SLs are synthesized via a sequential cleavage of alltrans-β-carotene by DWARF27 and the resulting 9-cis-β-carotene by MORE AXILLARY GROWTH3 and 4. The SL precursor carlactone is then transported through the xylem and biologically active SLs are formed by MAX1 and its homologs and LATERAL BRANCHING OXIDOREDUCTASE. Cumulative evidence supports the idea that the DWARF14 α/β-fold hydrolase functions as a SL receptor and is required for the perception of the SL signal in Petunia , rice , Arabidopsis and pea. Upon binding, D14 proteins hydrolyze SL by action of its conserved Ser-His-Asp catalytic triad, followed by thermal destabilization of the proteins. As a consequence, the structural rearrangement of D14 proteins in the presence of SL enables the protein to physically interact with the F-box proteins MAX2 and SMAX1-LIKE  family proteins SMXL 6, 7 and 8 to form a Skp-Cullin-Fbox ubiquitin ligase complex that polyubiquitinates SMXLs and targets them for degradation by the 26S proteasome. The subsequent signaling events are largely unknown, but tentatively the mechanism is similar to other systems employing targeted protein degradation. In Arabidopsis, two paralogs of AtD14 have been identified. One paralog, KARRIKIN INSENSITIVE2 was identified in a mutant in Ler background which showed insensitivity to karrikin , a butenolide-type germination stimulant from smoke water. 

Although both AtD14 and KAI2 signaling pathways converge upon MAX2 and might employ similar mechanisms to transduce the signal,planta de frambuesa en maceta the two proteins regulate separate physiological events. Unlike AtD14, KAI2 genetically interacts with the other members of the SMXL family , which redundantly regulate SLand KAR-related gene expression. KAI2 is required in Arabidopsis primarily for seed germination, normal seedling photomorphogenic responses, and leaf development ,while in rice KAI2 is essential to the perception of symbiotic signal needed for mycorrhizal association. This functional divergence suggested that KAI2 is a component of an SL-independent signaling pathway that perceives a hypothetical butenolide ligand, termed KL ,which is neither SL nor karrikin. Evidence supporting this hypothesis is that AtD14 shows high affinity toward both 5DS and the natural 5DS signal, while KAI2 stereospecifically binds and hydrolyzes only the non-natural 5DS SL. Comparisons of mutants from different ecotypes of Arabidopsis led to the isolation of a loss-of-function allele of KAI2 in Col-0 designated as htl-3. Very little is known about DWARF14-LIKE2 , the third member of the DWARF14 protein family, to which no physiological role has been assigned as yet. Arabidopsis dlk2 mutants in Col-0 background exhibit normal seed dormancy, photomorphogenic responses, and branching phenotypes , although in rice DLK2 may regulate mesocotyl elongation in the dark. DLK2 gene expression was recognized as an excellent marker for SL or KAR action , and as a karrikin-responsive transcript in germinating lettuce achenes. DLK2 is upregulated through the action of AtD14 or KAI2 in seedlings after SL or karrikin treatments, and its normal expression is highly dependent on MAX2 and KAI2. Interestingly, smxl1,2 double mutants exhibit increased DLK2 expression, indicating that KAR/KL signaling is constitutively activated in these mutants. This butenolide-dependent expression has been hypothesized to be a negative feedback system in which DLK2 plays a role as a strigolactone metabolic enzyme. 

Another scenario is that the high structural similarity imposes functional redundancy in the D14 family that affects SL or KL sensitivity and the resulting phenotypes. In this case, DLK2 could function as a SL/KL receptor that acts through the MAX2 core signaling pathway. Alternatively, parallel butenolide signaling pathways could interact, or DLK2 might mediate responses to an as yet unknown signal. Here we examine these hypotheses about DLK2 function and demonstrate that DLK2 is not involved in SL/KL perception and might act independently of the MAX2 pathway. PCR amplifications were accomplished with Phusion DNA Polymerase. cDNA for plasmid constructs was reverse transcribed with SuperScript III RT enzyme. GATEWAY compatible pGWB plasmids were kindly provided by Nakagawa et al.. The binary vectors were generated by the standard GATEWAY procedure using pDONR221 donor vector. Expression constructs were introduced into GV3101 Agrobacterium strain for floral dip transformation of Arabidopsis.For RNA extraction, seedlings were grown as described in the Section “Hypocotyl Elongation and Cotyledon Expansion Assay.” The stratified seeds on plates intended for dark grown seedlings were treated with red light for 10 min, kept in dark for 3 h, and then exposed to far red light for 10 min. All samples were harvested on day 4. Total RNA was isolated from at least 15 whole seedlings. RNA was isolated and DNAseI digested using RNEasy Plant Mini Kit. cDNA was reverse transcribed with Promega reverse transcriptase. The qRT-PCR analyses were performed as described previously with gene-specific DLK2 primers. The qRT-PCR results are presented as relative expression levels normalized against Arabidopsis ACTIN2. All real-time PCR reactions were performed in quadruplicates, and means ± SD were calculated for three biological replicates for each examined treatment. The protein structure – functional relationships of D14-family proteins have been studied in some detail. DLK2 protein shares 40 and 42% amino acid sequence identity with KAI2 and AtD14, respectively and presumably evolved from D14 by gene duplication. Several conserved sites can be aligned along the sequence, most importantly around the residues of the catalytic triad. Interestingly, DLK2 lacks conserved amino acid residues in positions 163 , 166 , 180 and 183 , which were shown to be essential for the interaction between AtD14 and D3. The predicted structure of DLK2 was compared with crystal structures of AtD14 and KAI2 using the I-TASSER server.

Based on the prediction, high structural similarity is present among the D14-family proteins. In particular, DLK2 contains a predicted ligand-binding pocket with the catalytic residues facing inward,cultivo frambuesa en maceta as is the case for its paralog proteins. The presence of the conserved catalytic triad prompted us to test whether DLK2 also binds and/or hydrolyses SLs in in vitro assays using recombinant proteins expressed in Escherichia coli. DSF has been established as a reliable method to infer alterations of protein thermal stability in the presence of a small-molecule interaction partner. In particular, DSF assays have been used to characterize the melting temperature shifts of DAD2, AtD14 and KAI2 in the presence of SLs. DSF data obtained from the positive control AtD14 was consistent with the previous findings , exhibiting a significant ligand concentration-dependent lowering of Tm in the presence of SLs regardless of the stereochemistry of the ligands. Under all conditions tested, DLK2 exhibited a characteristic two phase melting curve, suggesting that DLK2 either has two distinct phase transitions or can be present in monomer and dimer forms. Addition of 5DS did not cause a Tm shift for DLK2; however, in the presence of the highest concentration of 5DS, Tm shifted moderately lower implying that it can bind to DLK2 and destabilize it. Having demonstrated a stereo specific interaction between 5DS and DLK2, we tested the proposed hydrolytic function of DLK2 in vitro. The consumption of 5DS and 5DS and the production of a possible metabolite were monitored by HPLC , as described by Waters et al. with minor modifications. While SLs hydrolyzed spontaneously at a 10%/h rate, AtD14 hydrolyzed 100% of both substrates in 2 h. Consistent with the thermal stability assay, DLK2 exhibited moderate hydrolytic activity only against the nonnatural enantiomer 5DS. Stereospecific binding and hydrolysis suggests that recombinant DLK2 is not a receptor of tested SLs. In terms of hydrolytic activity and affinity toward the stereoisomers of deoxystrigol, DLK2 more closely resembles KAI2, which specifically binds and hydrolyzes only 5DS , suggesting that both proteins might have a non-SL butenolide ligand. Furthermore, as DLK2 does not hydrolyze natural SL 5DS, and only slowly hydrolyzes non-natural 5DS, we can conclude that DLK2 is not a SL metabolism enzyme as had been suggested by the positive feedback regulation of DLK2 expression. Binding and hydrolysis of the non-natural 5DS by DLK2 shows similarity to the same properties of KAI2 , raising the question whether the two proteins might interfere within the plant, possibly having the same natural ligand and redundantly regulating developmental responses. Thus, we tested whether absence or over expression of DLK2 results in any MAX2-related phenotypic alterations, and if so, whether crosstalk between the three D14 family related pathways is manifest in the phenotypes. dlk2 mutants in Col-0 background were reported to be normal with respect to seed dormancy, germination and shoot branching phenotypes. We assessed these traits and other SL-related phenotypes such as senescence and branching in dlk2 mutants as well as in DLK2-overexpressing lines. 

No obvious phenotypic differences in branching were observed in adult OE lines and mutant plants growing in long days and in rosettes grown in short days , nor in progress of senescence or seed germination characteristics. MAX2 acts as a promoter of seedling photomorphogenesis. To investigate whether DLK2 is involved in these MAX2-related signaling events, we tested dlk2 seedling responses to suboptimal light conditions, when the effect of max2 mutation is more prominent. Previous work found that Arabidopsis kai2 mutant seedlings showed distinct photomorphogenic phenotypes compared to wild type , while dlk2 mutants did not. Consistent with this finding, dlk2 mutants in Col-0 and Ler backgrounds exhibited normal photomorphogenic responses under low light conditions. To test whether functional redundancy exists among D14- family proteins affecting seedling phenotypes and SL sensitivity, we examined double and triple mutants of dlk2-3, d14-1, htl-3  and kai2-2 , grown under continuous low intensity white light in which mutants display distinct hypocotyl and cotyledon growth responses. Untreated dlk2-3, dlk2-4 and d14-1 single mutants seedlings displayed no significant differences from their wild types in hypocotyl elongation and cotyledon expansion, while htl-3 single mutants exhibited significantly greater hypocotyl elongation and decreased cotyledon expansion under low intensity light , consistent with several previous reports. Seedlings of the double mutant dlk2-3 htl-3, d14-1 htl-3 and the triple mutant d14-1 dlk2-3 htl-3 displayed increased hypocotyl length similar to those of the single htl-3 mutant, confirming that KAI2 contributes to inhibition of hypocotyl elongation in response to light. However, untreated dlk2-3 htl-3 seedlings exhibited slightly shorter hypocotyls than htl-3 seedlings, implying that DLK2 might be involved in the promotion of hypocotyl elongation by low light. Differences in inhibition of hypocotyl elongation by SLs were also evident among the mutant seedlings. Both 5DS and 5DS inhibited elongation of dlk2-3 mutant hypocotyls, while the htl-3 mutation alone or in combination with dlk2-3 exhibited growth inhibition only in the presence of 5DS. When combined with d14-1, the htl-3 mutation resulted in loss of sensitivity to both 5DS and 5DS, and presence of the dlk2 mutation did not affect substantially these effects of d14-1 and htl-3. In untreated seedlings, the dlk2-3 and d14-1 mutations alone or together had no effect on cotyledon expansion while the htl- 3 mutation in any combination reduced expansionby 40–70%. Similar to the action of racemic GR24 , 5DS and 5DS inhibited cotyledon expansion of Col-0 and Ler wild types while lines containing d14-1 were insensitive to these SLs. All multiple mutant lines containing htl-3 exhibited reduced cotyledon expansion similar to the htl- 3 single mutant, implying that KAI2 is the primary promoter of cotyledon expansion. Over expression of DLK2 driven by a 2 × 35S promoter in Ler plants [DLK2 OE ] or by a 35S promoter in dlk2-2 [DLK2 OE ] exhibited longer hypocotyls compared to wild type controls grown for 8 days under low light conditions. This finding suggests that DLK2 might promote hypocotyl elongation in low light. To test whether DLK2 dose might counteract SL inhibition on hypocotyl elongation, DLK2 OE plants were subjected to treatment with increased concentrations of SLs. However, rac-GR24-induced suppression of hypocotyl elongation was not affected by DLK2 over expression. It was reported that DLK2 is down regulated in d14 kai2 background ; thus, this double mutant might be regarded as a functional dlk2 mutant as well. To examine any possible phenotypes related to DLK2 and to exclude the effects of its paralogs, we generated DLK2 OE lines in the triple mutant background [DLK2 OE ] with the construct 35Spro:DLK2:sGFP. We found that DLK2 over expression resulted in a slightly more elongated hypocotyl in dlk2-3 d14-1 kai2-2 mutants. Furthermore, these plants exhibited more pronounced cotyledon expansion than triple mutants.