Limits...
Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus.

Payyavula RS, Tschaplinski TJ, Jawdy SS, Sykes RW, Tuskan GA, Kalluri UC - BMC Plant Biol. (2014)

Bottom Line: Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth.While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced.These results demonstrate that PdUGPase2 plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of Populus.

View Article: PubMed Central - PubMed

ABSTRACT

Background: UDP-glucose pyrophosphorylase (UGPase) is a sugar-metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and UTP. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in perennial woody plants is poorly understood.

Results: We characterized the functional role of a UGPase gene in Populus deltoides, PdUGPase2. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of PdUGPase2 results in perturbations in primary, as well as secondary metabolism, resulting in reduced sugar and starch levels and increased phenolics, such as caffeoyl and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced.

Conclusions: These results demonstrate that PdUGPase2 plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of Populus.

Show MeSH
Expression of selected sugar metabolism (A) and cellulose pathway genes (B) in young leaves of control andUGPase2overexpression lines. Relative expression was calculated based on the expression of reference genes Ubiquitin-conjugating enzyme E2 and 18S ribosomal RNA. SUT, Sucrose transporter; VIN, Vacuolar invertase; NIN, Neutral invertase; SPS, Sucrose phosphate synthase; CesA, Cellulose synthase; SuSy, Sucrose synthase; KOR, KORRIGAN. Accessions: SUT3, Potri.019G085800; SUT4, Potri.002G106900; VIN2, Potri.003G112600; VIN3, Potri.015G127100; NIN8, Potri.019G082000; SPS6, Potri.018G124700; CesA7b, Potri.018G103900; CesA8b, Potri.004G059600; KOR1, Poptri.003G151700; KOR2, Poptri.001G078900; SuSy1, Potri.018G063500; SuSy2, Potri.006G136700;18S, AF206999; UBCc, Potri.006G205700. Data represent means ± SE (n = 3). *indicates statistically significant, p ≤ 0.05 based on Student’s t-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4197241&req=5

Fig6: Expression of selected sugar metabolism (A) and cellulose pathway genes (B) in young leaves of control andUGPase2overexpression lines. Relative expression was calculated based on the expression of reference genes Ubiquitin-conjugating enzyme E2 and 18S ribosomal RNA. SUT, Sucrose transporter; VIN, Vacuolar invertase; NIN, Neutral invertase; SPS, Sucrose phosphate synthase; CesA, Cellulose synthase; SuSy, Sucrose synthase; KOR, KORRIGAN. Accessions: SUT3, Potri.019G085800; SUT4, Potri.002G106900; VIN2, Potri.003G112600; VIN3, Potri.015G127100; NIN8, Potri.019G082000; SPS6, Potri.018G124700; CesA7b, Potri.018G103900; CesA8b, Potri.004G059600; KOR1, Poptri.003G151700; KOR2, Poptri.001G078900; SuSy1, Potri.018G063500; SuSy2, Potri.006G136700;18S, AF206999; UBCc, Potri.006G205700. Data represent means ± SE (n = 3). *indicates statistically significant, p ≤ 0.05 based on Student’s t-tests.

Mentions: HPLC analysis of stem cell wall sugar composition showed that glucose and xylose were the most abundant sugars in all samples as expected (Table 1). Among glucose, xylose, galactose, arabinose and mannose, significant changes were observed only in mannose levels, with lower mannose levels in transgenic samples. While cellulose and lignin content appeared unaffected; the syringyl-to-guaiacyl (S/G) ratio of lignin was significantly reduced in transgenic lines (Figure 5A - C). In order to explore whether overexpression of UGPase2 has any effect on the transcription of carbohydrate metabolism or/and cellulose pathway, a subset of sugar metabolism and cellulose synthesis-related genes were studied (Figure 6). The panel of genes that we studied included, sucrose transporters, SUT3, linked previously to enhanced cellulose production [26], and SUT4, linked to carbon partitioning [27]; genes that encode sucrose hydrolyzing enzymes [1], vacuolar invertases, VIN2 and VIN3; neutral invertase, NIN8 [27]. Additionally, the secondary cell wall associated cellulose synthase (CesA) genes, CesA7B and CesA8B, and KORRIGAN (KOR) genes, KOR1 and KOR2, were included based on previously reports of proposed roles in cellulose biosynthesis [28-30]. SuSy1 and Susy2 transcripts and corresponding proteins have been shown to be elevated in tissue contexts with enhanced cellulose biosynthesis such as secondary xylem development and tension stress response [31,32]. Furthermore, a functional role for SuSy in supplying sugars for cellulose synthesis has been proposed [33]. The expression of most sugar metabolism genes was not significantly altered except SUT3 which was significantly higher in all the three transgenic lines (Figure 6A). Whether SUT3 has a direct role in sucrose transport and carbon partitioning in Populus is as yet unanswered. Of the surveyed cellulose pathway genes, CesA and SuSy isoforms were not significantly altered, but KOR1 and KOR2 were significantly increased in young leaves of transgenic lines (Figure 6B). These results suggest that overexpression of PdUGPase2 may be associated with modest changes in transcript levels of cellulose pathway-associated genes but that does not translate into changes in cellulose levels (Figure 5A). The unchanged cellulose levels in overexpression lines of PdUGPase2 contradicts with previous reports where cellulose levels were increased in AxUGPase overexpression lines [15]. While the implication of low sequence identity between the bacterial and Populus UGPase isoforms on enzyme activity is yet to be clarified, the functional activity of AxUGPase has shown to complement cellulose negative mutant phenotype by channeling sugar substrate for cellulose synthesis [34].Table 1


Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus.

Payyavula RS, Tschaplinski TJ, Jawdy SS, Sykes RW, Tuskan GA, Kalluri UC - BMC Plant Biol. (2014)

Expression of selected sugar metabolism (A) and cellulose pathway genes (B) in young leaves of control andUGPase2overexpression lines. Relative expression was calculated based on the expression of reference genes Ubiquitin-conjugating enzyme E2 and 18S ribosomal RNA. SUT, Sucrose transporter; VIN, Vacuolar invertase; NIN, Neutral invertase; SPS, Sucrose phosphate synthase; CesA, Cellulose synthase; SuSy, Sucrose synthase; KOR, KORRIGAN. Accessions: SUT3, Potri.019G085800; SUT4, Potri.002G106900; VIN2, Potri.003G112600; VIN3, Potri.015G127100; NIN8, Potri.019G082000; SPS6, Potri.018G124700; CesA7b, Potri.018G103900; CesA8b, Potri.004G059600; KOR1, Poptri.003G151700; KOR2, Poptri.001G078900; SuSy1, Potri.018G063500; SuSy2, Potri.006G136700;18S, AF206999; UBCc, Potri.006G205700. Data represent means ± SE (n = 3). *indicates statistically significant, p ≤ 0.05 based on Student’s t-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197241&req=5

Fig6: Expression of selected sugar metabolism (A) and cellulose pathway genes (B) in young leaves of control andUGPase2overexpression lines. Relative expression was calculated based on the expression of reference genes Ubiquitin-conjugating enzyme E2 and 18S ribosomal RNA. SUT, Sucrose transporter; VIN, Vacuolar invertase; NIN, Neutral invertase; SPS, Sucrose phosphate synthase; CesA, Cellulose synthase; SuSy, Sucrose synthase; KOR, KORRIGAN. Accessions: SUT3, Potri.019G085800; SUT4, Potri.002G106900; VIN2, Potri.003G112600; VIN3, Potri.015G127100; NIN8, Potri.019G082000; SPS6, Potri.018G124700; CesA7b, Potri.018G103900; CesA8b, Potri.004G059600; KOR1, Poptri.003G151700; KOR2, Poptri.001G078900; SuSy1, Potri.018G063500; SuSy2, Potri.006G136700;18S, AF206999; UBCc, Potri.006G205700. Data represent means ± SE (n = 3). *indicates statistically significant, p ≤ 0.05 based on Student’s t-tests.
Mentions: HPLC analysis of stem cell wall sugar composition showed that glucose and xylose were the most abundant sugars in all samples as expected (Table 1). Among glucose, xylose, galactose, arabinose and mannose, significant changes were observed only in mannose levels, with lower mannose levels in transgenic samples. While cellulose and lignin content appeared unaffected; the syringyl-to-guaiacyl (S/G) ratio of lignin was significantly reduced in transgenic lines (Figure 5A - C). In order to explore whether overexpression of UGPase2 has any effect on the transcription of carbohydrate metabolism or/and cellulose pathway, a subset of sugar metabolism and cellulose synthesis-related genes were studied (Figure 6). The panel of genes that we studied included, sucrose transporters, SUT3, linked previously to enhanced cellulose production [26], and SUT4, linked to carbon partitioning [27]; genes that encode sucrose hydrolyzing enzymes [1], vacuolar invertases, VIN2 and VIN3; neutral invertase, NIN8 [27]. Additionally, the secondary cell wall associated cellulose synthase (CesA) genes, CesA7B and CesA8B, and KORRIGAN (KOR) genes, KOR1 and KOR2, were included based on previously reports of proposed roles in cellulose biosynthesis [28-30]. SuSy1 and Susy2 transcripts and corresponding proteins have been shown to be elevated in tissue contexts with enhanced cellulose biosynthesis such as secondary xylem development and tension stress response [31,32]. Furthermore, a functional role for SuSy in supplying sugars for cellulose synthesis has been proposed [33]. The expression of most sugar metabolism genes was not significantly altered except SUT3 which was significantly higher in all the three transgenic lines (Figure 6A). Whether SUT3 has a direct role in sucrose transport and carbon partitioning in Populus is as yet unanswered. Of the surveyed cellulose pathway genes, CesA and SuSy isoforms were not significantly altered, but KOR1 and KOR2 were significantly increased in young leaves of transgenic lines (Figure 6B). These results suggest that overexpression of PdUGPase2 may be associated with modest changes in transcript levels of cellulose pathway-associated genes but that does not translate into changes in cellulose levels (Figure 5A). The unchanged cellulose levels in overexpression lines of PdUGPase2 contradicts with previous reports where cellulose levels were increased in AxUGPase overexpression lines [15]. While the implication of low sequence identity between the bacterial and Populus UGPase isoforms on enzyme activity is yet to be clarified, the functional activity of AxUGPase has shown to complement cellulose negative mutant phenotype by channeling sugar substrate for cellulose synthesis [34].Table 1

Bottom Line: Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth.While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced.These results demonstrate that PdUGPase2 plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of Populus.

View Article: PubMed Central - PubMed

ABSTRACT

Background: UDP-glucose pyrophosphorylase (UGPase) is a sugar-metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and UTP. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in perennial woody plants is poorly understood.

Results: We characterized the functional role of a UGPase gene in Populus deltoides, PdUGPase2. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of PdUGPase2 results in perturbations in primary, as well as secondary metabolism, resulting in reduced sugar and starch levels and increased phenolics, such as caffeoyl and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced.

Conclusions: These results demonstrate that PdUGPase2 plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of Populus.

Show MeSH