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Agrobacterium tumefaciens mediated transient expression of plant cell wall‐degrading enzymes in detached sunflower leaves
Author(s) -
Jung SangKyu,
Lindenmuth Benjamin E.,
McDonald Karen A.,
Hwang Min Sook,
Bui Mai Q. Nguyen,
Falk Bryce W.,
Uratsu Sandra L.,
Phu My L.,
Dandekar Abhaya M.
Publication year - 2014
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.1888
Subject(s) - cauliflower mosaic virus , agrobacterium tumefaciens , agroinfiltration , biology , sunflower , helianthus annuus , xylanase , jasmonic acid , agrobacterium , expression vector , tobacco mosaic virus , nicotiana benthamiana , plant cell , chitinase , biochemistry , genetically modified crops , horticulture , enzyme , transgene , recombinant dna , virus , salicylic acid , virology , gene
For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S‐driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA‐Based Overexpression Vector) systems. For 6‐day leaf incubations, codon‐optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two‐phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6‐fold. Production also increased up to 4.2‐fold when the leaf incubation temperature was elevated above the typical temperature, 20°C, to 30°C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co‐expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall‐degrading enzymes in sunflower. © 2014 American Institute of Chemical Engineers Biotechnol. Prog ., 30:905–915, 2014