Purification of Leaf Mitochondria from Arabidopsis thaliana Using Percoll Density Gradients

Tran, Huy Cuong; Aken, Olivier Van

doi:10.1007/978-1-0716-1653-6_1

Cited by 5 publications

(3 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, comparison to the distribution obtained for TIC110 reveals that the mitochondrial fraction contains a contamination of chloroplasts. Residual impurities of plant mitochondrial preparations by plastidal or peroxisomal membranes are unavoidable and have been repeatedly described ( Tran and Van Aken 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes

et al. 2023

View full text Add to dashboard Cite

All land plants encode two isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified two ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the two isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. Mitochondrial localization of PPO2 in Arabidopsis was ruled out. Since Arabidopsis PPO2 does not encode a cleavable transit peptide, integration of the protein into the chloroplast envelope must make use of a non-canonical import route. However, when a chloroplast transit peptide was fused to the N-terminus of PPO2, the enzyme was detected predominantly in thylakoid membranes and was able to fully complement ppo1. Thus, the two PPO isoforms in Arabidopsis are functionally equivalent but spatially separated. Their distinctive localizations within plastids thus enable the synthesis of discrete sub-pools of the PPO product protoporphyrin IX, which may serve different cellular needs.

show abstract

Section: Resultsmentioning

confidence: 99%

Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Stable transgenic and complementation lines were generated by transforming pB7FWG2-mTRAN1/2 into the WT or mtran1-2/ 2-2 by floral dipping, respectively. Plant mitochondria were purified as described in Tran et al (62) for further analysis, e.g., BN-PAGE, Western blot, MS/MS analysis, RIP-seq, and in organello translation assays. For whole genome RNA-seq analysis, 11-day-old WT and 18-dayold mtran1-2/2-2 seedlings grown on MS agar plates were harvested at developmental growth stage 1.04 (26).…”

Section: Materials and Methods Summarymentioning

confidence: 99%

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants

Tran,

Schmitt,

Lama

et al. 2023

Science

Self Cite

View full text Add to dashboard Cite

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that “mitochondrial translation factors” mTRAN1 and mTRAN2 are land plant–specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)–like RNA binding proteins of the mitoribosomal “small” subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5′ regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

show abstract

“…Mitochondria isolation was performed following the discontinuous Percoll gradient centrifugation method as previously described with modification (Heng et al., 2014; Tran & Van, 2022). Briefly, 100 g of the leaf sample was homogenized in 200 mL homogenization buffer (0.4 M mannitol, 5 mM EDTA (ethylenediamunetetraacetic acid disodium salt) 8 mM cysteine, 10 mM tricine, 1% BSA (bovine albumin), 1% polyvinyl‐pyrrolidone, and pH 7.8).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the CMS genetic regulation through comparative complete mitochondrial genome sequencing in Nicotiana tabacum

He,

Li,

Yuan

et al. 2023

The Plant Genome

View full text Add to dashboard Cite

Mitochondrial genomes (mitogenomes) of flowering plants vary greatly in structure and size, which can lead to frequent gene mutation, rearrangement, or recombination, then result in the cytoplasmic male sterile (CMS) mutants. In tobacco (Nicotiana tabacum), suaCMS lines are widely used in heterosis breeding; however, the related genetic regulations are not very clear. In this study, the cytological observation indicated that the pollen abortion of tobacco suaCMS(HD) occurred at the very early stage of the stamen primordia differentiation. In this study, the complete mitochondrial genomes of suaCMS(HD) and its maintainer HD were sequenced using the PacBio and Illumina Hiseq technology. The total length of the assembled mitogenomes of suaCMS(HD) and HD was 494,317 bp and 430,694 bp, respectively. Comparative analysis indicated that the expanded 64 K bases in suaCMS(HD) were mainly located in noncoding regions, and 23 and 21 big syntenic blocks (>5000 bp) were found in suaCMS(HD) and HD with a series of repeats. Electron transport chain‐related genes were highly conserved in two mitogenomes, except five genes (ATP4, ATP6, COX2, CcmFC, and SDH3) with substantial substitutions. Three suaCMS(HD)‐specific genes, orf261, orf291, and orf433, were screened. Sequence analysis and RT‐PCR verification showed that they were unique to suaCMS(HD). Further gene location analysis and protein property prediction indicated that all the three genes were likely candidates for suaCMS(HD). This study provides new insight into understanding the suaCMS mechanism and is useful for improving tobacco breeding.

show abstract

Purification of Leaf Mitochondria from Arabidopsis thaliana Using Percoll Density Gradients

Cited by 5 publications

References 8 publications

Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes

Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants

Characterization of the CMS genetic regulation through comparative complete mitochondrial genome sequencing in Nicotiana tabacum

Contact Info

Product

Resources

About