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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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T-DNA insertion sites of fln2 and their phenotypes.(A) The sketch map of the FLN2 gene, the T-DNA insertion sites of fln2 mutants. Boxes, exons; lines, introns. (B) The expression of FLN2 in WT (Col-0), fln2–3 and fln2–4. (C) Photographs of the fln2–3 and fln2–4 seedlings grown on MS medium for 7 days. (D) Ultrastructural analysis of chloroplasts from 7-day-old WT and fln2–4 seedlings grown on MS medium. Scale bars: 1 µm. (E) Phenotypes of WT and the fln2–4 complemented seedlings grown in soil.
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pone-0073092-g001: T-DNA insertion sites of fln2 and their phenotypes.(A) The sketch map of the FLN2 gene, the T-DNA insertion sites of fln2 mutants. Boxes, exons; lines, introns. (B) The expression of FLN2 in WT (Col-0), fln2–3 and fln2–4. (C) Photographs of the fln2–3 and fln2–4 seedlings grown on MS medium for 7 days. (D) Ultrastructural analysis of chloroplasts from 7-day-old WT and fln2–4 seedlings grown on MS medium. Scale bars: 1 µm. (E) Phenotypes of WT and the fln2–4 complemented seedlings grown in soil.

Mentions: To analyze the functional roles of FLN2 gene during plant growth and development, we obtained two T-DNA insertion lines, SALK_005734 and CS811853, from the Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu/). In these two lines, the T-DNA was inserted in the 3rd exon and the 5th exon of the FLN2 gene, respectively (Figure 1A). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the FLN2 transcript was absent in both mutants (Figure 1B). Due to the SALK_005734 line named as fln2–3[19], the CS811853 line in this work was termed as fln2–4. Both fln2–3 and fln2–4 displayed albino cotyledons and were seedling lethal on MS medium without sucrose (Figure 1C). The phenotypes of the two allelic mutants were similar; thereby the fln2–4 mutant was chosen for further analysis. Transmission electron microscopy (TEM) observations revealed that the chloroplasts in the 7-day-old fln2–4 mutants had a visible change in ultrastructural organization with irregular morphology and lacked internal membrane structures (Figure 1D). To confirm that the knockout of FLN2 was responsible for the defects in the fln2–4 phenotype, a construct containing the genomic sequence of the FLN2 gene, as well as 1517-base pair (bp) upstream and the FLAG sequence was introduced into the heterozygous plant (FLN2/fln2–4). A total of 53 transgenic plants were obtained. Six of them were identified to be homozygous for the T-DNA insertion, and exhibited normal morphology as the wild type (WT) (Figure 1E). These results demonstrate that the FLN2 gene is responsible for the defective phenotype in fln2–4 mutant, and FLN2 is important for chloroplast development and seedling growth.


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)

T-DNA insertion sites of fln2 and their phenotypes.(A) The sketch map of the FLN2 gene, the T-DNA insertion sites of fln2 mutants. Boxes, exons; lines, introns. (B) The expression of FLN2 in WT (Col-0), fln2–3 and fln2–4. (C) Photographs of the fln2–3 and fln2–4 seedlings grown on MS medium for 7 days. (D) Ultrastructural analysis of chloroplasts from 7-day-old WT and fln2–4 seedlings grown on MS medium. Scale bars: 1 µm. (E) Phenotypes of WT and the fln2–4 complemented seedlings grown in soil.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760890&req=5

pone-0073092-g001: T-DNA insertion sites of fln2 and their phenotypes.(A) The sketch map of the FLN2 gene, the T-DNA insertion sites of fln2 mutants. Boxes, exons; lines, introns. (B) The expression of FLN2 in WT (Col-0), fln2–3 and fln2–4. (C) Photographs of the fln2–3 and fln2–4 seedlings grown on MS medium for 7 days. (D) Ultrastructural analysis of chloroplasts from 7-day-old WT and fln2–4 seedlings grown on MS medium. Scale bars: 1 µm. (E) Phenotypes of WT and the fln2–4 complemented seedlings grown in soil.
Mentions: To analyze the functional roles of FLN2 gene during plant growth and development, we obtained two T-DNA insertion lines, SALK_005734 and CS811853, from the Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu/). In these two lines, the T-DNA was inserted in the 3rd exon and the 5th exon of the FLN2 gene, respectively (Figure 1A). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the FLN2 transcript was absent in both mutants (Figure 1B). Due to the SALK_005734 line named as fln2–3[19], the CS811853 line in this work was termed as fln2–4. Both fln2–3 and fln2–4 displayed albino cotyledons and were seedling lethal on MS medium without sucrose (Figure 1C). The phenotypes of the two allelic mutants were similar; thereby the fln2–4 mutant was chosen for further analysis. Transmission electron microscopy (TEM) observations revealed that the chloroplasts in the 7-day-old fln2–4 mutants had a visible change in ultrastructural organization with irregular morphology and lacked internal membrane structures (Figure 1D). To confirm that the knockout of FLN2 was responsible for the defects in the fln2–4 phenotype, a construct containing the genomic sequence of the FLN2 gene, as well as 1517-base pair (bp) upstream and the FLAG sequence was introduced into the heterozygous plant (FLN2/fln2–4). A total of 53 transgenic plants were obtained. Six of them were identified to be homozygous for the T-DNA insertion, and exhibited normal morphology as the wild type (WT) (Figure 1E). These results demonstrate that the FLN2 gene is responsible for the defective phenotype in fln2–4 mutant, and FLN2 is important for chloroplast development and seedling growth.

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