title: Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis.
abstract: BACKGROUND
  Alfalfa (Medicago sativa L.) is a widely adapted perennial forage crop that has high biomass production potential. Enhanced cellulose content in alfalfa stems would increase the value of the crop as a bioenergy feedstock. We examined if increased expression of sucrose synthase (SUS; EC 2.4.1.13) would increase cellulose in stem cell walls.
  
  
  RESULTS
  Alfalfa plants were transformed with a truncated alfalfa phosphoenolpyruvate carboxylase gene promoter (PEPC7-P4) fused to an alfalfa nodule-enhanced SUS cDNA (MsSUS1) or the β-glucuronidase (GUS) gene. Strong GUS expression was detected in xylem and phloem indicating that the PEPC7-P4 promoter was active in stem vascular tissue. In contrast to expectations, MsSUS1 transcript accumulation was reduced 75-90 % in alfalfa plants containing the PEPC7-P4::MsSUS1 transgene compared to controls. Enzyme assays indicated that SUS activity in stems of selected down-regulated transformants was reduced by greater than 95 % compared to the controls. Although SUS activity was detected in xylem and phloem of control plants by in situ enzyme assays, plants with the PEPC7-P4::MsSUS1 transgene lacked detectable SUS activity in post-elongation stem (PES) internodes and had very low SUS activity in elongating stem (ES) internodes. Loss of SUS protein in PES internodes of down-regulated lines was confirmed by immunoblots. Down-regulation of SUS expression and activity in stem tissue resulted in no obvious phenotype or significant change in cell wall sugar composition. However, alkaline/neutral (A/N) invertase activity increased in SUS down-regulated lines and high levels of acid invertase activity were observed. In situ enzyme assays of stem tissue showed localization of neutral invertase in vascular tissues of ES and PES internodes.
  
  
  CONCLUSIONS
  These results suggest that invertases play a primary role in providing glucose for cellulose biosynthesis or compensate for the loss of SUS1 activity in stem vascular tissue.
authors.affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. debby.samac@ars.usda.gov.|Department of Plant Pathology, University of Minnesota, St. Paul, MN, 55108, USA. debby.samac@ars.usda.gov.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Samac
authors.affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. bruna.bucciarelli@ars.usda.gov.
authors.forename: Bruna
authors.initials: B
authors.lastname: Bucciarelli
authors.affiliation: Department of Plant Pathology, University of Minnesota, St. Paul, MN, 55108, USA. sue.miller@ars.usda.gov.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Miller
authors.affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. sam.yang@monsanto.com.|Present address: Monsanto Company, Chesterfield, MO, 63017, USA. sam.yang@monsanto.com.
authors.forename: S Samuel
authors.initials: SS
authors.lastname: Yang
authors.affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. jamie.orourke@ars.usda.gov.|Present address: USDA-ARS-Corn Insects and Crop Genetics Research Unit, Ames, IA, 50011, USA. jamie.orourke@ars.usda.gov.
authors.forename: Jamie A
authors.initials: JA
authors.lastname: O'Rourke
authors.affiliation: Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, 55108, USA. shinx101@korea.kr.|Present address: National Institute of Crop Science, Iksan, 570-080, Korea. shinx101@korea.kr.
authors.forename: Sanghyun
authors.initials: S
authors.lastname: Shin
authors.affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. vance004@umn.edu.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, 55108, USA. vance004@umn.edu.
authors.forename: Carroll P
authors.initials: CP
authors.lastname: Vance
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D015536:Down-Regulation; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D000455:Medicago sativa / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018547:Plant Stems / Q000378:metabolism; D019076:Transgenes; D043324:beta-Fructofuranosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D002482:Cellulose; D005964:Glucosyltransferases; C022699:sucrose synthase; D043324:beta-Fructofuranosidase