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The reduced plastid-encoded polymerase-dependent plastid gene expression leads to the delayed greening of the Arabidopsis fln2 mutant.

Huang C, Yu QB, Lv RH, Yin QQ, Chen GY, Xu L, Yang ZN - PLoS ONE (2013)

Bottom Line: This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. fln2-4 exhibited delayed greening on sucrose-containing medium.Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1-FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in fln2-4.We proposed the partial PEP activity in the fln2 mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, P.R. China ; Department of Biology, School of Life Sciences, East China Normal University, Shanghai, P.R. China.

ABSTRACT
In Arabidopsis leaf coloration mutants, the delayed greening phenomenon is common. Nonetheless, the mechanism remains largely elusive. Here, a delayed greening mutant fln2-4 of FLN2 (Fructokinase-Like Protein2) was studied. FLN2 is one component of Transcriptionally Active Chromosome (TAC) complex which is thought to contain the complete plastid-encoded polymerase (PEP). fln2-4 displayed albino phenotype on medium without sucrose. The PEP-dependent plastid gene expression and chloroplast development were inhibited in fln2-4. Besides interacting with thioredoxin z (TRX z), we identified that FLN2 interacted with another two members of TAC complex in yeast including its homologous protein FLN1 (Fructokinase-Like Protein1) and pTAC5. This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. fln2-4 exhibited delayed greening on sucrose-containing medium. Comparison of the PEP-dependent gene expression among two complete albino mutants (trx z and ptac14), two yellow mutants (ecb2-2 and ys1) and the fln2-4 showed that fln2-4 remains partial PEP activity. FLN2 and FLN1 are the target proteins of TRX z involved in affecting the PEP activity. Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1-FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in fln2-4. We proposed the partial PEP activity in the fln2 mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.

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The relationships between FLN2 and other components of TAC.(A) Interactions of FLN2 with FLN1, pTAC5 and TRX z proteins in yeast. (B) GST pull-down assay revealed the existence of a physical interaction between FLN2 and its homologous protein FLN1. (C) In vitro GST pull-down assay for interaction between FLN2 and pTAC5. (D) FLN1 can interact with itself in yeast, while FLN2 can not. (E) Non-interaction existed between FLN2 and three essential subunits of TAC complex including RpoA, pTAC12 and pTAC14. (F) The primary working model for TRX z, FLN1, FLN2, pTAC5, pTAC7, pTAC10, pTAC12 and pTAC14.
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pone-0073092-g003: The relationships between FLN2 and other components of TAC.(A) Interactions of FLN2 with FLN1, pTAC5 and TRX z proteins in yeast. (B) GST pull-down assay revealed the existence of a physical interaction between FLN2 and its homologous protein FLN1. (C) In vitro GST pull-down assay for interaction between FLN2 and pTAC5. (D) FLN1 can interact with itself in yeast, while FLN2 can not. (E) Non-interaction existed between FLN2 and three essential subunits of TAC complex including RpoA, pTAC12 and pTAC14. (F) The primary working model for TRX z, FLN1, FLN2, pTAC5, pTAC7, pTAC10, pTAC12 and pTAC14.

Mentions: FLN2 belongs to the components of TAC complex in Arabidopsis[15]. To establish the relationship between FLN2 and the other components of TAC, we performed a yeast two-hybrid screen. FLN2 was fused to the GAL4 DNA-binding domain (BD) as a bait to screen a pool composed of the thirty-five reported TAC components fused to the GAL4 activation domain (AD). After yeast transformation, we randomly selected clones with blue appearance in selective dropout (SD) medium lacking trptophan (Trp), leucine (Leu), histidine (His) and adenine hemisulfate salt (Ade) with X-α-gal for PCR amplification. Sequencing the PCR products identified three genes including FLN1, pTAC5 and TRX z. Yeast two-hybrid experiments verified the interactions between FLN2 and the three proteins (Figure 3A). We subsequently performed pull-down assay to further confirm the interactions between FLN2 and FLN1 or pTAC5. Results showed that both the recombinant proteins GST-FLN1 and GST-pTAC5 were able to pull down FLN2-His (Figure 3B and C). FLN2 and FLN1 belong to the pfkB family, and share high peptide similarity [37]. To examine whether the FLN1 and FLN2 proteins form homodimers, we analyzed the interactions between them by yeast two-hybrid assay. Results showed that FLN1 can interact with itself, but FLN2 can not (Figure 3D). RpoA, pTAC12 and pTAC14 are the essential subunits in the PEP complex [18]. Yeast two-hybrid assay showed that FLN2 did not interact with these essential subunits in yeast (Figure 3E). Based on previous investigations [21], [22] and data in this study, a working model for partial TAC components is proposed (Figure 3F). In this model, pTAC7 interacts with FLN1, pTAC10, pTAC12 and pTAC14 while FLN2 interacts with FLN1, TRX z and pTAC5.


The reduced plastid-encoded polymerase-dependent plastid gene expression leads to the delayed greening of the Arabidopsis fln2 mutant.

Huang C, Yu QB, Lv RH, Yin QQ, Chen GY, Xu L, Yang ZN - PLoS ONE (2013)

The relationships between FLN2 and other components of TAC.(A) Interactions of FLN2 with FLN1, pTAC5 and TRX z proteins in yeast. (B) GST pull-down assay revealed the existence of a physical interaction between FLN2 and its homologous protein FLN1. (C) In vitro GST pull-down assay for interaction between FLN2 and pTAC5. (D) FLN1 can interact with itself in yeast, while FLN2 can not. (E) Non-interaction existed between FLN2 and three essential subunits of TAC complex including RpoA, pTAC12 and pTAC14. (F) The primary working model for TRX z, FLN1, FLN2, pTAC5, pTAC7, pTAC10, pTAC12 and pTAC14.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760890&req=5

pone-0073092-g003: The relationships between FLN2 and other components of TAC.(A) Interactions of FLN2 with FLN1, pTAC5 and TRX z proteins in yeast. (B) GST pull-down assay revealed the existence of a physical interaction between FLN2 and its homologous protein FLN1. (C) In vitro GST pull-down assay for interaction between FLN2 and pTAC5. (D) FLN1 can interact with itself in yeast, while FLN2 can not. (E) Non-interaction existed between FLN2 and three essential subunits of TAC complex including RpoA, pTAC12 and pTAC14. (F) The primary working model for TRX z, FLN1, FLN2, pTAC5, pTAC7, pTAC10, pTAC12 and pTAC14.
Mentions: FLN2 belongs to the components of TAC complex in Arabidopsis[15]. To establish the relationship between FLN2 and the other components of TAC, we performed a yeast two-hybrid screen. FLN2 was fused to the GAL4 DNA-binding domain (BD) as a bait to screen a pool composed of the thirty-five reported TAC components fused to the GAL4 activation domain (AD). After yeast transformation, we randomly selected clones with blue appearance in selective dropout (SD) medium lacking trptophan (Trp), leucine (Leu), histidine (His) and adenine hemisulfate salt (Ade) with X-α-gal for PCR amplification. Sequencing the PCR products identified three genes including FLN1, pTAC5 and TRX z. Yeast two-hybrid experiments verified the interactions between FLN2 and the three proteins (Figure 3A). We subsequently performed pull-down assay to further confirm the interactions between FLN2 and FLN1 or pTAC5. Results showed that both the recombinant proteins GST-FLN1 and GST-pTAC5 were able to pull down FLN2-His (Figure 3B and C). FLN2 and FLN1 belong to the pfkB family, and share high peptide similarity [37]. To examine whether the FLN1 and FLN2 proteins form homodimers, we analyzed the interactions between them by yeast two-hybrid assay. Results showed that FLN1 can interact with itself, but FLN2 can not (Figure 3D). RpoA, pTAC12 and pTAC14 are the essential subunits in the PEP complex [18]. Yeast two-hybrid assay showed that FLN2 did not interact with these essential subunits in yeast (Figure 3E). Based on previous investigations [21], [22] and data in this study, a working model for partial TAC components is proposed (Figure 3F). In this model, pTAC7 interacts with FLN1, pTAC10, pTAC12 and pTAC14 while FLN2 interacts with FLN1, TRX z and pTAC5.

Bottom Line: This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. fln2-4 exhibited delayed greening on sucrose-containing medium.Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1-FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in fln2-4.We proposed the partial PEP activity in the fln2 mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, P.R. China ; Department of Biology, School of Life Sciences, East China Normal University, Shanghai, P.R. China.

ABSTRACT
In Arabidopsis leaf coloration mutants, the delayed greening phenomenon is common. Nonetheless, the mechanism remains largely elusive. Here, a delayed greening mutant fln2-4 of FLN2 (Fructokinase-Like Protein2) was studied. FLN2 is one component of Transcriptionally Active Chromosome (TAC) complex which is thought to contain the complete plastid-encoded polymerase (PEP). fln2-4 displayed albino phenotype on medium without sucrose. The PEP-dependent plastid gene expression and chloroplast development were inhibited in fln2-4. Besides interacting with thioredoxin z (TRX z), we identified that FLN2 interacted with another two members of TAC complex in yeast including its homologous protein FLN1 (Fructokinase-Like Protein1) and pTAC5. This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. fln2-4 exhibited delayed greening on sucrose-containing medium. Comparison of the PEP-dependent gene expression among two complete albino mutants (trx z and ptac14), two yellow mutants (ecb2-2 and ys1) and the fln2-4 showed that fln2-4 remains partial PEP activity. FLN2 and FLN1 are the target proteins of TRX z involved in affecting the PEP activity. Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1-FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in fln2-4. We proposed the partial PEP activity in the fln2 mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.

Show MeSH
Related in: MedlinePlus