Limits...
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.

Show MeSH

Related in: MedlinePlus

Quantitative reverse transcriptase-PCR of MsSUS1 transcripts in elongating stem (ES) and post-elongation stem (PES) internodes.Expression values in MsSUS1 transformants (M17, M18) were calculated relative to SUS transcripts in controls. Control values represent the average of two plant lines (M22, M35). Values for M17 and M18 represent means ± standard error (n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Quantitative reverse transcriptase-PCR of MsSUS1 transcripts in elongating stem (ES) and post-elongation stem (PES) internodes.Expression values in MsSUS1 transformants (M17, M18) were calculated relative to SUS transcripts in controls. Control values represent the average of two plant lines (M22, M35). Values for M17 and M18 represent means ± standard error (n = 3)

Mentions: Primers specific to the MsSUS1 transcript were used in quantitative reverse transcriptase PCR (qRT-PCR) assays to measure MsSUS1 transcript accumulation in stems. A survey of ES internodes from 20 independent PEPC7-P4::SUS1 transformed lines showed that the MsSUS1 transcript was reduced 75 to 90 % compared to the mean transcript level in ES internodes of the control lines (M22, M35). Two transformed lines (M17, M18) that exhibited approximately 90 % down-regulation of the MsSUS1 transcript in both ES and PES internodes compared to the controls (Fig. 2) were selected for further study.Fig. 2


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)

Quantitative reverse transcriptase-PCR of MsSUS1 transcripts in elongating stem (ES) and post-elongation stem (PES) internodes.Expression values in MsSUS1 transformants (M17, M18) were calculated relative to SUS transcripts in controls. Control values represent the average of two plant lines (M22, M35). Values for M17 and M18 represent means ± standard error (n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Quantitative reverse transcriptase-PCR of MsSUS1 transcripts in elongating stem (ES) and post-elongation stem (PES) internodes.Expression values in MsSUS1 transformants (M17, M18) were calculated relative to SUS transcripts in controls. Control values represent the average of two plant lines (M22, M35). Values for M17 and M18 represent means ± standard error (n = 3)
Mentions: Primers specific to the MsSUS1 transcript were used in quantitative reverse transcriptase PCR (qRT-PCR) assays to measure MsSUS1 transcript accumulation in stems. A survey of ES internodes from 20 independent PEPC7-P4::SUS1 transformed lines showed that the MsSUS1 transcript was reduced 75 to 90 % compared to the mean transcript level in ES internodes of the control lines (M22, M35). Two transformed lines (M17, M18) that exhibited approximately 90 % down-regulation of the MsSUS1 transcript in both ES and PES internodes compared to the controls (Fig. 2) were selected for further study.Fig. 2

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