Specific function of a plastid sigma factor for ndhF gene transcription

Abstract

The complexity of the plastid transcriptional apparatus (two or three different RNA polymerases and numerous regulatory proteins) makes it very difficult to attribute specific function(s) to its individual components. We have characterized an Arabidopsis T-DNA insertion line disrupting the nuclear gene coding for one of the six plastid sigma factors (SIG4) that regulate the activity of the plastid-encoded RNA polymerase PEP. This mutant shows a specific diminution of transcription of the plastid ndhF gene, coding for a subunit of the plastid NDH [NAD(P)H dehydrogenase] complex. The absence of another NDH subunit, i.e. NDHH, and the absence of a chlorophyll fluorescence transient previously attributed to the activity of the plastid NDH complex indicate a strong down-regulation of NDH activity in the mutant plants. Results suggest that plastid NDH activity is regulated on the transcriptional level by an ndhF-specific plastid sigma factor, SIG4.

Figure 1

Characterization of the sig4 knock-out mutant. (A) Schematic presentation of the T-DNA insertion and the location of the primers. The exact position of the T-DNA insertion into the SIG4 gene (210) was determined by sequencing of the DNA fragments amplified by primer pairs 1/2 and 3/4. (B) The absence of SIG4 mRNA in the T-DNA insertion line was verified by RT–PCR. Total RNA prepared from 7-day-old WT and sig4 plantlets has been analysed in parallel for the presence of all six sigma factors. (C) WT and sig4 knock-out plants grown for 4 weeks under 16 h light/8 h dark cycle.

Figure 2

Analyses of ndhF, ndhG and ycf4 precursor RNAs. Total RNA was isolated from 7-day-old Arabidopsis plantlets that had been grown under 16 h light/8 h dark cycle. RNA was reverse transcribed and cDNAs were separated on 6% polyacrylamide gels under denaturing conditions. The accompanying sequence ladders are established with the same primer that was used for primer extension. (A) An aliquot of 10 µg (upper part) or 1 µg (lower part) of total RNA prepared from Arabidopsis WT (lanes 5 and 1, upper part and lower part, respectively) and sig4 plantlets (lanes 6 and 2, upper part and lower part, respectively) have been analysed by primer extension using a primer specific to ndhF (upper part) or by RT–PCR using primers specific for ndhF and psbN. (B) An aliquot of 10 µg of total RNA prepared from Arabidopsis WT (lane 5) and sig4 plantlets (lane 6) have been analysed by primer extension using a primer specific to ndhG. (C) An aliquot of 10 µg of total RNA prepared from Arabidopsis WT (lane 5) and sig4 plants (lane 6) have been analysed by primer extension using a primer specific to ycf4. (D) The nucleotide sequences upstream of the ndhF, ndhG and ycf4 precursor RNAs are aligned. Putative promoter sequences are underlined, and 5′ ends of transcripts [positions −320 (ndhF), −213 and −138 (ndhG), −326 and −226 (ycf4)] are marked in bold letters and by vertical arrows.

Figure 3

Characterization of the sig4 knock-out mutant by complementation and fluorescence induction. (A) An aliquot of 10 µg of total RNA prepared from 7-day-old Arabidopsis WT (lane 1), sig4 (lane 2) and three different 35S-SIG4 complemented lines (lanes 3–5) have been analysed by primer extension using the same primers as in Figure 2A (ndhF) and 2B (ndhG, only the b transcript is shown). (B) Rosette leaves of 6-week-old Arabidopsis plants have been used to measure transient chlorophyll fluorescence rise under non-actinic light following a 5 min illumination of WT, sig4 and 35S-SIG4 plants. (C) Thylakoid membranes have been prepared from 2-week-old WT, sig4 and 35S-SIG4 plants and 40 µg (lanes 1, 4 and 5), 10 µg (lane 2) and 4 µg (lane 3) of protein have been analysed by western immunoblotting using antibodies made against the NDHH subunit or the plastid terminal oxidase (PTOX).