The Direct Involvement of Dark-Induced Tic55 Protein in Chlorophyll Catabolism and Its Indirect Role in the MYB108-NAC Signaling Pathway during Leaf Senescence in Arabidopsis thaliana

Abstract

The chloroplast relies on proteins encoded in the nucleus, synthesized in the cytosol and subsequently transported into chloroplast through the protein complexes Toc and Tic (Translocon at the outer/inner membrane of chloroplasts). A Tic complex member, Tic55, contains a redox-related motif essential for protein import into chloroplasts in peas. However, Tic55 is not crucial for protein import in Arabidopsis. Here, a tic55-II-knockout mutant of Arabidopsis thaliana was characterized for Tic55 localization, its relationship with other translocon proteins, and its association with plant leaf senescence when compared to the wild type. Individually darkened leaves (IDLs) obtained through dark-induced leaf senescence were used to demonstrate chlorophyll breakdown and its relationship with plant senescence in the tic55-II-knockout mutant. The IDLs of the tic55-II-knockout mutant contained higher chlorophyll concentrations than those of the wild type. Our microarray analysis of IDLs during leaf senescence identified seven senescence-associated genes (SAGs) that were downregulated in the tic55-II-knockout mutant: ASP3, APG7, DIN2, DIN11, SAG12, SAG13, and YLS9. Real-time quantitative PCR confirmed the reliability of microarray analysis by showing the same expression patterns with those of the microarray data. Thus, Tic55 functions in dark-induced aging in A. thaliana by indirectly regulating downstream SAGs expression. In addition, the expression of four NAC genes, including ANAC003, ANAC010, ANAC042, and ANAC075 of IDL treated tic55-II-knockout mutant appeared to be downregulated. Yeast one hybrid assay revealed that only ANAC003 promoter region can be bound by MYB108, suggesting that a MYB-NAC regulatory network is involved in dark-stressed senescence.

Keywords: ANAC proteins; MYB108; Tic55 proteins of chloroplasts; dark-induced leaf senescence.

Figure 1

Molecular characterization of T-DNA insertion lines derived from SALK_086048. (A) RT-PCR analysis was performed using RT-F and RT-R primers as indicated in Table S1. Tic55 in A. thaliana is shown as atTic55. Internal control was atACT2, an actin gene in A. thaliana, used to normalize sample loading. (B) Protein gel blotting assay for both total protein and chloroplast protein was performed to detect Tic55 in the wild-type (WT) and knockout mutant (tic55-II) plant extracts by using a Tic55-specific antibody (αTic55) or the housekeeping protein porin (αPorin) and Tic40 (αTic40).

Figure 2

Phenotypes of the wild-type (WT) and tic55-II-knockout mutant lines. (A) WT and tic55-II-knockout (SALK_086048) mutant lines were grown on MS medium side by side for 10 days, and the appearance of seedlings were compared. White bar indicates 1 cm. (B) Comparison of root systems of 10-day-old WT and tic55-II-knockout mutant seedlings, respectively. White bar represents 1 cm. (C) Leaf tissues in (A) were photographed at closer distances for both WT and tic55-II-knockout mutant plants (upper panel). Lower panel shows the number of leaf hairs for both WT and tic55-II-knockout mutant plants. White bar (white line) indicates 1 mm. (D) Similar plants were grown in vitro for 10 days and then transferred to soil and grown for 30 days. White bar represents 2 cm.

Figure 3

Relationship of Tic55 and other translocon proteins. (A) Protein gel blot analysis demonstrating the location of the inner membrane proteins Tic110 and Tic40, the outer membrane protein Toc159, and the stoma protein Hsp93 in tic55-II-knockout or tic40-2 mutants and WT plants. (B) Location of Tic55 and other translocon proteins under redox environments. Chloroplast fraction was treated with oxidized glutathione (GSSG) and reduced glutathione (GSH). Control indicates no treatment. After ultracentrifugation, lipid-soluble (Pe) and water-soluble (Su) protein fractions were separated and examined using protein gel blots to explore the location and complex formation of Tic55 with other translocon proteins. IM: inner membrane. St: stroma. OM: outer membrane.

Figure 4

Tic55 could directly interact with other Tic proteins. Coimmunoprecipitation (Co-IP) analysis was used to determine complex formation between Tic55 and other translocon proteins. Isolated chloroplasts from wild-type A. thaliana were treated with ice for 15 min, followed by hypotonic treatment. Chloroplast membrane proteins were subsequently retrieved, and an immunoprecipitation (IP) assay was performed using either anti-Tic55 antibody or preimmune serum (negative control). Next, protein gel blots were conducted using specific antibodies αTic110, αHsp93, or αTic40 to detect whether a particular translocon protein could be coimmunoprecipitaed to indicate that the protein can form a complex with Tic55 protein.

Figure 5

Dark-induced aging and the importance of Tic55 in senescence. (A) Phenotypes of wild-type (WT) and tic55-II-knockout mutant lines without treatment on both Day 0 and Day 5 (Control Day 0 and Control Day 5, respectively) compared with the IDLs of WT and tic55-II-knockout mutant lines at Day 5 after dark treatment (IDL Day 5). The third and fourth rosette leaves of Day 0 plants were individually covered with foil soon after bolting, and the plants were grown for another five days. (B) Quantitative chlorophyll analysis: The fifth and sixth leaves of 19-day-old seedlings were collected as Day 0 samples. After five days, the fifth and sixth leaves of the control seedlings (without dark treatment) were gathered as Day 5 samples. The fifth and sixth leaves of dark-treated plants were collected five days after treatment started on Day 0 (IDL Day 5). Chlorophyll was extracted and quantified (µg/mL) in leaves gathered from each group. Solid bars represent WT and open bars indicate tic55-II-knockout mutant lines. Three independent experiments were performed and standard deviation was measured. Asterisk (*) depicts p < 0.05.

Figure 6

Regulated genes (log₂-fold change) assigned to functional protein categories based on the gene ontology (GO) classification scheme. These genes are involved in the biological processes (A), cellular components (B), or molecular functions (C). Positive and negative values on the scale indicate the numbers of significantly up- and downregulated genes, respectively.

Figure 7

Validation of microarray expression data by relative real-time quantitative RT-PCR. Validated leaf senescence-related genes, namely ASP3, APG7, DIN2, DIN11, SAG12, SAG13, and YLS9, showed downregulation in the tic55-II-knockout mutant after dark-induced leaf senescence compared with the wild-type (WT) in microarray analysis. Relative transcript levels of these genes were determined using three replicates, and signal intensities for each transcript were normalized with tubulin (internal control). Error bars represent standard deviation. Primers used in PCR reactions are listed in Table S1. Each experiment was repeated three times with similar results. Black and white boxes indicate the Columbia WT and tic55-II-knockout mutant, respectively.

Figure 8

ANAC domain structure of seven Arabidopsis NAC transcription factors (TFs). (A) Architecture of NAC containing domains of seven Arabidopsis NAC TFs. * indicates the identical amino acids aligned within the different ANAC proteins. (B) phylogenetic relationships of four downregulated ANAC proteins, including ANAC003, ANAC010, ANAC042, and ANAC075 with other ANAC proteins that have been previously published involved in plant senescence.

Figure 9

Downregulated expression of four ANAC genes accessed by relative real-time quantitative RT-PCR and MYB-NAC relationship determined by yeast one-hybrid assay. (A) Validated leaf NAC transcription factor genes, including ANAC003, ANAC010, ANAC042, and ANAC075 showed downregulation in the tic55-II-knockout mutant after dark-induced leaf senescence compared with the wild-type (WT) in microarray analysis. Relative transcript levels of these genes were determined using three replicates, and signal intensities for each transcript were normalized with tubulin (internal control). Error bars represent standard deviation. Primers used in PCR reactions are listed in Table S1. Black and white boxes indicate the Columbia WT and tic55-II-knockout mutant, respectively. (B) Interaction between MYB108 TF and promoters of different ANACs was analyzed. Promoter region of different ANAC genes was linked to the HIS3 reporter gene, resulting in different constructs: pHIS2.1-ANAC003p, pHIS2.1-ANAC010p, pHIS2.1-ANAC042p and pHIS2.1-ANAC075p. Each of these constructs was transformed into yeast cells either with plasmid carrying an Activation Domain (AD)-MYB108 TF fusion (pGADT7-MYB108) or pGADT7 plasmid as a negative control. Binding of MYB108 to the cis-elements of ANAC003, ANA010, ANAC042, or ANAC075 promoter region, resulting in the expression of reporter gene, showed the growth of yeast cells in SD/–Leu/–Trp/–His medium in the presence of 3-amino-1,2,4-triazol (3-AT).