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

Deborah A. Samac; Bruna Bucciarelli; Susan S. Miller; S. Samuel Yang; Jamie A. O'Rourke; Sanghyun Shin; Carroll P. Vance

doi:10.1186/s12870-015-0649-4

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

Deborah A. Samac, Bruna Bucciarelli, Susan S. Miller, S. Samuel Yang, Jamie A. O'Rourke, Sanghyun Shin, Carroll P. Vance

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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 postelongation 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.

Original language	English (US)
Article number	283
Journal	BMC plant biology
Volume	15
Issue number	1
DOIs	https://doi.org/10.1186/s12870-015-0649-4
State	Published - Dec 1 2015

Bibliographical note

Funding Information:
We are grateful to Prem Chourey, USDA-ARS, University of Florida, for providing the maize sucrose synthase (SS2) antibody. The authors would also like to thank Ted Jeo, USDA-ARS-Plant Science Research Unit, St. Paul, MN, for performing the cell wall analysis, Melinda Dornbusch for production of transgenic plants, and Jon Mack for plant maintenance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Funding for this research was provided by USDA-ARS CRIS Project 2640-12210-002-00D.

Publisher Copyright:
© 2015 Samac et al.

Keywords

Biofuels
Cell wall biosynthesis
Cellulose
Gene silencing
Phloem
Xylem

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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",

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 postelongation 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.",

keywords = "Biofuels, Cell wall biosynthesis, Cellulose, Gene silencing, Phloem, Xylem",

author = "Samac, {Deborah A.} and Bruna Bucciarelli and Miller, {Susan S.} and Yang, {S. Samuel} and O'Rourke, {Jamie A.} and Sanghyun Shin and Vance, {Carroll P.}",

note = "Funding Information: We are grateful to Prem Chourey, USDA-ARS, University of Florida, for providing the maize sucrose synthase (SS2) antibody. The authors would also like to thank Ted Jeo, USDA-ARS-Plant Science Research Unit, St. Paul, MN, for performing the cell wall analysis, Melinda Dornbusch for production of transgenic plants, and Jon Mack for plant maintenance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Funding for this research was provided by USDA-ARS CRIS Project 2640-12210-002-00D. Publisher Copyright: {\textcopyright} 2015 Samac et al.",

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T1 - Transgene silencing of sucrose synthase in alfalfa (medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

AU - Samac, Deborah A.

AU - Bucciarelli, Bruna

AU - Miller, Susan S.

AU - Yang, S. Samuel

AU - O'Rourke, Jamie A.

AU - Shin, Sanghyun

AU - Vance, Carroll P.

N1 - Funding Information: We are grateful to Prem Chourey, USDA-ARS, University of Florida, for providing the maize sucrose synthase (SS2) antibody. The authors would also like to thank Ted Jeo, USDA-ARS-Plant Science Research Unit, St. Paul, MN, for performing the cell wall analysis, Melinda Dornbusch for production of transgenic plants, and Jon Mack for plant maintenance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Funding for this research was provided by USDA-ARS CRIS Project 2640-12210-002-00D. Publisher Copyright: © 2015 Samac et al.

PY - 2015/12/1

Y1 - 2015/12/1

N2 - 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 postelongation 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.

AB - 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 postelongation 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.

KW - Biofuels

KW - Cell wall biosynthesis

KW - Cellulose

KW - Gene silencing

KW - Phloem

KW - Xylem

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SN - 1471-2229

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JO - BMC plant biology

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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

Abstract

Bibliographical note

Keywords

UN SDGs

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