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

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

BMC plant biology (2015-12-03)
Deborah A Samac, Bruna Bucciarelli, Susan S Miller, S Samuel Yang, Jamie A O'Rourke, Sanghyun Shin, Carroll P Vance
ABSTRACT

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. 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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Potassium hydroxide, ACS reagent, ≥85%, pellets
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Glycerol, ≥99.5%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Sucrose, ≥99.5% (GC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
Sucrose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
Glycerol, ACS reagent, ≥99.5%
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Glycerol, ReagentPlus®, ≥99.0% (GC)
Sigma-Aldrich
Sucrose, BioUltra, for molecular biology, ≥99.5% (HPLC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
Supelco
DL-Dithiothreitol solution, 1 M in H2O
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, for molecular biology, ≥97.0%
Sigma-Aldrich
Potassium hydroxide, reagent grade, 90%, flakes
Sigma-Aldrich
Sucrose, ≥99.5%
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Glycerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
Potassium hydroxide solution, 45 wt. % in H2O
Sigma-Aldrich
Sucrose, puriss., meets analytical specification of Ph. Eur., BP, NF
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide hydrate, ≥96.5% (HPLC), ≥96.5% (spectrophotometric assay), from yeast
Sigma-Aldrich
Sucrose, ACS reagent
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, ≥97.0%
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
Sigma-Aldrich
Potassium hydroxide, semiconductor grade, pellets, 99.99% trace metals basis (Purity excludes sodium content.)