Isolation of Mitochondria from Model and Crop Plants

Abstract

The ability to isolate intact and functional mitochondria has greatly deepened our understanding of mitochondrial structure and function. With the advancement of molecular biology techniques and progression into omics-based research over recent decades, mitochondrial research has shifted from crop species such as wheat, pea, and potato to genetically sequenced models such as Arabidopsis thaliana and rice. Although there are many attributes that make model species particularly appealing for plant research, they are often less than ideal for conducting biochemical investigations and as such, considerable modification to mitochondrial isolation methods has been made.As the cost of genome sequencing continues to decrease however, an increasing number of crop species are now being sequenced and with these new resources it appears that the research community is turning back toward crop research. In this chapter we present mitochondrial isolation methods using density gradient centrifugation for both model species such as Arabidopsis thaliana, rice, and Medicago and crop species including wheat, potato, and pea. In addition, we present a number of marker enzyme assays to confirm mitochondrial purity as well as respiratory assays to determine outer membrane integrity and respiratory function of isolated mitochondria.

Wheat mitochondrial respiration shifts from the tricarboxylic acid cycle to the GABA shunt under salt stress

“…Mitochondria were isolated from whole shoots by differential centrifugation followed by a 0–4.4% (w/v) PVP and self‐forming Percoll gradient based on the methods of Jacoby et al . () and Kerbler & Taylor (). Mitochondrial proteins were precipitated via acetone treatment, and the digested peptide samples were extracted using solid phase C‐18 macrospin columns.…”

Leaf specific overexpression of a mitochondrially-targeted glutamine synthetase in tomato increased assimilate export resulting in earlier fruiting and elevated yield

“…Mitochondria were isolated from 2-month-old tomato leaves as described by Kerbler and Taylor (2017). Briefly, ∼30 g of whole seedlings was disrupted in a precooled mortar and pestle using 150 mL of cold extraction buffer (0.3 M sucrose, 25 mM tetrasodiumpyrophosphate [10 H2O], 2 mM EDTA, 10 mM KH2PO4, 1% polyvinylpyrrolidone [PVP-40], 1% BSA, 20 mM ascorbic acid, 5 mM cysteine [pH 7.5]).…”

“…Mitochondria were isolated from 2-month-old tomato leaves as described by Kerbler and Taylor (2017). Briefly, ~30 g of whole seedlings was disrupted in a precooled mortar and pestle using 150 mL of cold extraction buffer (0.…”

Mitochondrial Complex I activity is inhibited by changes in the abundance of phosphatidylinositol and phosphatidylserine in wheat plants exposed to high temperatures

“…The trays were watered daily to avoid water limitation. Seventy to eighty grams of fresh wheat shoots were harvested following 14 days of growth, and mitochondria were isolated using PVP-Percoll gradient centrifugation following the method described by Kerbler & Taylor (2017). The mitochondrial pellet was stored at -80 ˚C.…”