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Title: Transgene silencing of sucrose synthase in alfalfa stem vascular tissue by a truncated phosphoenolpyruvate carboxylase: sucrose synthase construct

item Gronwald, John
item Yang, Suk
item Bucciarelli, Bruna
item Miller, Susan
item O`Rourke, Jamie
item SHIN, SANGHYUM - University Of Minnesota
item Samac, Deborah - Debby
item VANCE, CARROLL - Retired ARS Employee

Submitted to: Plant Biology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/30/2014
Publication Date: 7/12/2014
Citation: Gronwald, J.W., Yang, S.H., Bucciarelli, B., Miller, S.S., O'Rourke, J.A., Shin, S., Samac, D.A., Vance, C.P. 2014. Transgene silencing of sucrose synthase in alfalfa stem vascular tissue by a truncated phosphoenolpyruvate carboxylase: sucrose synthase construct. Plant Biology. July 12-16, 2014. Portland, OR.

Interpretive Summary:

Technical Abstract: An important role of sucrose synthase (SUS, EC in plants is to provide UDP-glucose needed for cellulose synthesis in cell walls. We examined if over-expressing SUS in alfalfa (Medicago sativa L.) would increase cellulose content of stem cell walls. Alfalfa plants were transformed with two constructs: a truncated alfalfa phosphoenolpyruvate carboxylase promoter (PEPC-4) fused to ß-glucuronidase (GUS), and PEPC-4 fused with alfalfa nodule-enhanced SUS cDNA (MsSUS1). GUS histochemical staining in stems of transformants indicated that the PEPC-4 promoter was active in phloem and xylem. In contrast to what was expected, SUS transcript levels measured in stems of transformants were reduced by 75-90%. Two control transformants (M22, M35) containing the PEPC-4::GUS construct and two PEPC-4::SUS transformants (M17, M18) that exhibited 85-90% reduction of MsSUS1 transcript in ES (elongating stem) and PES (post-elongating stem) internodes were selected for further study. Immunoblotting proteins in the soluble fraction (16,000g supernatant) from stems of controls with a polyclonal antibody from maize indicated the presence of three SUS polypeptides in ES internodes and one SUS polypeptide in PES internodes. The major SUS polypeptide was absent from both ES and PES internodes in the SUS down-regulated transformants. Staining for in situ SUS activity in stems of control plants (M22) revealed that SUS activity was located in phloem and xylem. SUS activity as measured by in situ assays was significantly reduced in phloem and xylem of M18 plants. Down-regulation of SUS activity in M17 and M18 had only minor effects on plant dry weight or cell wall composition of stems. Neutral invertase in situ assays showed activity in stem vascular tissue with similar levels of activity in the control (M22) and the SUS down-regulated line (M18). The results suggest that neutral invertase activity in stem vascular tissue compensated for the down-regulation of SUS.