Yukio KAWAMURA - Plant Physiology - ykawa@

I. Research achievements
1. Understanding the mechanism of chilling-induced cytoplasmic acidification in a chilling-sensitive plant
Chilling injury proceeds in two distinct processes: an early reversible process which is caused by the primary sites of chilling injury and a latter irreversible process. For better understanding of the chilling injury mechanism, it is important to reveal the primary sites vulnerable to low temperatures. In chilling-sensitive plants, the vacuolar membrane is estimated to be a primary site of chilling injury in the dark. Chilling rapidly results in cytoplasmic acidification which is due to H+-leakage from the vacuole; however, the detailed mechanism of the acidifcation has been unclear. In nature, plant cells always suffer quick changes of environmental conditions (e.g., temperature) and may not be able to deal with the quick changes only by the expression of genes. To keep intracellular conditions stable during the quick changes of environmental conditions, the biochemical adjustment system, for example, pH-stat system, must be present in plant cells. It is estimated that the cytoplasmic pH-stat system is tightly related to stabilizing delta pH between the cytoplasm and the vacuole (delta pHvac), referred to as delta pHvac-stat system. Finally, our results showed PPi-dependent delta pHvac-stat system might keep the stable delta pHvac, and stable cytoplasmic pH as well. Chilling led to the increase of PPi-dependent H+-permeability and the decrease of H+-influx activity by V-PPase, and consequently, PPi-dependent delta pHvac-stat system could not keep enough delta pHvac. Thus, cytoplasmic acidification may be directly caused by the breakdown of PPi-dependent delta pHvac-stat system.

2. Identification of putative plasma membrane proteins associated with cold acclimation.
Enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, but the molecular mechanism for the increased cryostability of plasma membrane is still unclear. Although there have been many studies on cold-acclimation-associated changes in lipid composition in the plasma membrane and their roles in enhancement of freezing tolerance, similar studies with plasma membrane proteins are still lacking. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.

II. Goals in the COE Program
The membrane repair related protein of synaptotagmin-like protein which quickly increased during cold acclimation was identified in the plasma membrane fraction using MALDI-TOF MS. Rapid membrane repair is general physiological phenomenon in animal cells which inhabit mechanically stressful environments, although nobody has studied this mechanism in plant cells. The mechanical disruption of plasma membrane results in the incursion of extracellular Ca2+ into cytoplasm, and consequently, endomembranes are delivered to and fuse into the damaged plasma membrane through the exocytosis manner which is related with SNAREs and synaptotagmin family. This idea may be adapted to the plant mechanical stress, too. When the plasma membrane is disrupted, for example, by the ice formation or by the freeze-induced dehydration, if the plant cells rapidly can repair or reseal the disruption, they may survive the extreme mechanical stress induced by the freezing dependently on the extracellular Ca2+ at least. At present, our result suggested that the freezing tolerance of Arabidopsis protoplasts isolated from control and cold-acclimated samples remarkably decrease in the buffer without Ca2+. Finally, our results will lead to the better understanding of the plant cold/freezing adaptation mechanisms.

III. Related website
http://news7a1.atm.iwate-u.ac.jp/~crcdbbt/index.htm (Japanese only)

Back