Differential transcriptome profiling of chilling stress response between shoots and rhizomes of <i>Oryza longistaminata</i> using RNA sequencing

Rice (Oryza sativa) is very sensitive to chilling stress at seedling and reproductive stages, whereas wild rice, O. longistaminata, tolerates non-freezing cold temperatures and has overwintering ability. Elucidating the molecular mechanisms of chilling tolerance (CT) in O. longistaminata should thus provide a basis for rice CT improvement through molecular breeding. In this study, high-throughput RNA sequencing was performed to profile global transcriptome alterations and crucial genes involved in response to long-term low temperature in O. longistaminata shoots and rhizomes subjected to 7 days of chilling stress. A total of 605 and 403 genes were respectively identified as up- and down-regulated in O. longistaminata under 7 days of chilling stress, with 354 and 371 differentially expressed genes (DEGs) found exclusively in shoots and rhizomes, respectively. GO enrichment and KEGG pathway analyses revealed that multiple transcriptional regulatory pathways were enriched in commonly induced genes in both tissues; in contrast, only the photosynthesis pathway was prevalent in genes uniquely induced in shoots, whereas several key metabolic pathways and the programmed cell death process were enriched in genes induced only in rhizomes. Further analysis of these tissue-specific DEGs showed that the CBF/DREB1 regulon and other transcription factors (TFs), including AP2/EREBPs, MYBs, and WRKYs, were synergistically involved in transcriptional regulation of chilling stress response in shoots. Different sets of TFs, such as OsERF922, OsNAC9, OsWRKY25, and WRKY74, and eight genes encoding antioxidant enzymes were exclusively activated in rhizomes under long-term low-temperature treatment. Furthermore, several cis-regulatory elements, including the ICE1-binding site, the GATA element for phytochrome regulation, and the W-box for WRKY binding, were highly abundant in both tissues, confirming the involvement of multiple regulatory genes and complex networks in the transcriptional regulation of CT in O. longistaminata. Finally, most chilling-induced genes with alternative splicing exclusive to shoots were associated with photosynthesis and regulation of gene expression, while those enriched in rhizomes were primarily related to stress signal transduction; this indicates that tissue-specific transcriptional and post-transcriptional regulation mechanisms synergistically contribute to O. longistaminata long-term CT. Our findings provide an overview of the complex regulatory networks of CT in O. longistaminata.栽培稻（Oryza sativa）在苗期与生殖生长期对低温胁迫极为敏感，而长雄蕊野生稻（O. longistaminata）可耐受非冻结低温且具备越冬能力。阐明长雄蕊野生稻的耐寒性（chilling tolerance, CT）分子机制，可为通过分子育种改良栽培稻的耐寒性提供理论基础。本研究采用高通量RNA测序（high-throughput RNA sequencing）技术，对经7天低温胁迫处理的长雄蕊野生稻地上部与根茎中参与长期低温响应的全局转录组变化及关键基因进行了分析。在7天低温胁迫下，长雄蕊野生稻中共鉴定出605个上调表达基因与403个下调表达基因，其中分别有354个、371个差异表达基因（differentially expressed genes, DEGs）仅在地上部和根茎中被检测到。基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析显示，两类组织中共同诱导的基因富集于多条转录调控通路；与之相对，地上部特异诱导的基因仅显著富集于光合作用通路，而根茎特异诱导的基因则富集于多条关键代谢通路及程序性细胞死亡过程。对这些组织特异性差异表达基因的进一步分析表明，CBF/DREB1调控子及其他转录因子（transcription factors, TFs，包括AP2/EREBPs、MYBs与WRKYs）可协同参与地上部低温胁迫响应的转录调控。而在长期低温处理下，根茎中仅特异性激活了另一组转录因子（如OsERF922、OsNAC9、OsWRKY25与WRKY74）以及8个编码抗氧化酶的基因。此外，两类组织中均高度富集多种顺式调控元件（cis-regulatory elements），包括ICE1结合位点、参与光信号调控的GATA元件以及WRKY结合的W盒，这证实了长雄蕊野生稻耐寒性的转录调控涉及多种调控基因与复杂调控网络。最后，多数仅在地上部发生的低温诱导可变剪接基因与光合作用及基因表达调控相关，而根茎中富集的低温诱导可变剪接基因则主要参与胁迫信号转导，这表明组织特异性的转录与转录后调控机制协同赋予了长雄蕊野生稻长期耐寒性。本研究结果全面解析了长雄蕊野生稻耐寒性的复杂调控网络。