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Increased biomass, seed yield and stress tolerance is conferred in Arabidopsis by a novel enzyme from the resurrection grass Sporobolus stapfianus that glycosylates the strigolactone analogue GR24.

Islam S, Griffiths CA, Blomstedt CK, Le TN, Gaff DF, Hamill JD, Neale AD - PLoS ONE (2013)

Bottom Line: Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted in the identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT).Here, we examine the effects of introducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana.Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme acting downstream of ABA to reduce drought-induced senescence.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Monash University, Melbourne, Victoria, Australia.

ABSTRACT
Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted in the identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT). Here, we examine the effects of introducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana. Results show that Arabidopsis plants constitutively over-expressing SDG8i exhibit enhanced growth, reduced senescence, cold tolerance and a substantial improvement in protoplasmic drought tolerance. We hypothesise that expression of SDG8i in Arabidopsis negatively affects the bioactivity of metabolite/s that mediate/s environmentally-induced repression of cell division and expansion, both during normal development and in response to stress. The phenotype of transgenic plants over-expressing SDG8i suggests modulation in activities of both growth- and stress-related hormones. Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme acting downstream of ABA to reduce drought-induced senescence. Analysis of the in vitro activity of the UGT recombinant protein product demonstrates that SDG8i can glycosylate the synthetic strigolactone analogue GR24, evoking a link with strigolactone-related processes in vivo. The large improvements observed in survival of transgenic Arabidopsis plants under cold-, salt- and drought-stress, as well as the substantial increases in growth rate and seed yield under non-stress conditions, indicates that overexpression of SDG8i in crop plants may provide a novel means of increasing plant productivity.

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Analysis of the enzyme activity of SDG8i in vitro.(A) The glycosyltransferase activity of SDG8i recombinant protein extract using the plant hormones gibberillin (GA3), salicylic acid, kinetin, auxin (IAA), methyl jasmonate, ABA([±]-cis, trans-abscisic acid and the synthetic strigolactone analogue GR24 as substrate (p < 0.05). The coupled enzyme assay [9] was conducted at pH 7.4 using 25μg of protein extract with a final substrate concentration of 1mM. Rates were calculated per mg total protein extract. GR24 was obtained from Chiralix B.V. Nijmegen,The Netherlands. All other hormones were obtained from Sigma St. Louis, MO.(B) A Lineweaver-Burke plot of SDG8i activity with varying concentrations of GR24 indicating a Km of 0.349mM and a Vmax of 5.67 μmole/min/mg. Rates were calculated per mg total protein extract.(C) Level of stimulation of germination of Orobanche seeds in response to root induction by wildtype Col-O and SDG8i transgenic Arabidopsis, sorghum and S. stapfianus seedlings in vitro. The percent germination was calculated by counting the number of seeds having an emerged radicle. Values are the means ± SE of 5 replicates.(D) Effect of GR24 (0 or 5 µM) on bud outgrowth of wild-type Col-0, SDG8i transgenic and max2 Arabidopsis plants. Plants were treated with GR24 on the rosette shoot meristem, axillary buds and leaf axils every third day for 20 days and the number of branches was counted after 43 days. Data are means ± 4 replicates.
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pone-0080035-g006: Analysis of the enzyme activity of SDG8i in vitro.(A) The glycosyltransferase activity of SDG8i recombinant protein extract using the plant hormones gibberillin (GA3), salicylic acid, kinetin, auxin (IAA), methyl jasmonate, ABA([±]-cis, trans-abscisic acid and the synthetic strigolactone analogue GR24 as substrate (p < 0.05). The coupled enzyme assay [9] was conducted at pH 7.4 using 25μg of protein extract with a final substrate concentration of 1mM. Rates were calculated per mg total protein extract. GR24 was obtained from Chiralix B.V. Nijmegen,The Netherlands. All other hormones were obtained from Sigma St. Louis, MO.(B) A Lineweaver-Burke plot of SDG8i activity with varying concentrations of GR24 indicating a Km of 0.349mM and a Vmax of 5.67 μmole/min/mg. Rates were calculated per mg total protein extract.(C) Level of stimulation of germination of Orobanche seeds in response to root induction by wildtype Col-O and SDG8i transgenic Arabidopsis, sorghum and S. stapfianus seedlings in vitro. The percent germination was calculated by counting the number of seeds having an emerged radicle. Values are the means ± SE of 5 replicates.(D) Effect of GR24 (0 or 5 µM) on bud outgrowth of wild-type Col-0, SDG8i transgenic and max2 Arabidopsis plants. Plants were treated with GR24 on the rosette shoot meristem, axillary buds and leaf axils every third day for 20 days and the number of branches was counted after 43 days. Data are means ± 4 replicates.

Mentions: The phenotype of the transgenic plants suggests that SDG8i UGT activity is influencing hormone homeostasis. To investigate the catalytic function of SDG8i, the UGT activity of Nicotiana benthamiana leaf extracts infiltrated with an actin (AtACT2)-promoter driven SDG8i construct, using a viral-based system [19], was tested against a number of plant hormones as substrates and compared with UGT activity in extracts infiltrated with a vector-only control. The substrates used were chosen for their known ability to affect plant growth and stress responses. The SDG8i extract showed substantial glycosylation activity of the strigolactone analogue GR24 (Figure 6A) with a Km of 0.349 mM and a Vmax of 5.67 μmole/min/mg (Figure 6B). The activity observed with the other substrates showed very little increase over background endogenous NADH oxidase activity. Similarly, the vector-only control extract showed no substantial activity above background with GR24 or with any of the other substrates. The results indicate that SDG8i encodes a glucosyltransferase with in vitro activity against a strigolactone-like compound.


Increased biomass, seed yield and stress tolerance is conferred in Arabidopsis by a novel enzyme from the resurrection grass Sporobolus stapfianus that glycosylates the strigolactone analogue GR24.

Islam S, Griffiths CA, Blomstedt CK, Le TN, Gaff DF, Hamill JD, Neale AD - PLoS ONE (2013)

Analysis of the enzyme activity of SDG8i in vitro.(A) The glycosyltransferase activity of SDG8i recombinant protein extract using the plant hormones gibberillin (GA3), salicylic acid, kinetin, auxin (IAA), methyl jasmonate, ABA([±]-cis, trans-abscisic acid and the synthetic strigolactone analogue GR24 as substrate (p < 0.05). The coupled enzyme assay [9] was conducted at pH 7.4 using 25μg of protein extract with a final substrate concentration of 1mM. Rates were calculated per mg total protein extract. GR24 was obtained from Chiralix B.V. Nijmegen,The Netherlands. All other hormones were obtained from Sigma St. Louis, MO.(B) A Lineweaver-Burke plot of SDG8i activity with varying concentrations of GR24 indicating a Km of 0.349mM and a Vmax of 5.67 μmole/min/mg. Rates were calculated per mg total protein extract.(C) Level of stimulation of germination of Orobanche seeds in response to root induction by wildtype Col-O and SDG8i transgenic Arabidopsis, sorghum and S. stapfianus seedlings in vitro. The percent germination was calculated by counting the number of seeds having an emerged radicle. Values are the means ± SE of 5 replicates.(D) Effect of GR24 (0 or 5 µM) on bud outgrowth of wild-type Col-0, SDG8i transgenic and max2 Arabidopsis plants. Plants were treated with GR24 on the rosette shoot meristem, axillary buds and leaf axils every third day for 20 days and the number of branches was counted after 43 days. Data are means ± 4 replicates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818285&req=5

pone-0080035-g006: Analysis of the enzyme activity of SDG8i in vitro.(A) The glycosyltransferase activity of SDG8i recombinant protein extract using the plant hormones gibberillin (GA3), salicylic acid, kinetin, auxin (IAA), methyl jasmonate, ABA([±]-cis, trans-abscisic acid and the synthetic strigolactone analogue GR24 as substrate (p < 0.05). The coupled enzyme assay [9] was conducted at pH 7.4 using 25μg of protein extract with a final substrate concentration of 1mM. Rates were calculated per mg total protein extract. GR24 was obtained from Chiralix B.V. Nijmegen,The Netherlands. All other hormones were obtained from Sigma St. Louis, MO.(B) A Lineweaver-Burke plot of SDG8i activity with varying concentrations of GR24 indicating a Km of 0.349mM and a Vmax of 5.67 μmole/min/mg. Rates were calculated per mg total protein extract.(C) Level of stimulation of germination of Orobanche seeds in response to root induction by wildtype Col-O and SDG8i transgenic Arabidopsis, sorghum and S. stapfianus seedlings in vitro. The percent germination was calculated by counting the number of seeds having an emerged radicle. Values are the means ± SE of 5 replicates.(D) Effect of GR24 (0 or 5 µM) on bud outgrowth of wild-type Col-0, SDG8i transgenic and max2 Arabidopsis plants. Plants were treated with GR24 on the rosette shoot meristem, axillary buds and leaf axils every third day for 20 days and the number of branches was counted after 43 days. Data are means ± 4 replicates.
Mentions: The phenotype of the transgenic plants suggests that SDG8i UGT activity is influencing hormone homeostasis. To investigate the catalytic function of SDG8i, the UGT activity of Nicotiana benthamiana leaf extracts infiltrated with an actin (AtACT2)-promoter driven SDG8i construct, using a viral-based system [19], was tested against a number of plant hormones as substrates and compared with UGT activity in extracts infiltrated with a vector-only control. The substrates used were chosen for their known ability to affect plant growth and stress responses. The SDG8i extract showed substantial glycosylation activity of the strigolactone analogue GR24 (Figure 6A) with a Km of 0.349 mM and a Vmax of 5.67 μmole/min/mg (Figure 6B). The activity observed with the other substrates showed very little increase over background endogenous NADH oxidase activity. Similarly, the vector-only control extract showed no substantial activity above background with GR24 or with any of the other substrates. The results indicate that SDG8i encodes a glucosyltransferase with in vitro activity against a strigolactone-like compound.

Bottom Line: Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted in the identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT).Here, we examine the effects of introducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana.Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme acting downstream of ABA to reduce drought-induced senescence.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Monash University, Melbourne, Victoria, Australia.

ABSTRACT
Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted in the identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT). Here, we examine the effects of introducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana. Results show that Arabidopsis plants constitutively over-expressing SDG8i exhibit enhanced growth, reduced senescence, cold tolerance and a substantial improvement in protoplasmic drought tolerance. We hypothesise that expression of SDG8i in Arabidopsis negatively affects the bioactivity of metabolite/s that mediate/s environmentally-induced repression of cell division and expansion, both during normal development and in response to stress. The phenotype of transgenic plants over-expressing SDG8i suggests modulation in activities of both growth- and stress-related hormones. Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme acting downstream of ABA to reduce drought-induced senescence. Analysis of the in vitro activity of the UGT recombinant protein product demonstrates that SDG8i can glycosylate the synthetic strigolactone analogue GR24, evoking a link with strigolactone-related processes in vivo. The large improvements observed in survival of transgenic Arabidopsis plants under cold-, salt- and drought-stress, as well as the substantial increases in growth rate and seed yield under non-stress conditions, indicates that overexpression of SDG8i in crop plants may provide a novel means of increasing plant productivity.

Show MeSH
Related in: MedlinePlus