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

Samac DA, Bucciarelli B, Miller SS, Yang SS, O'Rourke JA, Shin S, Vance CP - BMC Plant Biol. (2015)

Bottom Line: In contrast to expectations, MsSUS1 transcript accumulation was reduced 75-90 % in alfalfa plants containing the PEPC7-P4::MsSUS1 transgene compared to controls.Down-regulation of SUS expression and activity in stem tissue resulted in no obvious phenotype or significant change in cell wall sugar composition.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.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. debby.samac@ars.usda.gov.

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.

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Comparison of in situ sucrose synthase activity in stem transverse-sections of elongating stem (ES) and post-elongating stem (PES) internodes of control (M22) and the SUS down-regulated (M18) transformant. Purple coloration in cells indicates enzyme activity. a ES internodes of M22; (c), ES internodes of M18; (e), PES internode of M22; (g), PES internode of M18. b, d, f, h are negative controls (no sucrose in assay medium) for a, c, e, g, respectively. Abbreviations: PF, phloem fibers; P, phloem; C, cambium; XV, xylem vessel
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Fig4: Comparison of in situ sucrose synthase activity in stem transverse-sections of elongating stem (ES) and post-elongating stem (PES) internodes of control (M22) and the SUS down-regulated (M18) transformant. Purple coloration in cells indicates enzyme activity. a ES internodes of M22; (c), ES internodes of M18; (e), PES internode of M22; (g), PES internode of M18. b, d, f, h are negative controls (no sucrose in assay medium) for a, c, e, g, respectively. Abbreviations: PF, phloem fibers; P, phloem; C, cambium; XV, xylem vessel

Mentions: In stems of the control (M22) alfalfa line, SUS enzyme activity was found to be 1.6-fold higher in PES compared to ES internodes (Fig. 3). In the PEPC7-P4::SUS1 transformed lines (M17, M18) SUS activity in ES internodes was below the level of detection (Fig. 3a) and SUS activity was reduced by more than 95 % in PES internodes compared to the control line M22 (Fig. 3b). SUS activity was detected by in situ enzyme assays in the phloem and xylem tissue in the control line with greater activity in the PES internodes than ES internodes (Fig. 4a, e). The in situ enzyme assays showed that SUS activity was greatly reduced in ES internodes of the PEPC7-P4::MsSUS1 transformant (Fig. 4c) and was below the level of detection in PES internodes (Fig. 4g).Fig. 3


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

Samac DA, Bucciarelli B, Miller SS, Yang SS, O'Rourke JA, Shin S, Vance CP - BMC Plant Biol. (2015)

Comparison of in situ sucrose synthase activity in stem transverse-sections of elongating stem (ES) and post-elongating stem (PES) internodes of control (M22) and the SUS down-regulated (M18) transformant. Purple coloration in cells indicates enzyme activity. a ES internodes of M22; (c), ES internodes of M18; (e), PES internode of M22; (g), PES internode of M18. b, d, f, h are negative controls (no sucrose in assay medium) for a, c, e, g, respectively. Abbreviations: PF, phloem fibers; P, phloem; C, cambium; XV, xylem vessel
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666122&req=5

Fig4: Comparison of in situ sucrose synthase activity in stem transverse-sections of elongating stem (ES) and post-elongating stem (PES) internodes of control (M22) and the SUS down-regulated (M18) transformant. Purple coloration in cells indicates enzyme activity. a ES internodes of M22; (c), ES internodes of M18; (e), PES internode of M22; (g), PES internode of M18. b, d, f, h are negative controls (no sucrose in assay medium) for a, c, e, g, respectively. Abbreviations: PF, phloem fibers; P, phloem; C, cambium; XV, xylem vessel
Mentions: In stems of the control (M22) alfalfa line, SUS enzyme activity was found to be 1.6-fold higher in PES compared to ES internodes (Fig. 3). In the PEPC7-P4::SUS1 transformed lines (M17, M18) SUS activity in ES internodes was below the level of detection (Fig. 3a) and SUS activity was reduced by more than 95 % in PES internodes compared to the control line M22 (Fig. 3b). SUS activity was detected by in situ enzyme assays in the phloem and xylem tissue in the control line with greater activity in the PES internodes than ES internodes (Fig. 4a, e). The in situ enzyme assays showed that SUS activity was greatly reduced in ES internodes of the PEPC7-P4::MsSUS1 transformant (Fig. 4c) and was below the level of detection in PES internodes (Fig. 4g).Fig. 3

Bottom Line: In contrast to expectations, MsSUS1 transcript accumulation was reduced 75-90 % in alfalfa plants containing the PEPC7-P4::MsSUS1 transgene compared to controls.Down-regulation of SUS expression and activity in stem tissue resulted in no obvious phenotype or significant change in cell wall sugar composition.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.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA. debby.samac@ars.usda.gov.

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.

Show MeSH
Related in: MedlinePlus