DEAD-box RNA helicases belong to an RNA helicase family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation and RNA decay. This study investigated a DEAD-box RNA helicase, AtRH7/PRH75, in Arabidopsis. Expression of AtRH7/PRH75 was ubiquitous; however, the levels of mRNA accumulation were increased in cell division regions and were induced by cold stress. The phenotypes of two allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled auxin-related developmental defects that were exhibited in several ribosomal protein mutants, and were more severe under cold stress. Northern blot and circular reverse transcription-PCR (RT-PCR) analyses indicated that unprocessed 18S pre-rRNAs accumulated in the atrh7 mutants. The atrh7 mutants were hyposensitive to the antibiotic streptomycin, which targets ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. In addition, the atrh7-2 and atrh7-3 mutants displayed cold hypersensitivity and decreased expression of CBF1, CBF2 and CBF3, which might be responsible for the cold intolerance. The present study indicated that AtRH7 participates in rRNA biogenesis and is also involved in plant development and cold tolerance in Arabidopsis.

The Arabidopsis mutant heat-intolerant 4-1 (hit4-1) was isolated from an ethyl methanesulphonate-mutagenized M2 population on the basis of its inability to withstand prolonged heat stress (4 days at 37°C). Further characterization indicated that hit4-1 was impaired specifically in terms of basal but not acquired thermotolerance. Map-based cloning revealed that the HIT4 gene encoded a plant-specific protein for which the molecular function has yet to be studied. To investigate the cellular role of HIT4 and hence elucidate better its protective function in heat tolerance in plants, a GFP-HIT4 reporter construct was created for a protoplast transient expression assay. Results showed that fluorescently tagged HIT4 was localized to the chromocentre, a condensed heterochromatin domain that harbours repetitive elements for which transcription is normally suppressed by transcriptional gene silencing (TGS). DAPI-staining analysis and FISH with a probe that targeted centromeric repeats showed that heat-induced chromocentre decondensation was inhibited in nuclei of hit4-1 subjected to direct heat treatment, but not in those that were allowed to acquire thermotolerance. Moreover, heat reactivation of various TGS loci, regardless of whether they were endogenous or transgenic, or existed as a single copy or as repeats, was found to be attenuated in hit4-1. Meanwhile, the levels of transcripts of heat shock protein genes in response to heat stress were similar in both hit4-1 and wild-type plants. Collectively, these results demonstrated that HIT4 defines a new TGS regulator that acts at the level of heterochromatin organization and is essential for basal thermotolerance in plants.

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