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The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice.

Shi J, Hu H, Zhang K, Zhang W, Yu Y, Wu Z, Wu P - J. Exp. Bot. (2013)

Bottom Line: In vitro and in vivo protein-protein interaction analyses indicated that these two proteins can form homodimers and heterodimers, also implying their functional redundancy.Genetic interaction analysis indicated that SPX3/5 are functional repressors of OsPHR2 (PHR2), the rice orthologue of the central regulator AtPHR1 for Pi homeostasis and Pi signalling.These results suggest that the evolution of the additional redundant paralogous SPX genes is beneficial to plants recovering Pi homeostasis after Pi starvation by PHR2 pathway.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
The importance of SPX-domain-containing proteins to phosphate (Pi) homeostasis and signalling transduction has been established in plants. In this study, phylogenetic analysis revealed that OsSPX3 and OsSPX5 (SPX3/5) are paralogous SPX genes ( SYG1/Pho81/XPR1) in cereal crops. SPX3/5 are specifically responsive to Pi starvation at both the transcriptional and post-transcriptional levels. Similar tissue expression patterns of the two genes and proteins were identified by in situ hybridization and the transgenic plants harbouring SPX3pro-SPX3-GUS or SPX5pro-SPX5-GUS fusions, respectively. Both SPX3/5 are localized in the nucleus and cytoplasm in rice protoplasts and plants. SPX3/5 negatively regulate root-to-shoot Pi translocation with redundant function. The data showed that the Pi-starvation-accumulated SPX3/5 proteins are players in restoring phosphate balance following phosphate starvation. In vitro and in vivo protein-protein interaction analyses indicated that these two proteins can form homodimers and heterodimers, also implying their functional redundancy. Genetic interaction analysis indicated that SPX3/5 are functional repressors of OsPHR2 (PHR2), the rice orthologue of the central regulator AtPHR1 for Pi homeostasis and Pi signalling. These results suggest that the evolution of the additional redundant paralogous SPX genes is beneficial to plants recovering Pi homeostasis after Pi starvation by PHR2 pathway.

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SPX3 and SPX5 redundantly function on Pi homeostasis. (A and B) Phenotypic performances of 30-d-old wild-type (WT) and spx3/RiSPX5 plants under 200 µM Pi (A) and 20 µM Pi (B) conditions; bar, 20cm. (C and D) Dried biomass of 30-d-old WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants. (E and G) Cellular Pi concentration in the shoots (E) and roots (G) of WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants grown under 200 or 20 µM Pi conditions. (F) Uptake rate of [33P] Pi in WT and spx3/RiSPX5 plants. (H) The shoot-to-root ratio of [33P] Pi concentration of WT and spx3/RiSPX5 plants. Plants were supplied with 33P-labelled Pi (H333PO4) for 6, 12, and 24h. Values represent mean ± standard deviation of five biological replicates. Data significantly different from the corresponding wild-type controls are indicated *P < 0.05; (**P < 0.01, Student’s t-test).
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Figure 4: SPX3 and SPX5 redundantly function on Pi homeostasis. (A and B) Phenotypic performances of 30-d-old wild-type (WT) and spx3/RiSPX5 plants under 200 µM Pi (A) and 20 µM Pi (B) conditions; bar, 20cm. (C and D) Dried biomass of 30-d-old WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants. (E and G) Cellular Pi concentration in the shoots (E) and roots (G) of WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants grown under 200 or 20 µM Pi conditions. (F) Uptake rate of [33P] Pi in WT and spx3/RiSPX5 plants. (H) The shoot-to-root ratio of [33P] Pi concentration of WT and spx3/RiSPX5 plants. Plants were supplied with 33P-labelled Pi (H333PO4) for 6, 12, and 24h. Values represent mean ± standard deviation of five biological replicates. Data significantly different from the corresponding wild-type controls are indicated *P < 0.05; (**P < 0.01, Student’s t-test).

Mentions: This work isolated a spx3 mutant (Nipponbare, ALNE05) from the CIRAD T-DNA insertion library (Supplementary Fig. S3A–C). Because the spx5 mutant is unavailable, the transgenic plants (Nipponbare) with repression of SPX5 were developed using RNAi (Supplementary Fig. S3D, G). The plants with repression of SPX5 (RiSPX5) under the spx3 mutant background were developed by a cross between the homozygous spx3 mutant and plants harbouring a single copy of a plasmid containing the SPX5-RNAi vector (designated spx3/RiSPX5). The wild-type plants (Nipponbare), spx3 mutants, two independent lines of RiSPX5 plants (RiSPX5-1 and RiSPX5-3), and spx3/RiSPX5 plants were used for cellular Pi concentration and Pi signalling analyses in hydroponic cultures. Under both high Pi (200 µM Pi) and low Pi (20 µM Pi) conditions, no significant phenotypic difference between wild-type plants and spx3 mutants or RiSPX5 plants was observed (data not shown), while the growth of spx3/RiSPX5 plants was significantly inhibited compared to wild-type plants (Fig. 4A–D). A significantly higher shoot Pi concentration was observed in spx3/RiSPX5 plants compared to wild-type plants, but not in spx3 mutants or RiSPX5 plants (Fig. 4E). No statistically significant difference was observed in root Pi concentration between wild-type and spx3/RiSPX5 plants under either Pi condition (Fig. 4G). The significantly higher Pi-uptake ability and shoot-to-root ratio of Pi in spx3/RiSPX5 plants compared to wild-type plants were confirmed by 33P-labelled Pi uptake and concentration ratio of shoots to roots (Fig. 4F, H). These results indicated the redundant negative effect of SPX3 and SPX5 on Pi homeostasis.


The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice.

Shi J, Hu H, Zhang K, Zhang W, Yu Y, Wu Z, Wu P - J. Exp. Bot. (2013)

SPX3 and SPX5 redundantly function on Pi homeostasis. (A and B) Phenotypic performances of 30-d-old wild-type (WT) and spx3/RiSPX5 plants under 200 µM Pi (A) and 20 µM Pi (B) conditions; bar, 20cm. (C and D) Dried biomass of 30-d-old WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants. (E and G) Cellular Pi concentration in the shoots (E) and roots (G) of WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants grown under 200 or 20 µM Pi conditions. (F) Uptake rate of [33P] Pi in WT and spx3/RiSPX5 plants. (H) The shoot-to-root ratio of [33P] Pi concentration of WT and spx3/RiSPX5 plants. Plants were supplied with 33P-labelled Pi (H333PO4) for 6, 12, and 24h. Values represent mean ± standard deviation of five biological replicates. Data significantly different from the corresponding wild-type controls are indicated *P < 0.05; (**P < 0.01, Student’s t-test).
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Figure 4: SPX3 and SPX5 redundantly function on Pi homeostasis. (A and B) Phenotypic performances of 30-d-old wild-type (WT) and spx3/RiSPX5 plants under 200 µM Pi (A) and 20 µM Pi (B) conditions; bar, 20cm. (C and D) Dried biomass of 30-d-old WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants. (E and G) Cellular Pi concentration in the shoots (E) and roots (G) of WT, spx3, RiSPX5-1, RiSPX5-3, and spx3/RiSPX5 plants grown under 200 or 20 µM Pi conditions. (F) Uptake rate of [33P] Pi in WT and spx3/RiSPX5 plants. (H) The shoot-to-root ratio of [33P] Pi concentration of WT and spx3/RiSPX5 plants. Plants were supplied with 33P-labelled Pi (H333PO4) for 6, 12, and 24h. Values represent mean ± standard deviation of five biological replicates. Data significantly different from the corresponding wild-type controls are indicated *P < 0.05; (**P < 0.01, Student’s t-test).
Mentions: This work isolated a spx3 mutant (Nipponbare, ALNE05) from the CIRAD T-DNA insertion library (Supplementary Fig. S3A–C). Because the spx5 mutant is unavailable, the transgenic plants (Nipponbare) with repression of SPX5 were developed using RNAi (Supplementary Fig. S3D, G). The plants with repression of SPX5 (RiSPX5) under the spx3 mutant background were developed by a cross between the homozygous spx3 mutant and plants harbouring a single copy of a plasmid containing the SPX5-RNAi vector (designated spx3/RiSPX5). The wild-type plants (Nipponbare), spx3 mutants, two independent lines of RiSPX5 plants (RiSPX5-1 and RiSPX5-3), and spx3/RiSPX5 plants were used for cellular Pi concentration and Pi signalling analyses in hydroponic cultures. Under both high Pi (200 µM Pi) and low Pi (20 µM Pi) conditions, no significant phenotypic difference between wild-type plants and spx3 mutants or RiSPX5 plants was observed (data not shown), while the growth of spx3/RiSPX5 plants was significantly inhibited compared to wild-type plants (Fig. 4A–D). A significantly higher shoot Pi concentration was observed in spx3/RiSPX5 plants compared to wild-type plants, but not in spx3 mutants or RiSPX5 plants (Fig. 4E). No statistically significant difference was observed in root Pi concentration between wild-type and spx3/RiSPX5 plants under either Pi condition (Fig. 4G). The significantly higher Pi-uptake ability and shoot-to-root ratio of Pi in spx3/RiSPX5 plants compared to wild-type plants were confirmed by 33P-labelled Pi uptake and concentration ratio of shoots to roots (Fig. 4F, H). These results indicated the redundant negative effect of SPX3 and SPX5 on Pi homeostasis.

Bottom Line: In vitro and in vivo protein-protein interaction analyses indicated that these two proteins can form homodimers and heterodimers, also implying their functional redundancy.Genetic interaction analysis indicated that SPX3/5 are functional repressors of OsPHR2 (PHR2), the rice orthologue of the central regulator AtPHR1 for Pi homeostasis and Pi signalling.These results suggest that the evolution of the additional redundant paralogous SPX genes is beneficial to plants recovering Pi homeostasis after Pi starvation by PHR2 pathway.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
The importance of SPX-domain-containing proteins to phosphate (Pi) homeostasis and signalling transduction has been established in plants. In this study, phylogenetic analysis revealed that OsSPX3 and OsSPX5 (SPX3/5) are paralogous SPX genes ( SYG1/Pho81/XPR1) in cereal crops. SPX3/5 are specifically responsive to Pi starvation at both the transcriptional and post-transcriptional levels. Similar tissue expression patterns of the two genes and proteins were identified by in situ hybridization and the transgenic plants harbouring SPX3pro-SPX3-GUS or SPX5pro-SPX5-GUS fusions, respectively. Both SPX3/5 are localized in the nucleus and cytoplasm in rice protoplasts and plants. SPX3/5 negatively regulate root-to-shoot Pi translocation with redundant function. The data showed that the Pi-starvation-accumulated SPX3/5 proteins are players in restoring phosphate balance following phosphate starvation. In vitro and in vivo protein-protein interaction analyses indicated that these two proteins can form homodimers and heterodimers, also implying their functional redundancy. Genetic interaction analysis indicated that SPX3/5 are functional repressors of OsPHR2 (PHR2), the rice orthologue of the central regulator AtPHR1 for Pi homeostasis and Pi signalling. These results suggest that the evolution of the additional redundant paralogous SPX genes is beneficial to plants recovering Pi homeostasis after Pi starvation by PHR2 pathway.

Show MeSH
Related in: MedlinePlus