditions.
Consequently, TEMs seem to play a pivotal role as repressors in floral induction Fig. Open in new tab Download slide Floral transition model in Arabidopsis thaliana. TEM1 and TEM2 genes play a central role in regulating the flowering process by repressing at least the photoperiod and gibberellin pathways under inductive and non-inductive daylengths, in leaves and apical meristems. TEM1 transcript levels follow a diurnal oscillation, such that TEM1 abundance is low during the daytime and peaks at dusk.
Similar developmental and circadian regulations were observed for TEM1 and TEM2, supporting the proposed redundant role of both genes Castillejo and Pelaz, ; Osnato et al. Moreover, TEM1 mRNA abundance is very high during early stages of seedling development but a pronounced decline takes place just before floral transition.
CO expression remains almost unaltered throughout development, although a subtle increase occurs during the transition to flowering Castillejo and Pelaz, In addition in wild-type plants, FT mRNA remains at basal levels until the transition to flowering, at days 10—12, when there is a pronounced increase in FT accumulation. However, FT expression increases from day 6 in the tem1—1 tem2—2 double mutant, when plants had only formed the first two true leaves Osnato et al.
The identical precocious flowering phenotypes of 35S::CO and tem1 tem2 plants suggested strongly that only when TEM levels drop drastically can CO activate FT to reach the threshold level necessary to trigger the floral transition under inductive photoperiods Castillejo and Pelaz, The combination of tem1—1 and ft mutants confirmed the epistatic relationship between both genes, as the double mutant tem1—1 ft flowers at the same time as ft alone Castillejo and Pelaz, TEM1 expression is detected in all vegetative tissues Castillejo and Pelaz, RAV binding motifs Kagaya et al.
Therefore, precise control of flowering time could be explained if the CO and TEM proteins compete for their respective binding sites to directly regulate FT accumulation. Moreover, the in vivo physical interactions of these proteins were found to take place in the nucleus but not in the cytosol Sawa and Kay, These authors also showed that GI activates FT expression independently of CO through direct binding to FT promoter regions alone or in a complex with another protein.
Under SDs, tem1—1 tem2—2 double mutants still flower much earlier than wild-type plants. By contrast, 35S::TEM1 plants flower extremely late under SDs, most of them remaining at the vegetative phase and producing leaves indefinitely. In this photoperiod, TEM mRNA levels are low during the light period, start to increase at dusk and peak early in the night in wild-type plants. These are phenotypes typical of GA-deficient mutants, such as ga3ox1—3 and the double mutant ga3ox1 ga3ox2 Eriksson et al.
These results indicate a clear effect of TEM on the enzymes that catalyse the last step of GA4 biosynthesis. In conclusion, TEM genes link the photoperiod- and GA-dependent flowering pathways, controlling the floral transition under inductive and non-inductive daylengths Fig. However, it has not been shown whether the full-length antisense construct used to generate these antisense lines is specific for RAV1.
It is also possible that RAV1 antisense plants flower a few days earlier than wild-type plants as a result of differences in the rate of leaf production Hu et al. They proposed that the repression of flowering by GmRAV in tobacco may indirectly result from negative effects on photosynthesis and other aspects of plant physiology.
Further research should determine whether GmRAV is a regulator of flowering. Several AP2 family genes are targets of the miRNA miR and encode floral repressors that act in the photoperiod- and the age-dependent flowering pathways.
However, TEM1 mRNA levels are not altered in the leaves and the shoot meristem of an activation-tagged smz-D mutant, which flowers later than the wild type Mathieu et al. The action of TPL and its homologues in mammals and yeast involves histone deacetylation and chromatin condensation Long et al.
Ambient temperature pathway Changes in ambient temperature affect flowering and low temperatures delay the floral transition in arabidopsis Blazquez et al. This suggests that the repression of flowering by ELF3 may be mediated at least in part by an increase in TEM2 expression. Both are involved in responses to ambient temperature. SVP is important for the repression of flowering at low ambient temperature, while FLC suppresses the induction of flowering by high temperatures Balasubramanian et al.
FLC also plays an important role in vernalization, a response to long periods of cold that induces flowering after winter has passed Song et al. In addition, FLC acts in the autonomous flowering pathway Simpson, The mechanism of this positive regulation remains unknown, but probably contributes to reinforce the repression of flowering under unfavourable conditions. Brassinosteroids Brassinosteroids BRs are a class of steroid hormones that regulate many developmental processes throughout plant life, such as vascular development, senescence and flowering.
Mutants with altered content in endogenous BRs, such as deetiolated2 or dwarf4, flower late, indicating that components of the BR pathway also affect flowering time reviewed by Li et al. Light intensity and quality In addition to photoperiod, light intensity and quality affect floral induction, as well as many other aspects of plant development and growth Chen et al.
Several results indicate that RAV genes may be involved in light responses. In addition, HY5 represses flowering, as shown by the early flowering of hy5 mutants Goto et al. Therefore, TEM2 is a good candidate to link HY5 with the regulation of flowering in response to light signals. Moreover, TEM2 expression is induced by a short exposure to far-red light Tepperman et al.
These data indicate that RAV genes show specificity in their response to different light conditions in different organs. Consistent with this, tem mutants and plants overexpressing TEM1 have longer and shorter hypocotyls than wild-type plants, respectively, under SD Osnato et al.
Also, GmRAV causes a reduction in chlorophyll content and photosynthetic rate when overexpressed in tobacco Zhao et al. A detailed analysis of plant growth in loss-of-function tem mutants and GmRAV-silenced lines would be useful to demonstrate whether these genes play a role as growth regulators.
Again, silencing of GmRAV in soybean would help to determine its biological function. Although overexpression of RAV1 causes a reduction in the number of lateral roots and probably in the rate of leaf production, suggesting that RAV1 may be a negative regulator of plant growth, down-regulation of RAV1 by an antisense construct does not have a significant effect on these processes Hu et al. RAV1 might regulate leaf senescence Leaf senescence, a physiological mechanism affected by many internal and external factors Lim et al.
This maximum plant fitness is obtained by remobilizing nutrients from senescent leaves Woo et al. RAV1 expression is triggered at a mature stage, reaching maximum expression at an early senescence stage and decreasing at later stages. These similar expression patterns during leaf development and senescence suggest a possible redundant role among this family in this aspect.
However, neither single loss-of-function mutants of these genes nor the rav1 tem1 and rav1 rav1l double mutants show any significant alteration of the senescence process. By contrast, arabidopsis plants overexpressing RAV1 under a constitutive promoter show an early age-dependent leaf senescence phenotype as well as one induced by artificial dark Woo et al. The main senescence-associated physiological markers, such as the degree of leaf yellowing, chlorophyll content and photochemical efficiency, are altered.
Similar results are found in transgenic plants that express RAV1 under an inducible promoter.