Lon1 Inactivation Downregulates Autophagic Flux and Brassinosteroid Biogenesis, Modulating Mitochondrial Proportion and Seed Development in Arabidopsis
Arabidopsis Lon1 serves dual roles as a mitochondrial protease and chaperone. Loss of Lon1 function reduced autophagy flux at transcriptional, protein and cellular levels. Meanwhile, changes occurred in the levels of cellular subunit proteins, especially a significant upregulation of mitochondrial proteins overall in lon1 mutants. Lon1 is a key enzyme in maintaining mitochondrial protein homeostasis. In lon1 mutants, other mitochondrial proteases including mitochondrial autophagy receptor proteins showed significant increases in abundance. Mitophagy is an important mechanism for cellular quality control. lon1-2atg5-1 double mutant were constructed to investigate how they cooperatively regulate mitochondrial protein homeostasis. Transcriptome sequencing results revealed that Lon1 inactivation led to widespread unfolded protein responses (UPRs) pathway activation in lon1-2atg5-1, consistent with lon1-2, while loss function of ATG5 inactivation did not affect mitochondrial homeostasis. The double mutant also exhibited independent plant phenotypes from single parents, yet more severe seed number and embryo development issues, suggesting an additive effect of lon1-2 and atg5-1. Protein Storage Vacuole (PSV) and oil body phenotypes also showed more severe developmental damage in the double mutants. Brassinosteroid (BR) acts as a key plant hormone controlling seed and embryo development. GO and KEGG analysis of genes simultaneously downregulated in lon1-2, atg5-1, and lon1-2atg5-1 revealed significant downregulation of genes involved in BR biosynthesis and its homeostasis, indicating that this may be one of the key factors leading to abnormal seed and embryo development in the mutants. In general, the loss function of Lon1 reduces autophagy flux and induces UPRs to regulate protein homeostasis. Meanwhile, the simultaneous absence of Lon1 and autophagy significantly down-regulates the expression of genes related to BR biosynthesis and homeostasis, thereby leading to pronounced blockage in the development of double mutant seeds and embryos. Overall design: Arabidopsis thaliana is Columbia-0 (Col-0) background. T-DNA insertion mutant lon1-2 (AT5G26860, SALK_012797), atg5-1 (AT5G17290, SAIL_129_B07) and lon1-2atg5-1 double mutant are owned by our laboratory. Arabidopsis grown best under 16/8 hours light / dark cycle at room temperature (18-22 ° C), with light intensity of 120-150 µ mol / m2 (normal conditions). Seven-day-old seedlings were harvested in biological triplicate under normal conditions. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer's protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) = 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Then these libraries were sequenced on the Illumina sequencing platform (DNBSEQ-T7) and 125bp/150bp paired-end reads were generated.