TY - JOUR

T1 - How to Document the Purity of Mitochondria Prepared from Green Tissue of Pea, Tobacco and Arabidopsis thaliana

AU - Hausmann, Nicole

AU - Werhahn, Wolf

AU - Huchzermeyer, Bernhard

AU - Braun, Hans Peter

AU - Papenbrock, Jutta

PY - 2003/7/21

Y1 - 2003/7/21

N2 - It is a difficult task to isolate mitochondria from green tissue, especially from the widely used model plant Arabidopsis thaliana. However, for several physiological queries very pure and physiologically intact mitochondria are needed. From three plant species, Pisum sativum L., Nicotiana tabacum L. and Arabidopsis thaliana HEYNH., mitochondria were isolated according to different purification protocols described before or by an own optimised protocol. The methods involved differential centrifugation and density gradient centrifugation on self-generating gradients or step-gradients of Percoll. The quality of the purified mitochondria were tested by different methods to prove intactness of the membrane system, the physiological activity and the contamination by other organelles. In summary, it was possible to isolate mitochondria from the three different plant species according to all criteria investigated. However, the reliability of the organelle preparations and the purity and intactness varied between the methods. To demonstrate the quality of the purified mitochondria with respect to different traits a combination of several tests is recommendable. Determination of chlorophyll contents and enzyme activities, functional analysis of the respiratory chain, and the use of the powerful technique of Blue-native/Tricine SDS polyacrylamide gel electrophoresis are necessary to fully characterize the mitochondria. Especially combining a visual result by comparing the separation of protein complexes in gels with physiological measurements seems to be very useful for a comprehensive characterization of the organelle preparation.

AB - It is a difficult task to isolate mitochondria from green tissue, especially from the widely used model plant Arabidopsis thaliana. However, for several physiological queries very pure and physiologically intact mitochondria are needed. From three plant species, Pisum sativum L., Nicotiana tabacum L. and Arabidopsis thaliana HEYNH., mitochondria were isolated according to different purification protocols described before or by an own optimised protocol. The methods involved differential centrifugation and density gradient centrifugation on self-generating gradients or step-gradients of Percoll. The quality of the purified mitochondria were tested by different methods to prove intactness of the membrane system, the physiological activity and the contamination by other organelles. In summary, it was possible to isolate mitochondria from the three different plant species according to all criteria investigated. However, the reliability of the organelle preparations and the purity and intactness varied between the methods. To demonstrate the quality of the purified mitochondria with respect to different traits a combination of several tests is recommendable. Determination of chlorophyll contents and enzyme activities, functional analysis of the respiratory chain, and the use of the powerful technique of Blue-native/Tricine SDS polyacrylamide gel electrophoresis are necessary to fully characterize the mitochondria. Especially combining a visual result by comparing the separation of protein complexes in gels with physiological measurements seems to be very useful for a comprehensive characterization of the organelle preparation.

KW - Arabidopsis thaliana

KW - Blue-native gel electrophoresis

KW - Mitochondria

KW - Nicotiana tabacum

KW - Pisum sativum

UR - http://www.scopus.com/inward/record.url?scp=0042512002&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:0042512002

VL - 43

SP - 215

EP - 229

JO - Phyton - Annales Rei Botanicae

JF - Phyton - Annales Rei Botanicae

SN - 0079-2047

IS - 1

ER -