Bibenzyl synthesis in Cannabis sativa L.

This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the 'bibenzyls'. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this 'bibenzyl synthase' enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing 'parts prospecting' inventory that supports the rational metabolic engineering of natural product therapeutics.

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco.

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.

Characterization of a putative grapevine Zn transporter, VvZIP3, suggests its involvement in early reproductive development in Vitis vinifera L.

BACKGROUND: Zinc (Zn) deficiency is one of the most widespread mineral nutritional problems that affect normal development in plants. Because Zn cannot passively diffuse across cell membranes, it must be transported into intracellular compartments for all biological processes where Zn is required. Several members of the Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) gene family have been characterized in plants, and have shown to be involved in metal uptake and transport. This study describes the first putative Zn transporter in grapevine. Unravelling its function may explain an important symptom of Zn deficiency in grapevines, which is the production of clusters with fewer and usually smaller berries than normal. RESULTS: We identified and characterized a putative Zn transporter from berries of Vitis vinifera L., named VvZIP3. Compared to other members of the ZIP family identified in the Vitis vinifera L. genome, VvZIP3 is mainly expressed in reproductive tissue - specifically in developing flowers - which correlates with the high Zn accumulation in these organs. Contrary to this, the low expression of VvZIP3 in parthenocarpic berries shows a relationship with the lower Zn accumulation in this tissue than in normal seeded berries where its expression is induced by Zn. The predicted protein sequence indicates strong similarity with several members of the ZIP family from Arabidopsis thaliana and other species. Moreover, VvZIP3 complemented the growth defect of a yeast Zn-uptake mutant, ZHY3, and is localized in the plasma membrane of plant cells, suggesting that VvZIP3 has the function of a Zn uptake transporter. CONCLUSIONS: Our results suggest that VvZIP3 encodes a putative plasma membrane Zn transporter protein member of the ZIP gene family that might play a role in Zn uptake and distribution during the early reproductive development in Vitis vinifera L., indicating that the availability of this micronutrient may be relevant for reproductive development.

The promoter of the TLC1.1 retrotransposon from Solanum chilense is activated by multiple stress-related signaling molecules.

The LTR retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. The promoter of the TLC1.1 retrotransposon from Solanum chilense contains two primary ethylene-responsive elements (PERE boxes) that are essential for its response to ethylene and for the stress-induced expression. Here, we describe that a 270 bp fragment (P270), derivative of this retroelement promoter, is also able to activate the transcription of the GUS reporter gene in transgenic plants in response to salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA), hydrogen peroxide (H2O2) and the synthetic auxin 2,4-D. PERE box-dependent and independent routes are involved in the response of P270 to these signal molecules. MeJA, H2O2 and 2,4-D activate this promoter through cis-acting elements other than PERE boxes, whereas ABA and SA act via a PERE box-independent pathway but require this element for maximal activation. Three putative cis-acting elements MRE, GCN4 and GT1/TCA identified in the P270 promoter may be involved in the PERE box-independent activation pathway. These results suggest that the promoter of TLC1.1 may act as an integrator of different signal transduction pathways, allowing this member of the TLC1 retrotransposon family to be activated in response to multiples challenges.