Genetic engineering of maize towards desiccation tolerance: Electroporation with the trehalose gene

Trehalose is a non-reducing disaccharide of glucose that occurs in a large number of organisms such as bacteria, fungi, nematodes or crustaceans. Trehalose plays an important role in desiccation and heat stress protection since it has been shown to stabilize proteins and cell membranes under these stress conditions. Trehalose accumulation has proven to be an effective way of increasing drought tolerance in both model plants such as tobacco and important crops such as potato or rice. In this work we aim to improve desiccation tolerance in maize, one of the, most agronomical important crops, by increasing trehalose accumulation through transformation with the Arabidopsis thaliana trehalose phosphate synthase gene (AtTPSl) is involved in trehaIose-6-phosphate synthase and hence on trehalose biosynthesis. A cassette harboring the AtTPSl gene under the control of the CaMV35S promoter and the Bialaphos resistance gene Bar as a selective agent (conferring resistance to the PPT) was inserted in the plasmid vector pGreenO229 and used to transform maize inbred line Pa91. Immature zygotic embryos were collected 14-20 days after pollination and embryogenic calli culture were initiated. Embryogénie calli were electroporated with 20µg of plasmid DNA using a Biorad Gene Puiser II at 374 V, for 1 second. Embryogenic calli were electroporated and selected PPT. Eighty putative transgenic plants were obtained and analyzed by PCR for the presence of the AtTPS1 gene

Proteomic and transcriptomic analysis of rice tranglutaminase and chloroplast-related proteins

The recently cloned rice transglutaminase gene (tgo) is the second plant transglutaminase identified to date (Campos et al. Plant Sci. 205–206 (2013) 97–110). Similarly to its counterpart in maize (tgz), this rice TGase was localized in the chloroplast, although in this case not exclusively. To further characterise plastidial tgo functionality, proteomic and transcriptomic studies were carried out to identify possible TGO-related proteins. Some LHCII antenna proteins were identified as TGO related using an in vitro proteomic approach, as well as ATPase and some PSII core proteins by mass spectrometry. To study the relationship between TGO and other plastidial proteins, a transcriptomic in vivo Dynamic Array (Fluidigm™) was used to analyse the mRNA expression of 30 plastidial genes with respect to that of tgo, in rice plants subjected to different periods of continuous illumination. The results indicated a gene-dependent tendency in the expression pattern that was related to tgo expression and to the illumination cycle. For certain genes, including tgo, significant differences between treatments, principally at the initiation and/or at the end of the illumination period, connected with the day/night cycling of gene expression, were observed. The tgo expression was especially related to plastidial proteins involved in photoprotection and the thylakoid electrochemical gradient.This study was supported by the Spanish projects MEC BFU2006-15115-01/BMC, BFU2009-08575 and a collaborative project (TRANSBIO 2011) with the Biotechnology Department of Neiker Technalia, financed by the Environmental, Territorial Planification, Agriculture and Fish Dept., basque Gov, Spain. N. Campos had a pre-doctoral fellowship from the Agencia Española de Cooperación Internacional para el Desarrollo (AECID). The authors would especially like to thank Oriol Casagran (CRAG Genomic Services), Sami Irar (CRAG Proteomic Services), and Shirley Burgess (English correction). The authors would also like to thank the Spanish Ministry of Science and Innovation, Consolider-Ingenio 2010 Programme. CSD2007-00036 “CRAG” and the Xarxa de Referencia en Biotecnologia of the Generalitat de Catalunya.Peer reviewe