Physiological characterization and cold-responsive miRNA profiling of oil palm (Elaeis guineensis Jacq.) under cold stress

Introduction Cold stress seriously restricts the sustainable development of China’s oil palm industry. MicroRNAs (miRNAs), a class of small non-coding RNAs, act as key post-transcriptional regulators of gene expression and play essential roles in plant growth, development, and stress adaptation. Although they have been extensively characterized in various plant species under adverse environmental conditions, their regulatory functions in oil palm ( Elaeis guineensis Jacq.) during cold stress responses remain largely unexplored. Methods Seedlings of the thin shelled Tenera oil palm genotype were used in this study. Uniform aged seedlings were subjected to cold treatment at 8 °C, with sampling at 0 (26 °C), 0.5, 1, 2, 4, and 8 h, three biological replicates were included for each time point. We assessed physiological injury indicators including malondialdehyde, electrolyte leakage, and chlorophyll content. Eighteen independent small RNA libraries were sequenced on the Illumina HiSeq Xten platform, and cold responsive miRNAs and their predicted target genes were identified. Expression of 17 candidate miRNAs was further validated by qPCR. Results and discussion Physiological measurements showed that with increasing cold stress, malondialdehyde accumulation and electrolyte leakage rate in leaves increased significantly, whereas chlorophyll content decreased continuously, indicating progressive impairment of cell membrane integrity. To this end, 18 independent small RNA sequencing libraries (three biological replicates for each of six experimental groups, including one normal temperature control (26 °C) and five cold-treatment time points (0.5, 1, 2, 4, and 8 h under 8 °C)) were constructed for small RNA profiling. A total of 191 unfiltered known miRNA candidates and 98 predicted novel miRNA candidates were initially identified by sequencing. Functional enrichment analysis revealed that the target genes of differentially expressed miRNAs were mainly enriched in transcriptional regulation pathways and sesquiterpenoid and triterpenoid biosynthesis. qPCR confirmed the expression dynamics of 17 cold responsive miRNAs, among which the negative regulation between the miR166 family and its predicted target genes ATHB9-like and ATHB12 - like was observed based on expression correlation. Our data support the inference that miRNA dependent post transcriptional reprogramming participates in oil palm cold stress adaptation, rather than resolving the complete molecular mechanism. Further analysis indicated that temporal expression shifts of cold responsive miRNAs were consistent with dynamic changes in plant morphological, physiological, and biochemical indicators. Intersection analysis across the five cold treatment comparisons defined core cold responsive and stage specific sets for both conserved and novel miRNAs, and unbiased Mfuzz time series clustering classified all cold-responsive miRNAs into six temporal expression modules.