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Title: Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

Author
item Samac, Deborah - Debby
item BUCCIARELLI, BRUNA
item Miller, Susan - Sue
item YANG, SUK
item O`Rourke, Jamie
item SHIN, SANGHYUN - UNIVERSITY OF MINNESOTA
item VANCE, CARROLL

Submitted to: Biomed Central (BMC) Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2015
Publication Date: 12/1/2015
Citation: Samac, D.A., Bucciarelli, B., Miller, S.S., Yang, S.H., O'Rourke, J.A., Shin, S., Vance, C.P. 2015. Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis. Biomed Central (BMC) Plant Biology. 15:283. doi:10.1186/s12870-015-0649-4.

Interpretive Summary: There is a need for the U.S. to develop renewable energy sources to reduce dependence on foreign oil supplies. Alfalfa is a widely grown crop that offers advantages as a bioenergy feedstock for the production of cellulosic ethanol. Compared to other bioenergy feedstocks, alfalfa makes its own nitrogen fertilizer, improves the soil, and can be readily incorporated into a rotation with corn. The model for development of alfalfa as a bioenergy crop involves separating leaves from stems at harvest. The leaves would be used as a protein supplement for livestock while the stems would be used for cellulosic ethanol production. In cellulosic ethanol production, cellulose, a major component in plant cell walls, is converted to ethanol. Increasing the amount cellulose in cell walls of alfalfa stems would increase the efficiency of ethanol production. Most evidence suggests that the protein sucrose synthase plays a major role in making cellulose in plant cell walls. To test the importance of sucrose synthase in cellulose synthesis, we inserted an extra sucrose synthase gene into alfalfa stems. Contrary to what was expected, the result was a large reduction in the amount of sucrose synthase in stems. This occurred because of the phenomenon called gene silencing where an inserted gene expressed at high levels can sometimes turn off all similar genes. Although the stems containing the extra alfalfa sucrose synthase gene had low levels of this protein, the amount of cellulose in cell walls was not affected. We found that invertase, another protein that makes cellulose, was increased in plants that had low levels of sucrose synthase. The results indicate that under conditions where sucrose synthase is low, the plant can compensate by producing more invertase. Further research will be needed to define the relative contributions of sucrose synthase and invertase to cellulose synthesis in plants. The research generated new knowledge of proteins that are involved in the synthesis of cellulose in plants. This knowledge can be used by molecular biologists and plant breeders to advance the development of alfalfa and other crops as bioenergy feedstocks.

Technical Abstract: Alfalfa (Medicago sativa L.) plants were transformed with two constructs: (1) a truncated phosphoenolpyruvate carboxylase promoter isolated from alfalfa nodules (PEPC-4) fused to GUS; and (2) PEPC-4 fused with sucrose synthase (SUS) isolated from alfalfa nodules. Histochemical staining for GUS in stems of transformants indicated that the PEPC-4 promoter was expressed in phloem and xylem. Of the 20 PEPC-4::SUS transformants analyzed, SUS transcript levels in stems were reduced by an average of 75%. Two control transformants (M22, M35) containing the PEPC-4::GUS construct and two PEPC-4::SUS transformants (M17, M18) that exhibited 90% reduction of SUS transcript in stems were selected for further study. Immunoblotting the soluble fraction from stems of controls with a polyclonal antibody from maize indicated the presence of a major SUS isoform of approximately 90 kDa. This isoform was absent from the SUS down regulated transformants. In situ staining for SUS activity in stems of control plants (M22) revealed that enzyme activity was located in phloem, developing xylem, and xylem parenchyma. In plants where SUS transcript was down regulated (M18), in situ SUS activity was significantly reduced in phloem and xylem parenchyma. Down regulation of SUS in stems of M18 was associated with up regulation of acid invertase transcript. The SUS down regulated lines did not exhibit an obvious shoot phenotype. In addition, there was no reduction in cellulose concentration in stems of down regulated lines. Expressing the PEPC-4::SUS construct in alfalfa stems results in post transcriptional silencing of a soluble SUS isoform located in phloem and xylem. Up-regulation of acid invertase may partially compensate for the down regulation of SUS.