Building up of the plastid transcriptional machinery during germination and early plant development

Abstract

The plastid genome is transcribed by three different RNA polymerases, one is called plastid-encoded RNA polymerase (PEP) and two are called nucleus-encoded RNA polymerases (NEPs). PEP transcribes preferentially photosynthesis-related genes in mature chloroplasts while NEP transcribes preferentially housekeeping genes during early phases of plant development, and it was generally thought that during plastid differentiation the building up of the NEP transcription system precedes the building up of the PEP transcription system. We have now analyzed in detail the establishment of the two different transcription systems, NEP and PEP, during germination and early seedling development on the mRNA and protein level. Experiments have been performed with two different plant species, Arabidopsis (Arabidopsis thaliana) and spinach (Spinacia oleracea). Results show that the building up of the two different transcription systems is different in the two species. However, in both species NEP as well as PEP are already present in seeds, and results using Tagetin as a specific inhibitor of PEP activity demonstrate that PEP is important for efficient germination, i.e. PEP is already active in not yet photosynthetically active seed plastids.

Figure 1.

Analysis of mRNA levels of NEP and PEP components during germination and early Arabidopsis seedling development. A, Developmental stages of Arabidopsis that have been used in B and C. B, mRNA levels coding for NEP and PEP core subunits. C, PEP transcription factors of the σ type have been analyzed by semiquantitative RT-PCR. The principal developmental stages that have been analyzed are as follows: dry seeds (0, lane 1), seeds after imbibition and vernalization (0+, lane 2), and 1 to 6 d after germination (lanes 3–8). 18S RNA in B was analyzed as internal constitutive control standard and RNA preparations have been routinely tested for the absence of DNA by performing PCR directly without prior RT (shown as example for rpoA in C, Control [−RT]).

Figure 2.

Analysis of protein levels of NEP and PEP components during germination and early Arabidopsis seedling development. A, Characterization of specific RPOTp and RPOTmp peptide antibodies. Thirty microgram aliquots of either total leaf proteins (lanes 1–3 and 5–7) or chloroplast proteins (lane 4) have been separated on 12% polyacrylamide gels and Nitrocellulose blots have been probed with either preimmune serum (PI, lanes 1 and 5) or RPOTp and RPOTmp antisera before (lanes 2 and 6) and after (lanes 3, 4, and 7) affinity purification of specific IgG fractions. B, Exclusion of antibody cross-reaction between RPOTmp and RPOTp. Different concentrations (as indicated in ng on the left side of the figure) of peptides AtRPOTmp-a (lanes 1 and 5), AtRPOTmp-b (lanes 2 and 6), AtRPOTp-a (lanes 3 and 7), and AtRPOTp-b (lanes 4 and 8) have been spotted in equal volumes to Nitrocellulose filters and probed either with RPOTp (left-hand side) or RPOTmp (right-hand side) antibodies. C, Immunological cross-reaction of two different antibodies prepared against E. coli RNA polymerase with PEP subunits in Arabidopsis protein extracts. Thirty microgram aliquots of total leaf proteins (lanes 1–3) or chloroplast proteins (lane 4) have been separated on 12% SDS-polyacrylamide gels and Nitrocellulose blots have been probed with the A antibodies (lane 1 and bottom part of lanes 3 and 4) and the B antibodies (lane 2 and top part of lanes 3 and 4). D, Protein patterns of the protein extracts prepared from dry seeds (0), seeds after vernalization (0+), and from seedlings 1 to 6 d after germination (1–6) as revealed by Ponceau staining of Nitrocellulose membranes after SDS gel separation on 10% acrylamide gels and blotting. E, Immunoblot analysis of Nitrocellulose blots as described in D by the different antibodies that are characterized in A, B, and C.

Figure 3.

Analysis of mRNA levels of NEP and PEP components during germination and early seedling development of spinach. A, Stages of spinach development that have been used for analyzing the mRNA and protein levels. B, mRNA levels of stages presented in A have been analyzed by semiquantitative RT-PCR as described in “Materials and Methods.” 18S RNA has been analyzed as internal constitutive control standard.

Figure 4.

Analysis of protein levels of NEP and PEP components during germination and early spinach seedling development. A, Characterization of the spinach SIG2 antibodies on recombinant SoSIG2 protein. The cDNA of SoSIG2 has been cloned into pBAD-ThioTOPO and the thioredoxin fusion protein has been analyzed in total E. coli protein extracts before (lanes 2 and 4) and after (lanes 3 and 5) induction of protein expression by Arabinose using the SoSIG2 antibodies (lanes 2 and 3) and the antithioredoxin antibodies (lanes 4 and 5). B, Characterization of affinity purified SoSIG2 antibodies on spinach total protein extracts. SoSIG2 antibodies have been affinity purified on either the SoSIG2-a peptide (P1, lane 1) or the SoSIG2-b peptide (P2, lane 2) and probed on 50 μg aliquots of total spinach proteins. Lane 3 shows a control reaction with preimmune serum. C, Characterization of SoRPOA antibodies on recombinant RPOA protein and on spinach protein extracts. The cDNA of SoRPOA has been cloned into pET32a and the His-fusion protein has been analyzed in total E. coli protein extracts before (lanes 1, 3, and 5) and after (lanes 2, 4, and 6) induction of protein expression by IPTG using preimmune serum (lanes 1 and 2), the SoRPOA antibodies (lanes 3 and 4), and the anti-His antibodies (lanes 5 and 6). Thirty microgram aliquots of total leaf proteins (lane 7) and chloroplast proteins (lane 8) have been separated on a 12% SDS-polyacrylamide gel and analyzed by anti-SoRPOA antibodies after transfer to nitrocellulose. D, Analysis of NEP and PEP protein levels during germination and early seedling development. Nitrocellulose blots of proteins from developmental stages shown in Figure 3A have been probed by the different antibodies that are characterized in Figure 4, A to C. The RPOTp antibody has been characterized previously (Azevedo et al., 2006).

Figure 5.

Delay of germination by Tagetin. A, Arabidopsis seeds of the tt2-1 mutant have been germinated on moistened filter paper in the absence (lane 1) and in the presence of 1 μm (lane 2), 10 μm (lane 3), and 100 μm (lane 4) of Tagetin. B, Tagetin has no influence on 5S RNA levels. 5S RNA has been analyzed from Arabidopsis plantlets shown in A by in vitro capping of isolated total RNA, separation on 6% denaturing polyacrylamide gels, and autoradiography. C, Arabidopsis seeds were photographed after 24 h vernalization and 36 h incubation at 23°C under continuous light in the absence (water) and presence (Tagetin) of 100 μm Tagetin. D, Analysis of the effect of Tagetin on the time course of seed germination. Seeds were germinated as described in “Materials and Methods” in the absence (blue) and in the presence (pink) of 100 μm Tagetin. Germination is expressed as percentage of seeds showing radicle protrusion at the indicated time points. The time scale does not include the first 24 h after the vernalization period. E, RNA and protein have been isolated from water (lanes 2 and 4) and Tagetin (lanes 1 and 3) treated seeds at the end of the imbibition period (0+) and 42 h after cold release. RNA levels have been analyzed by semiquantitative RT-PCR for 18S rRNA, rbcL, and rpoA mRNAs and protein levels have been analyzed by immunoblotting for RPOA and RPOC2 using anti-E. coli B antibodies.