Plastid retrograde regulation of miRNA expression in response to light stress.

BACKGROUND: MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs that play a pivotal role in the regulation of plant development and responses to the surrounding environment. Despite the efforts made to elucidate their function in the adaptation of plants to many abiotic and biotic stresses, their role in high light (HL) stress is still vague. HL stress often arises upon plant exposure to full sunlight. Subsequent changes in nuclear gene expression are triggered by chloroplast-derived retrograde signals. RESULTS: In this study, we show that HL is involved in miRNA-dependent regulation in Arabidopsis thaliana rosettes. Microtranscriptomic screening revealed a limited number of miRNAs reacting to HL. To explain the miRNA regulation mechanisms at the different biogenesis stages, chemical and genetic approaches were applied. First, we tested the possible role of plastoquinone (PQ) redox changes using photosynthetic electron transport chain inhibitors. The results suggest that increased primary transcript abundance (pri-miRNAs) of HL-regulated miRNAs is dependent on signals upstream of PQ. This indicates that such signals may originate from photosystem II, which is the main singlet oxygen (1O2) source. Nevertheless, no changes in pri-miRNA expression upon a dark-light shift in the conditional fluorescent (flu) mutant producing 1O2 were observed when compared to wild-type plants. Thus, we explored the 1O2 signaling pathway, which is initiated independently in HL and is related to ß-carotene oxidation and production of volatile derivatives, such as ß-cyclocitral (ß-CC). Pri-miRNA induction by ß-CC, which is a component of this 1O2 pathway, as well as an altered response in the methylene blue sensitivity 1 (mbs1) mutant support the role of 1O2 signaling in miRNA regulation. CONCLUSIONS: We show that light stress triggers changes in miRNA expression. This stress response may be regulated by reactive oxygen species (ROS)-related signaling. In conclusion, our results link ROS action to miRNA biogenesis, suggesting its contribution to inconsistent pri- and mature miRNA dynamics.

Light Regulates Plant Alternative Splicing through the Control of Transcriptional Elongation.

Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.

Mutual interdependence of splicing and transcription elongation.

Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis.

The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5' and 3' splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.