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, 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. 

Notably, lateral root primordia displayed no GUS signal,macetas de 10 litros 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. There is compelling evidence that at least two butenolide signaling pathways exist in vascular plants. 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 stereospecific 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 over expressing lines might be the consequence of KL metabolism by excess DLK2 and the elongated hypocotyl phenotype of DLK2 OE lines resembles the htl-3hypocotyl 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, 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 protein degradation. 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,medidas maceta 30 litros suggesting a post transcriptional effect. It cannot be ruled out that other but enolides 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. Over expression 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.Pesticides are natural or synthetic chemicals used to control pests. In order to support an expanding population there is a continuous need for pesticides. Worldwide, there are thousands of pests including insects, weeds, fungi, bacteria, viruses, mycoplasma and nematodes that destroy crops, transmit diseases and compete for resources. One of the first written records of pesticide use was from around 1000 B.C. when Homer described the use of sulfur to control pests by farmers. Many natural pesticides and botanicals were used since that initial discovery: arsenic, mercury, lead, nicotine, pyrethrum and rotenone. However, insect resistance and safety issues for these inorganics and botanicals led to the production and use of the first synthetic organic insecticide, dichlorodiphenyltrichloroethane discovered in 1939 by Paul Müller. Currently, there are over 40,000 different pesticide products for retail sales with different formulations and control mechanisms. Since the major discovery of DDT, advances have continued with the synthesis and commercialization of hundreds of pesticides including five major neuroactive insecticide classes all with unique toxicity profiles and target sites: chlorinated hydrocarbons , pyrethroids, carbamates, organophosphates and neonicotinoids. Chlorinated hydrocarbons and pyrethroids are insecticidal through their ability to destabilize voltage-gated sodium ion channels receptor for some chlorinated hydrocarbons. DDT has low acute toxicity to mammals, but is persistent in the environment which ultimately led to it being banned in the US in 1972. Other problems from DDT include its potential carcinogenicity, thinning of bird eggshells and fish death. Pyrethroids, modeled from natural pyrethrin compounds from the Chrysanthemum flower, are relatively non-toxic and are less stable in the environment than DDT. Carbamates and organophosphates both inhibit acetylcholinesterase leading to accumulation of acetylcholine and over stimulation of the nervous system. Carbaryl was at one time the most commonly used carbamate with low mammalian toxicity and broad-spectrum use and selectivity. Organophosphates are related to potent nerve agents. Often highly toxic to mammals, they are metabolized and detoxified readily. Neonicotinoids, the most important class of insecticides, have favorable mammalian and environmental toxicology and now account for approximately 25 percent of the worldwide insecticide market value. In an attempt to understand the mechanism of action of nicotine, Izuru Yamamoto discovered that insecticidal activity depends on ionization or basicity of the nitrogen of nicotine and all nicotine-related compounds. Yamamoto and colleagues realized that although ionization prevents penetration of the CNS of insects which decreases insecticidal activity, these insecticides needed to be ionized to interact with the nAChR. The search began for synthetic insecticides with high insecticidal activity, low mammalian toxicity and the ability to penetrate the insect CNS yet basic enough to interact with the nAChR. Nithiazine, a nitromethylene heterocycle, was the first neonicotinoid prototype developed by Shell Development Company in 1978. It had excellent insecticidal activity, good systemic action in plants and low mammalian toxicity. However, nithiazine was highly photolabile. Shinzo Kagabu and colleagues modified the structure of nithiazine and synthesized a series of compounds with varying ring structures and sub-stituents and screened them for insecticidal activity against the major rice pest, the green rice leaf hopper. This led to the discovery of the first highly active neonicotinoid, imidacloprid , in 1985. IMI has 12 times higher insecticidal activity than nicotine, is more systemic and photostable and therefore was commercialized by Bayer in 1991. Other first-generation chloropyridinyl-containing neonicotinoids include nitenpyram , acetamiprid and thiacloprid commercialized in 1995, 1996 and 2000, respectively. Further derivatization and optimization lead to the discovery of the two second-generation neonicotinoids, thiamethoxam and clothianidin by Novartis and Takeda, respectively. Finally, the only tetrahydrofuranyl-containing neonicotinoid, dinotefuran , was commercialized by Mitsui Chemical Company in 2002.