Identification and partial characterisation of allene oxide synthase (EC 4.2.1.92) from Vitis vinifera L. Sauvignon blanc, a key enzyme in the jasmonic acid biosynthetic pathway, whose manipulation may confer increased natural resistance to Botrytis cinerea infections

Pathogen infection or plant disease cause major losses in crop production across many species. In grapevine, in particular, there is an ongoing need to decrease dependence on chemical agents as a method to control or manage pathogen infection. Therefore, new approaches need to be explored to provide effective methodologies or approaches to minimise the impacts of pathogen infections. Jasmonic acid is known to be an important compound in plants that orchestrates both wound and plant defence responsiveness against a range of plant herbivores and pathogens. Jasmonic acid, via complex signalling cascades, induces plant defence genes such as those encoding proteinase inhibitors (involved in the protection of plant from insect damage), defensins and thionin (involved in the production of antimicrobials), and a raft of biosynthetic genes that lead to the accumulation of antimicrobial secondary metabolite such as alkaloids, terpenoids, flavonoids, and glucosinolates. Furthermore, jasmonic acid also facilitates the interaction between other defence signalling pathways such as those mediated by salicylic acid and ethylene to acquire the most effective ways to combat herbivore and pathogen attacks. Allene oxide synthase is the first committed biosynthetic step in the formation of jasmonic acid. Previous studies indicate that genetic variation within allene oxide synthase that alter its biosynthetic capacity have the potential to confer to the host plant increased resistance to attack from fungal pathogens. Therefore characterisation of grapevine allene oxide synthase function and genetic variation is an important step in ascertaining the potential this enzyme to contribute to increased tolerance to a wide range of fungal pathogens. Allene oxide synthase (hydroperoxide dehydratase; EC 4.2.1.92) is an enzyme belonging to the cytochrome P-450 (CYP74A) that known to catalyse the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived hydroperoxides. A functional study of grapevine allene oxide synthase has not been previously reported. Therefore in this study we focused on the identification and functional characterization of the putative allene oxide synthase from Vitis vinifera L. Sauvignon blanc via complementation of an Arabidopsis allene oxide synthase null mutant. We investigated the relationships between allene oxide synthase and the other members of the CYP74 family in grapevine, in terms of sequence similarities, subcellular localisations and transcriptional regulation, both spatially and in response to mechanical wounding. We also determined the range of genetic variation of the grapevine allene oxide synthase within a commercial grapevine population. Our findings clearly demonstrate that there is a single allene oxide synthase gene in grapevine and that this gene is able to function in a heterologous system (Arabidopsis) to compliment a null mutation in allene oxide synthase. We show that grapevine allene oxide synthase is localised within the chloroplast and likely associated with chloroplast membranes. In addition the remaining members of the grapevine CYP74 family are found to be localised in varying cellular locations, not necessarily those predicted by in silico sequence analysis. The members of the CYP74 family show differential spatial and developmental transcript accumulation in grapevine. In order to assess the potential for increasing allene oxide synthase levels to increase biochemical flux through to jasmonic acid we overexpressed both the grapevine and Arabidopsis allene oxide synthases in a wild type Arabidopsis background. Our findings suggest that grapevine AOS might not be the only limitation in production of enhanced levels of jasmonic acid in response to wounding or pathogen attack. While we obtained increased levels of allene oxide synthase transcription, this did not result in a concomitant increase in jasmonic acid and consequently increases in the transcription of jasmonate regulated genes. However, while the alterations in jasmonate levels in the transgenic lines was below expectations, we did note that increased levels of jasmonate as a result of overexpression of allene oxide synthase did result in a limited and transient increase in tolerance to Botrytis infection. Investigation of the potential levels of genetic diversity of allene oxide synthase locus in grapevine indicated that this locus is highly conserved with no variation being evident among 100 vines in a commercial vineyard. While the levels of genetic variation strongly suggest that identification of suitable genetic variation in allene oxide synthase that would contribute to increased jasmonate accumulation from within existing grapevine populations is uneconomically practical or efficient. In conclusion our data suggests that to increase jasmonate mediated resistance against fungal disease in grapevine would likely require a coordinated alteration in allene oxide synthase as well as downstream genes in the biosynthetic pathway such as allene oxide cyclase and 12-oxophytodienoic acid reductase. To achieve such an alteration without resorting to transgenic approaches would require the use of a hybridization/breeding approach (which is currently unpalatable to industry) or identification of a suitable gain-of-function mutation from the native transposon mutation population that our group is currently producing

Functional characterization of the grapevine γ-glutamyl transferase/transpeptidase (E.C. 2.3.2.2) gene family reveals a single functional gene whose encoded protein product is not located in either the vacuole or apoplast

γ-glutamyl transferases/transpeptidases (E.C. 2.3.2.2, GGTs) are involved in the catabolism of many compounds that are conjugated to glutathione (GSH), which have a variety of roles. GSH can act as storage and transport vehicle for reduced sulfur; it is involved in the detoxification of xenobiotics and also acts as a redox buffer by utilizing its thiol residue to protect against reactive oxygen species, which accumulate in response to biotic and abiotic stress. Furthermore, many distinctive flavor and aroma compounds in Sauvignon blanc wines originate from odorless C5- and C6-GSH conjugates or their GGT catabolized derivatives. These precursors are then processed into their volatile forms by yeast during fermentation. In many plant species, two or more isoforms of GGTs exist that target GSH-conjugates to either the apoplast or the vacuole. A bioinformatics approach identified multiple GGT candidates in grapevine (Vitis vinifera). However, only a single candidate, VvGGT3, has all the conserved residues needed for GGT activity. This is intriguing given the variety of roles of GSH and GGTs in plant cells. Characterization of VvGGT3 from cv. Sauvignon blanc was then undertaken. The VvGGT3 transcript is present in roots, leaves, inflorescences, and tendril and at equal abundance in the skin, pulp, and seed of mature berries and shows steady accumulation over the course of whole berry development. In addition, the VvGGT3 transcript in whole berries is upregulated upon Botrytis cinerea infection as well as mechanical damage to leaf tissue. VvGGT3-GFP fusion proteins transiently over-expressed in onion cells were used to study subcellular localization. To confirm VvGGT3 activity and localization in vivo, the fluorescent γ-glutamyl-7-amido-4-methylcoumarin substrate was added to Nicotiana benthamiana leaves transiently over-expressing VvGGT3. In combination, these results suggest that the functional VvGGT3 is associated with membrane-like structures. This is not consistent with its closely related functionally characterized GGTs from Arabidopsis, radish and garlic

First Report of Verticillium Wilt Caused by Verticillium dahliae Infection on Chinese Cabbage in Korea

Chinese cabbage (Brassica rapa L.) is an important vegetable in Korea as the main ingredient for kimchi. In June 2014, symptoms of leaf wilt, drying, and drop off were observed in a Chinese cabbage farm located at Taebeak (37°26′50.7″N, 128°95′50.0″E), Gangwon province, Korea. This disease was observed on ∼35% of plants in the field, causing an almost 10% decrease in production