Lon1 Inactivation Downregulates Autophagic Flux and Brassinosteroid Biogenesis, Modulating Mitochondrial Proportion and Seed Development in Arabidopsis

Abstract

Mitochondrial protein homeostasis is crucially regulated by protein degradation processes involving both mitochondrial proteases and cytosolic autophagy. However, it remains unclear how plant cells regulate autophagy in the scenario of lacking a major mitochondrial Lon1 protease. In this study, we observed a notable downregulation of core autophagy proteins in Arabidopsis Lon1 knockout mutant lon1-1 and lon1-2, supporting the alterations in the relative proportions of mitochondrial and vacuolar proteins over total proteins in the plant cells. To delve deeper into understanding the roles of the mitochondrial protease Lon1 and autophagy in maintaining mitochondrial protein homeostasis and plant development, we generated the lon1-2atg5-1 double mutant by incorporating the loss-of-function mutation of the autophagy core protein ATG5, known as atg5-1. The double mutant exhibited a blend of phenotypes, characterized by short plants and early senescence, mirroring those observed in the individual single mutants. Accordingly, distinct transcriptome alterations were evident in each of the single mutants, while the double mutant displayed a unique amalgamation of transcriptional responses. Heightened severity, particularly evident in reduced seed numbers and abnormal embryo development, was observed in the double mutant. Notably, aberrations in protein storage vacuoles (PSVs) and oil bodies were evident in the single and double mutants. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of genes concurrently downregulated in lon1-2, atg5-1, and lon1-2atg5-1 unveiled a significant suppression of genes associated with brassinosteroid (BR) biosynthesis and homeostasis. This downregulation likely contributes to the observed abnormalities in seed and embryo development in the mutants.

Keywords: Arabidopsis; Lon1; autophagy; brassinosteroid; mitochondrion.

Figure 1

The disruption of Arabidopsis Lon1 downregulates autophagic flux by reducing the expression of autophagy core genes. (A) Log-transformed fold changes in the transcript levels of genes encoding transcription factors (TGA1, TGA9, and TGA10), autophagy core proteins involved in autophagy membrane delivery (ATG2), phagophore extension (ATG8A, E, F, and G), and tonoplast fusion (VTI12 and CFS1) are presented. All displayed genes exhibit a significant decrease (FC < 1.5⁻¹, p < 0.05, Student’s t-test) in lon1-2 compared with Col-0. (B,C) Autophagic flux assay in 7-day-old seedlings measured by Western blotting, represented by the difference in GFP-ATG8 between Col-0 and lon1 mutants. The GFP-ATG8 relative protein level in lon1 mutant lines was acquired by normalizing to Col-0. Ponceau staining serves as a control for equal protein loading. Error bars represent standard deviations from three biological replicates. Statistical significance was determined using Student’s t-test (** indicates p < 0.01).

Figure 2

Lon1 loss of function modulates relative proportion of proteins in cellular compartments. (A) Relative proportion of proteins in different subcellular compartments obtained by label-free quantification (LFQ) mass spectrometry analysis using root total proteins extracted from ten-day-old Col-0 and lon1 lines. Error bars represent standard deviations from three biological replicates. Statistical significance was determined using Student’s t-test (* indicates p < 0.05, ** indicates p < 0.01). (B) Log-transformed fold changes in protein abundance of mitochondrial proteases and mitophagy-associated proteins in lon1 mutants compared with Col-0 (* indicates p < 0.05, ** indicates p < 0.01). Proteins with fold changes greater than 2 in lon1 lines are highlighted in bold fonts.

Figure 3

RNA deep sequencing reveals independent changes in transcript levels between lon1-2 and atg5 mutant lines. (A) Principal component analysis (PCA) performed using RNA deep sequencing data from Col-0, atg5-1, lon1-2, and lon1-2atg5-1. (B) Log-transformed fold changes in transcript levels of mitochondrial proteases in mutant lines compared with Col-0 are presented. (C–E) Log-transformed fold changes in transcript levels showing statistically significant induction (FC > 1.5, p < 0.05) in lon1-2 for mitochondrial unfolded protein responses, including UPR^mt [26,27], UPR^cp [28], and UPR^er [29] target genes that are extracted and presented. Transcripts with fold changes greater than 2 in lon1-2 are highlighted in bold. The symbol # denotes transcripts with fold changes greater than 2 in lon1-2, atg5-1, and lon1-2atg5-1. Genes encoding mitochondrial proteases, ethylene biosynthesis and signaling proteins, and chaperones are annotated. Blue-yellow color gradient represents the log₂FC values between mutants and Col-0. * indicate p < 0.05, ** indicate p < 0.01.

Figure 4

lon1-2atg5-1 double mutant exhibits additive growth phenotype compared to single mutants. (A) Presentation of five-week-old soil-grown Arabidopsis plants of Col-0, lon1-2, atg5, and lon1-2atg5. (B–D) Measurement and presentation of rosette diameter, plant height, and numbers of senescent leaves as column graphs. Error bars indicate standard deviation across five biological replicates. Different letters in grouping of different lines indicate statistically significant differences based on one-way ANOVA test. The dots represent biological replicates.

Figure 5

lon1-2atg5-1 double mutant displays increased severity in abnormal seed development and nutrient storage. (A) Observations reveal embryo development retardation, reduction in number of seeds per silique, and seed shape changes in lon1-2, atg5, and lon1-2atg5 compared with Col-0. Red arrows indicate abnormal seeds. Scale bar indicates 20 μm for embryos, 2 mm for siliques, and 1 mm for seeds. (B,C) Number of seeds per silique and abnormality in seed shape are displayed for Col-0 and mutant lines. Different letters in grouping of different lines indicate statistically significant differences determined by one-way ANOVA. The dots represent biological replicates. (D) Observation of protein storage vacuoles (PSVs) with autofluorescence and Nile red-stained oil bodies in cotyledons using fluorescence confocal microscopy. Scale bar indicates 10 μm.

Figure 6

Commonly downregulated genes in lon1-2, atg5-1, and lon1-2atg5-1 are enriched with the brassinosteroid biosynthesis pathway. (A) A total of 166 genes were commonly downregulated in lon1-2, atg5-1, and lon1-2atg5-1 and were subjected to an enrichment of Gene Ontology (GO) analysis. The horizontal bars represent the counts for biological processes (BPs), cell compartments (CCs), and molecular functions (MFs), while the blue–red color bar indicates the range of Log-transformed p values. (B) The KEGG pathway enrichment analysis of downregulated genes in all three mutants. The size of the bubbles corresponds to the number of genes involved, while the green–red color bar indicates the range of Log-transformed p values. (C) Log-transformed fold changes in transcript levels for genes related to brassinosteroids in lon1-2, atg5-1, and lon1-2atg5-1 (* indicates p < 0.05, and ** indicates p < 0.01).