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. Overexpression 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 overexpression 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 . As DLK2 binds and weakly hydrolyzes 5DS in vitro, we tested whether the compound would inhibit growth of DLK2 OE hypocotyls. DLK2 OE lines were unresponsive to both 5DS and 5DS, indicating that DLK2 does not transduce 5DS signal . To elucidate the spatio-temporal regulation of DLK2 expression induced by dark and SLs, we generated a transcriptional fusion of a 1023 bp DLK2 promoter fragment with the GUS genecoding region. We assayed for GUS expression in at least seven representative T4 homozygous Arabidopsis Col-0 lines.

In young control seedlings grown on 0.5 × MS plates, GUS stain was detected first in the cotyledons which progressively intensified with the onset of the cotyledon expansion and subsequently was detected also in the roots . In seedlings grown on plates supplemented with 10 µM racGR24, a specific and strong GUS signal appeared at the basal end of the hypocotyl . In accordance with the real-time PCR data,vertical grow tables dark-grown seedlings displayed intensive GUS accumulation , especially in the hypocotyl. In the aerial parts of adult plants, GUS signal was strong in primary and mature leaves and petals . No GUS activity was detected in mature hypocotyl, petiole vasculature and non-elongating, mature stems , while the axillary buds and the vascular bundles of elongating stem segments adjacent to the cauline leaves displayed intensive GUS staining. Interestingly, DLK2 promoter activity was strong in buds and the vascular cells connecting the stipules with the vasculature of the petiole . In the root system of adult plants, GUS activity was strong in the differentiation zone and the GUS signal gradually faded away toward the primary root tip . DLK2 promoter activity was the strongest in root hairs and in the cortex of adult plants. Notably, lateral root primordia displayed no GUS signal, while DLK2 promoter activity was detected in young lateral root tips . These findings indicate that DLK2 expression pattern is tissue specific and regulated by SLs or dark directly.To assess whether DLK2 is degraded upon SL treatment and to further characterize DLK2 expression in tissues and at the subcellular level, we generated translational fusions of DLK2 cDNA to sGFP, driven by the 1023-bp DLK2 promoter in DLK2pro:DLK2:sGFP or DLK2 cDNA driven by the constitutive CaMV35S promoter in a 35Spro:DLK2:sGFP construct. Consistent with the PSORT prediction , DLK2 localizes in the cytoplasm and nucleus . In DLK2pro:DLK2:sGFP plants, a strong GFP signal has been observed in guard cells . When plants harboring a DLK2 promoter-driven sGFP construct were subjected to rac-GR24 treatment, a transient increase in GFP signal intensities was detected, while the constitutive 35S promoter resulted in higher GFP expression in the epidermal cells that did not change in response to rac-GR24 . Consistent with this, immunoblot assays using constitutively expressing lines showed that DLK2 was not targeted for degradation after rac-GR24 treatments . Instead, DLK2 protein slightly accumulated after 6 h in rac-GR24-treated 21-day-old whole 35Spro:DLK2:sGFP plants .

In DLK2pro:DLK2:sGFP plants, DLK2 accumulated upon rac-GR24 treatments confirming that SLs induce upregulation of DLK2 as shown earlier . These findings suggest that unlike AtD14 or KAI2 , DLK2 protein degradation is not promoted by rac-GR24 SLs , instead, DLK2 remains stable upon rac-GR24 treatment.The ancient KAI2 pathway has an as yet unknown butenolide ligand , which is neither SL nor karrikin. During the course of evolution, KAI2 underwent a gene duplication event which resulted in the D14 clade . The D14 pathway perceives the canonical SL ligand and diverged from the KAI2 clade both evolutionarily and physiologically . The question then emerges, how does DLK2 relate to these MAX2-dependent signaling pathways? We showed that recombinant DLK2 does not hydrolyze 5DS and is not destabilized in the presence of 5DS , indicating that DLK2 is not an SL receptor nor an SL hydrolase that functions in a negative feedback system to remove excess SL. This is further supported by the sensitivity of dlk2 mutants to 5DS and rac-GR24 and DLK2 OE lines do not show a SL-deficient phenotype . On the other hand, compared to AtD14, DLK2 shows weaker stereo specific binding and hydrolysis toward 5DS , a non-natural SL which, along with karrikins, oddly substitutes for the unknown endogenous KAI2 ligand . It is intriguing to consider that DLK2 might be a receptor or hydrolase for the enigmatic KL. The structure of KL is unknown; therefore, it is hard to draw a parallel between DLK2 and KAI2 ligand-binding mechanisms, and SL binding does not necessarily result in physiological effects . The light hyposensitivity of DLK2 overexpressing lines might be the consequence of KL metabolism by excess DLK2 and the elongated hypocotyl phenotype of DLK2 OE lines resembles the htl-3 hypocotyl phenotype, however, other htl-3-related traits, such as suppressed cotyledon expansion or broad leaves were not observed in these lines. Furthermore, dlk2 mutants are sensitive to 5DS and to karrikin treatment , suggesting that DLK2 is not involved in KL signaling, although 5DS and karrikin do not necessarily mimic KL action.

We propose that DLK2 neither perceives nor hydrolyzes the natural ligand of D14 and KAI2. A remaining question is whether DLK2 should be regarded as a component of a separate signaling pathway, or is its function merely to regulate other MAX2-dependent pathways through the sequestration of the signaling molecules. The known pathways related to the D14 family diverge at the level of SMXL-family proteins. Intuitively,flower pot wholesale the weakly characterized members of the SMXL/D53 family, SMXL3, -4 and -5 might be co-opted by DLK2. SMXL4, originally referred to as AtHSPR , plays a role in abiotic stress responses and displays a vascular bundle-specific expression , as does DLK2 in elongating stem segments. It was shown recently that smxl4 smxl5 double mutants are defective in carbohydrate accumulation and phloem transport and SMXL3, -4 and -5 are essential for phloem formation . In SMXL3, -4 and -5, the RGKT motif needed for MAX2-mediated protein degradation of D53/SMXL7 is absent , and SMXL5 is not degraded upon rac-GR24 application , suggesting that these proteins may not be degraded through MAX2. Intriguingly, DLK2 lacks the residues required for the physical interaction with MAX2. A recent publication also suggested that DLK2 homologues presumably do not interact with MAX2 . The glycine residue in position 158 is required to form a π-turn structure, which is a prerequisite of proper conformational changes of the D14 lid during SL activation . Other substitutions that reportedly do disrupt D14–MAX2 interactions , and are conserved in KAI2, are not present in DLK2 .

Furthermore, DLK2 is not degraded upon rac-GR24 application suggesting that DLK2 does not interact with MAX2; however, its expression regulation is mostly accomplished through MAX2 . It was previously shown that upon binding their proposed ligand, AtD14 and KAI2 underwent substrate-induced proteindegradation. AtD14 is degraded in a MAX2-dependent manner through the 26S proteasome system , and KAI2 is degraded independently of MAX2 and 26S proteasomes . The immunoblot analysis showed a slight increase in the amount of DLK2:sGFP protein even in 35Spro:DLK2:sGFP plants, suggesting a post transcriptional effect . It cannot be ruled out that other butenolides or the proposed KL might promote DLK2 degradation. A potential future direction of DLK2 research could be the elucidation of the relationship between DLK2 and SMXL3, SMXL4, and SMXL5. We demonstrated that KAI2 is a principal promoter of cotyledon expansion in the D14 family, although interactions can be observed. Overexpression of DLK2 in wt, dlk2-2 and dlk2-3 d14-1 kai2-2 backgrounds results in more elongated hypocotyls and expanded cotyledons under low light conditions , suggesting that DLK2 is indeed capable of regulating these physiological responses per se. However, dlk2 mutants do not display the opposite phenotypes, and the phenotype of the OE lines does not correlate with the transcript level , so neomorphic or hypermorphic effects of DLK2 over expression cannot be ruled out. We propose that DLK2 can promote hypocotyl elongation under sub-optimal light conditions, although this effect is modulated by other members of the D14 family. This finding is in conflict with the interpretation of an earlier report , where the authors suggested that the shorter mesocotyls of KAI2– RNAi d14 seedlings compared to those of the d3 mutant in rice is due to suppression by DLK2. However, differences between species might also contribute to this effect, and, as the authors noted, this finding should be interpreted with caution as there was residual KAI2 expression in the RNAi lines. We demonstrated that apart from the well documented SL and karrikin responsiveness, DLK2 expression is also down-regulated by light. Dark adaptation promotes DLK2 expression especially in the hypocotyl, and DLK2 upregulation in dark-kept seedlings is accomplished through MAX2 and KAI2 . DLK2 expression is suppressed in the pif Q mutant either in light or dark, indicating that light signaling regulates DLK2 transcription via PIFs. It is also noteworthy that the spatial DLK2 expression pattern is regulated by rac-GR24 , suggesting a dynamic adaptation of DLK2 transcription to hormonal and environmental changes. DLK2 activity is strong in root hair and cortex, implying that DLK2 might be involved in the physiological processes linked to these tissues, such as water and nutrient uptake and edaphic stress responses . DLK2 expression was strong in axillary buds and the adjacent vascular bundles might also suggest that DLK2 plays a role in the regulation of nutrient distribution. In summary, the results herein show that although it is structurally similar to its paralog D14 family proteins, DLK2 only weakly binds or hydrolyzes natural and unnatural SL ligands. DLK2 is widely expressed in seedlings and has a role in the promotion of hypocotyl elongation. These data together with the knowledge accumulated so far on DWARF14 family suggest that DLK2 represents a divergent member of the family. The fine details of DLK2 regulation, signaling and its role in adult plant life are the subject of future investigations.Cannabis sativa has a long history of cultivation for a variety of uses including food, fifibre, medicine, and recreational drugs . Cannabis produces many different secondary compounds such as cannabinoids, flavonoids, stilbenoids, alkaloids, lignanamides, and phenolic amides . D9 -Tetrahydrocannabinolic acid , a product of the cannabinoid class, is the primary psychoactive agent. This compound is produced as an acid in the glandular trichomes of inflorescence bracts and undergoes decarboxylation with age or heating to D9 -tetrahydrocannabinol . Cannabis cultivars differ substantially in economic traits that range from marijuana, arguably the most widespread illicit drug, to hemp fibre derived from the stems of the plant. Marijuana consists of the dried female inflorescences in which the quantity of THC exceeds that of cannabidiol, and potency varies among cultivars by several orders of magnitude .