The crucial role of Φ- and K-segments in the in vitro functionality of Vitis vinifera dehydrin DHN1a | A comparative analysis of the two DHN1a splicing variants from Vitis vinifera reveals the crucial role of the Φ- and K-segments in VvcDHN1a in vitro functionality

Dehydrins (DHNs), group II LEA (Late Embryogenesis Abundant) proteins, are among the most commonly observed proteins which accumulate in plants in response to cold and any other environmental factors, causing the dehydration of cells. In previous studies, we isolated a YSK2-type VvcDHN1a gene from table grapes (Vitis vinifera cv. Cardinal) which presented two spliced variants (the spliced, DHN1a_s and the unspliced, DHN1a_u). Their expression was induced by low temperature storage and CO2, although with different accumulation patterns. DHN1a_u codifies for a truncated YS protein lacking ¿- and K-segments, which might affect its functionality. In this work, we expressed both DHN1a_s and DHN1a_u recombinant proteins in Escherichia coli. We carried out a number of in vitro assays to analyze the implications that ¿- and K-segments have in the protective role of VvcDHN1 against different abiotic stresses and their antifungal activity against the fungal pathogen Botrytis cinerea. Our results showed that unlike DHN1a_u, DHN1a_s has a potent cryoprotective effect on lactate dehydrogenase activity, protects malate dehydrogenase against dehydration and partially inhibits B. cinerea growth. Moreover, the DHN1a promoter presented cis-regulatory elements related to cold and drought, as well as biotic stress-related elements. We also observed that both spliced variants interact weakly with DNA, suggesting that K-segments are not involved in DNA binding. Overall, this work highlights the crucial role of ¿- and K-segments in DHNs function in plant response to abiotic stress showing for the first time, the potential role of the V. vinifera DHN1a_s in the protection against freezing and dehydration as well as inhibiting B. cinerea growth.

This work was supported by CICYT project AGL2011-26742 and by the European Union under the 7th Framework Programme FP7-PEOPLE-2012-CIG No. 321694. R.R. and I.R. were supported by a postdoctoral JAE contract from the CSIC and a postdoctoral Juan de la Cierva contract from the MICINN, respectively. The work benefited from the networking activities within the European COST Action FA1106-QualityFruit.

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