Guard cells are specialized epidermal cells that control stomata aperture to adjust
transpiration, carbon fixation and therefore also the water use efficiency and
photosynthetic performance of a plant.
For this, they have to change their cell volume, which is accomplished by transport of ions and
other osmolytes by active, energy consuming transport across the plasma membrane and the
tonoplast.
How this high energy demand is covered remains a matter of debate.
Guard cells harbor chloroplasts of increased size and number compared to their epidermal surrounding,
however, their number and size is considerably decreased compared to the mesophyll.
Consequently, their photosynthetic activity and ATP production is also lower compared to the
situation in the mesophyll.
It can therefore be expected that mitochondrial energy metabolism is especially important in these cell
types and may be characterized by unique features compared to the mitochondria operating in the
mesophyll.
To test this hypothesis, we employed a modern affinity purification of mitochondria (Mito-AP), allowing
cell type specific enrichment of organelles within only half an hour and subsequent investigation of
their protein complex proteome by Blue Native-Polyacrylamide Gel Electrophoresis assisted
Complexome Profiling.
For this, mitochondria were solubilized on beads in 5% [w/v] digitonin and 185 µg of protein were separated for
organelle fractions containing: mesophyll derived mitochondria (MDM),
guard cell derived mitochondria (GDM) and total leaf mitochondria (TLM), serving as a control.
Each gel was cut into 39 fractions, subjected to tryptic in-gel digestion and peptide mixtures in each fraction
were analyzed by LC-tims-TOF-MS using a nanoElute2 equipped with a 15 cm C18 Aurora Elite
column connected to a timsTOF Pro MS that was operated in DDA-PASEF using a pre-installed
0.5 s cycle time method.
Protein abundance was estimated using iBAQ values computed by the MaxQuant software and aided to
estimate proportion of mitochondrial proteins (49% in TLM; 55% in MDM; 63% in GDM) using the SUBAcon
algorithm.
Abundance profiles were filtered for mitochondrial proteins (636 in TLM; 596 in MDM; 503 in GDM) and
hierarchically clustered by NOVA. Clustered mitochondrial abundance
profiles can be manually
inspected here. A search function enables
to look for candidate proteins and their potential interaction partners.
