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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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Related in: MedlinePlus

Immunoblot of sucrose synthase proteins in elongating stem (ES) and post-elongating stem (PES) internodes of control lines (M22, M35) and the lines containing the PEPC7-P4::MsSUS1 construct (M17, M18). Each lane contains 40 μg of soluble protein from ES or PES internodes. Numbers at the side of the blot indicate the molecular mass of the protein markers in kDa
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Fig5: Immunoblot of sucrose synthase proteins in elongating stem (ES) and post-elongating stem (PES) internodes of control lines (M22, M35) and the lines containing the PEPC7-P4::MsSUS1 construct (M17, M18). Each lane contains 40 μg of soluble protein from ES or PES internodes. Numbers at the side of the blot indicate the molecular mass of the protein markers in kDa

Mentions: Immunoblotting was conducted to examine SUS protein in the soluble fraction (16,000 x g supernatant fraction) of stem extracts from the controls (M22, M35) and the MsSUS1 down-regulated transformants (M17, M18). SUS antiserum produced against maize sucrose synthase 2 [21] was used to probe the immunoblot. The results showed that three immunoreactive polypeptides were detected in ES internodes in the control lines, one major polypeptide of approximately 90 kDa and two minor polypeptides of slightly higher molecular weight (Fig. 5). In the ES internodes of the SUS down-regulated transformed lines the major 90 kDa SUS polypeptide band was absent, although the two minor bands of higher molecular weight remained. In contrast, the PES internodes of control lines showed the presence of only one major SUS polypeptide band at approximately 90 kDa. The PES internodes of the SUS down-regulated lines showed no immunoreactive polypeptides.Fig. 5


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)

Immunoblot of sucrose synthase proteins in elongating stem (ES) and post-elongating stem (PES) internodes of control lines (M22, M35) and the lines containing the PEPC7-P4::MsSUS1 construct (M17, M18). Each lane contains 40 μg of soluble protein from ES or PES internodes. Numbers at the side of the blot indicate the molecular mass of the protein markers in kDa
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Immunoblot of sucrose synthase proteins in elongating stem (ES) and post-elongating stem (PES) internodes of control lines (M22, M35) and the lines containing the PEPC7-P4::MsSUS1 construct (M17, M18). Each lane contains 40 μg of soluble protein from ES or PES internodes. Numbers at the side of the blot indicate the molecular mass of the protein markers in kDa
Mentions: Immunoblotting was conducted to examine SUS protein in the soluble fraction (16,000 x g supernatant fraction) of stem extracts from the controls (M22, M35) and the MsSUS1 down-regulated transformants (M17, M18). SUS antiserum produced against maize sucrose synthase 2 [21] was used to probe the immunoblot. The results showed that three immunoreactive polypeptides were detected in ES internodes in the control lines, one major polypeptide of approximately 90 kDa and two minor polypeptides of slightly higher molecular weight (Fig. 5). In the ES internodes of the SUS down-regulated transformed lines the major 90 kDa SUS polypeptide band was absent, although the two minor bands of higher molecular weight remained. In contrast, the PES internodes of control lines showed the presence of only one major SUS polypeptide band at approximately 90 kDa. The PES internodes of the SUS down-regulated lines showed no immunoreactive polypeptides.Fig. 5

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