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. 2010 Sep;154(1):55-66.
doi: 10.1104/pp.110.157776. Epub 2010 Jul 6.

Virus-induced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduces drought stress tolerance in Nicotiana benthamiana

Gavin M George  1 , Margaretha J van der Merwe, Adriano Nunes-Nesi, Rolene Bauer, Alisdair R Fernie, Jens Kossmann, James R Lloyd
Affiliations

Virus-induced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduces drought stress tolerance in Nicotiana benthamiana

Gavin M George et al. Plant Physiol. 2010 Sep.

Abstract

The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes.

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Figures

Figure 1.
Figure 1.
Pyrophosphatase activity measurements and PPi levels in transiently repressed psPPase in N. benthamiana leaves. A, In-gel assays for soluble inorganic pyrophosphatase activity of N. benthamiana plastid-enriched fraction (lane 1), crude protein extract (lane 2), infiltrated TRV2 control (lane 3), and TRV2-psPPase-silenced plants (lane 4). B, Immunoblotting of 30 μg of crude protein extracts for cytosolic sPPase expression in TRV2 control and psPPase-silenced plants. C, Maximal catalytic pyrophosphatase activity of soluble crude protein extract from TRV2 control and TRV2-psPPase-silenced leaves. D, Total cellular PPi concentrations of TRV2 control and TRV2-psPPase-silenced leaves. FW, Fresh weight. Values are presented as means ± se of five individual plants; values with an asterisk were determined by Student’s t test to be significantly different (P < 0.05) from the TRV2 control.
Figure 2.
Figure 2.
Phenotypical characteristics and physiological assessment of carbon assimilation of down-regulated soluble plastidial pyrophosphatase activity in N. benthamiana source leaves. A and B, TRV2 control (A) and TRV2-psPPase-silenced (B) leaves grown under prevailing greenhouse conditions. C, Photosynthetic carbon assimilation at both ambient (380 μmol mol−1; black bars) and saturated (1,000 μmol mol−1; gray bars) intercellular CO2 concentrations in TRV2 control and TRV2-psPPase-silenced plants. Values are presented as means ± se of four individual plants; values with an asterisk were determined by Student’s t test to be significantly different (P < 0.05) from the respective TRV2 control.
Figure 3.
Figure 3.
Alterations in starch, protein, and Rubisco transcript levels in down-regulated soluble plastidial pyrophosphatase activity in N. benthamiana leaves. A, Change in starch content of dark-adapted leaf discs from TRV2 control and silenced plants after 6 h (black bars) and 24 h (gray bar) of incubation on 1.5% (w/v) Suc. B, Maximal catalytic activity of ADP-Glc pyrophosphorylase (AGPase) from TRV2 control and TRV2-psPPase leaf protein extracts expressed in relation to fresh weight (black bars) and total soluble protein (gray bars). FW, Fresh weight. C, Total soluble protein content of leaf discs of TRV2 control and silenced plants. D, Soluble protein amounts of control and psPPase-silenced 64-mm2 leaf discs, extracted in equal volumes of buffer and 60 μL of supernatant separated on either 10% (w/v; left) or 12% (w/v; right) SDS-PAGE, and stained with Coomassie Brilliant Blue. Molecular mass markers are indicated on the left of each gel. The large (rbcL) and small (rbcS) subunits of Rubisco are indicated by arrows. E, Relative mRNA accumulation of rbcS (black bars) and rbcL (gray bars) subunits in TRV2 and TRV2-psPPase leaves determined by semiquantitative RT-PCR. Transcript levels are represented as the expression ratio of the respective rbc subunit and β-actin gene. Values are presented as means ± se of four/five individual plants; values with an asterisk were determined by Student’s t test to be significantly different (P < 0.05) from the TRV2 control.
Figure 4.
Figure 4.
Relative metabolite content of TRV2 control and TRV2-psPPase plants. Metabolite levels were determined by GC-MS technology. Data are normalized with respect to the mean response calculated for the TRV2 unstressed control (to allow statistical assessment in the same way). Values are presented as means ± se of five individual plants per genotype/treatment, and the data were analyzed using one-way ANOVA followed by Fisher’s lsd test; bars that do not share letters are significantly different (P < 0.05) from each other. FW, Fresh weight; 3-PGA, 3-phosphoglyceraldehyde; 1-Pyrr 2-carboxylate, 1-pyrroline 2-carboxylate.
Figure 5.
Figure 5.
Uridinylate and adenylate levels of VIGS-repressed psPPase activity in N. benthamiana leaves. UDP-Glc (A), ATP (B), UDP (C), and ADP (D) levels were determined by HPLC. Data are presented as means ± se of four individual plants per treatment. The data were analyzed using one-way ANOVA followed by Fisher’s lsd test; bars that do not share letters are significantly different (P < 0.05) from each other. FW, Fresh weight.
Figure 6.
Figure 6.
Physiological and biochemical assessment of drought tolerance in TRV2 control and TRV2-psPPase plants. A, Wilting response of TRV2 control (left) and TRV2-psPPase-silenced (right) plants. B, Stomatal conductance measurements under well-watered and 12-h drought-induced conditions in TRV2 control and TRV2-psPPase-silenced plants. C, PPi levels in TRV2 control and TRV2-psPPase-stressed plants. D to F, ABA (D), GA3 (E), and indole 3-acetic acid (IAA; F) concentrations measured in the leaves under similar conditions under well-watered and stress conditions. Values are presented as means ± se of five individual plants, statistically analyzed using one-way ANOVA followed by Fisher’s lsd; bars that do not share letters are significantly different (P < 0.05) from each other except for the PPi analysis, where a value with an asterisk was determined by Student’s t test to be significantly different (P < 0.05) from the stressed TRV2 control. FW, Fresh weight.
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References

    1. Aharoni A, Jongsma MA, Bouwmeester HJ. (2005) Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Sci 10: 594–602 - PubMed
    1. Amir J, Cherry JH. (1972) Purification and properties of adenosine diphosphoglucose pyrophosphorylase from sweet corn. Plant Physiol 49: 893–897 - PMC - PubMed
    1. Baltscheffsky M, Schultz A, Baltscheffsky H. (1999) H+-PPases: a tightly membrane-bound family. FEBS Lett 457: 527–533 - PubMed
    1. Bradford MM. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72: 248–252 - PubMed
    1. Chappell J. (2002) The genetics and molecular genetics of terpene and sterol origami. Curr Opin Plant Biol 5: 151–157 - PubMed

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