Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development

Abstract

We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a "buffering" mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleus.

Keywords: RNA trafficking; anterograde regulation; cytoplasmic male sterility (CMS); plant mitochondria; retrograde regulation; ribozyme; signaling.

Figure 1

Structure of the ribozyme-PKTLS chimeric RNAs targeted to mitochondria in the present study. (a) Structure of the Rznad9-L-PKTLS RNA. The trans-cleaving ribozyme Rznad9 directed against the A. thaliana mitochondrial nad9 mRNA (Atnad9) is attached to the 5’-end of the PKTLS shuttle via a 40 nucleotide linker (L) selected from a pool of random sequences. The Rznad9 hammerhead sequence is annealed to its target sequence motif in the Atnad9 mRNA. The Rzsdh3 (b), Rzcox3 (c) and Rzatp9 (d) ribozymes, directed against the N. tabacum sdh3 (Ntsdh3) mRNA, the A. thaliana cox3 (Atcox3) mRNA and the A. thaliana atp9 (Atatp9) mRNA are attached to the same L-PKTLS moiety to generate the Rzsdh3-L-PKTLS, Rzcox3-L-PKTLS and Rzatp9-L-PKTLS, respectively. Ribozyme cleavage sites are indicated in the target sequence motifs by thick arrows. Their precise location is after position 421 in the A. thaliana nad9 coding sequence (24662-25234 in accession JF729201), after position 216 in the N. tabacum sdh3 coding sequence (77198-77524 in accession BA000042, complementary strand), after position 685 in the A. thaliana cox3 coding sequence (328926-329723 in accession JF729201, complementary strand), and after position 99 in the A. thaliana atp9 coding sequence (269920-270177 in accession JF729201, complementary strand). The Rzsdh3 and Rzatp9 ribozymes are as described earlier in Sultan et al. [21] and Val et al. [20], respectively.

Figure 2

Structure of the orf77-PKTLS RNA targeted to mitochondria in the present study. The Zea mays CMS-S-specific orf77 coding sequence (initiation codon and termination codon in bold) with its four nucleotide upstream and 92 nucleotide downstream sequences (italics) is directly attached to the 5’-end of the PKTLS shuttle. Short additional sequences deriving from the HindIII and BamHI restriction sites introduced for cloning purposes are underlined. The chimeric RNA was expressed from a nuclear transgene and driven into the organelles by the PKTLS moiety.

Figure 3

Chimeric ribozyme expression and knockdown of steady-state levels of mitochondrial target RNAs in transformed seedlings at different stages of growth. (a–h) A. thaliana control seeds and seeds carrying the Rzatp9-L-PKTLS, Rznad9-L-PKTLS or Rzcox3-L-PKTLS transgene were germinated in the light on solid MS-agar medium. Plants at early stage (upper panel) or intermediate stage (lower panel) of development were transferred at Day 0 to wells in culture plates containing liquid medium supplemented with estradiol for transgene induction. Kinetics of induced expression of the Rzatp9-L-PKTLS (a), Rznad9-L-PKTLS (d) or Rzcox3-L-PKTLS (g) RNA and of the steady-state level of the mitochondrial atp9 (b,f), nad9 (c,e) or cox3 (h) target RNA were analyzed by RT-qPCR with total RNA from plant samples collected each day from Day 0 to Day 4 post-induction. (i,j) Transformant N. tabacum carrying the Rzsdh3-L-PKTLS transgene was germinated in the light on solid MS-agar medium and transferred at intermediate stage of development to liquid medium supplemented with estradiol for transgene induction. Kinetics of induced expression of the Rzsdh3-L-PKTLS RNA (i) and of the steady-state level of the mitochondrial sdh3 target RNA (j) were analyzed by RT-qPCR with total RNA from transformed plant samples collected each day from Day 0 to Day 4 post-induction. Data from three independent biological replicates were analyzed with the Student’s t-test; NS = not significant; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

Figure 4

Chimeric ribozyme expression and knockdown of steady-state levels of mitochondrial target RNAs in transformed seedlings in different conditions. (a–e) A. thaliana control seedlings and seedlings carrying the Rznad9-L-PKTLS, or the Rzatp9-L-PKTLS transgene were grown in the light (upper panel) or in the dark (lower panel) on solid MS-agar medium. Plants at bolting stage of development (upper panel) were transferred at Day 0 to wells in culture plates containing liquid medium supplemented with estradiol for transgene induction. Kinetics of induced expression of the Rznad9-L-PKTLS RNA (a), of the steady-state level of the mitochondrial nad9 target RNA (b) and of the steady-state level of the mitochondrial atp9 target RNA (c) were analyzed by RT-qPCR with total RNA from plant samples collected each day from Day 0 to Day 4 post-induction. Plates with ten-day-old seedlings grown in the dark (lower panel) were overlayed with liquid medium supplemented with estradiol for transgene induction. Kinetics of induced expression of the Rznad9-L-PKTLS RNA (d) and of the steady-state level of the mitochondrial nad9 target RNA (e) were analyzed by RT-qPCR with total RNA from plant samples collected each day from Day 0 to Day 4 post-induction. Data from three independent biological replicates were analyzed with the Student’s t-test; NS = not significant; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

Figure 5

Synthetic scheme representing the impact of orf77-PKTLS expression on the mitochondrial transcriptome in transformed A. thaliana plants at different stages of growth in the light and at 10 days of growth in the dark. Plants were grown to the appropriate stage and the transgene was induced with estradiol as in Figure 3 and Figure 4. Following transgene induction, samples were collected each day from Day 0 to Day 4 post-induction and RNAs were analyzed by RT-qPCR. The total numbers of positively or negatively affected transcripts combining all daily samples for a given growth stage are indicated and represented by proportional circular areas. Detailed day-by-day and gene-by-gene results are given in Table 1. Data from three independent biological replicates were analyzed with the Graph Pad Prism version 7.01 software.