The Effect Of Osmotic Stress-induced Changes Of Plasma Membrane Proteome In Arabidopsis Cio4Deletion

When plants receive the external stimulus, they will transmit external environmental stimulationsacross the plasma membrane into the cytosol via the membrane receptor. The membrane receptor can induce a series of phosphorylation reactions on the membrane (mainly on G proteins, phospholipases) to activate the calcium ion channels. This results in the release of Ca2+intothe cytoplasm from calcium store, relaying the signal from external membrane to internal membrane by increasing Ca2+concentration in the cytoplasm. Many important progresses have been made regarding how the environmental stress triggers signal transduction mechanisms within plant cells. However, research on how environmental signalsinducethe intracellular secondary messenger, Ca2+release through the receptors has been lacking.In this study, the homozygous calcium-insensitive mutant cio4was obtained by using hybridization technique and Ca+image analysis-based high-throughput screening.Theplasma membrane of Col/AQ and cio4were purified with discontinuous sucrose gradient centrifugation and aqueous two-phase partition. We then usetwo-dimensional electrophoresis (2-DE) and mass spectrometric to analyze the plasma membrane proteomes of cio4and Col/AQ. There is a total of230different points after analyzing2-DE electrophoresis. We chose41different points withthe most significant changefor protein peptide mass fingerprint (PMF) analysis, and successfully identified36points. Some important protein points are as follows:(1) Two kinases involved in Ca2+-dependent signaling are up-regulated in the plasma membrane of wildtype Col/AQ during osmotic treatment.Under infiltration treatment, we identified protein kinase family protein (point L5) and calmodulin-binding proteins (point L6). The two kinases play an important role in the Ca2+signaling pathway. The two proteins (L5, L6) are both up-regulated in the wildtype Col/AQ, while protein point L5cannot be detected and point L6is expressed at a very low level in mutant cio4.(2) The accumulation of enzymes involving stress-tolerant enhanced in mutant cio4compared to wildtype under osmotic treatment. The wildtype Col/AQ and mutant cio4both accumulate a lot of resistant materials such as some enzymes involved in reactive oxygen species (ROS) scavenging, ascorbic acid reductase (M3) temperature adjustable lipid carrier protein (M20) peroxide reductase (L3) key protein disulfide isomerase (L18), and the other enzymes involving resistant materials such as jacalin-like lectin domain-containing protein(L16) ATP synthase subunitbeta-1(M4) etal in the plasma membrane under osmotic stress. These enzymes are up-regulated in mutant cio4. The some expression levels in wildtype Col/AQ were lower than mutant cio4, while the others are down-regulated.In conclusion, two important kinases involved in Ca2+signaling pathway are up-regulated obviously in the wild type Col/AQ, while their plasma membrane levels in mutant cio4are very low. It is probable that the mutant cio4are unable to induce the expression or localization to plasma membrane of the two kinases due to defect in calcium signaling.(2) The level of enzymes involving stress-tolerant as enzymes involved in ROS scavenging pathways and other enzymes such as jacalin-like lectin domain-containing protein (L16) ATP synthase subunit beta-1(M4) GDSL lipase (M9,M13) elongation factor (M7) were accumulated higher in cio4than that in Col/AQ. The cio4mutant suffers from the disruption in homeostasis of intracellular physiological environment due to deficient Ca2+signaling, and thus instead enhances the accumulation of intracellular resistance material as compared to the wildtype Col/AQ in a short period of time.Those results indicate: Ca2+-dependent signaling plays an important role in maint aining cellular homeostais in response to stressresistance, while the disruption of Ca2+-dependent signaling results in the ectopic expression of stress-related enzymes and th us the transient dysregulation of plant metabolism and the accumulation of stress-relat ed substances; alternatively, the accumulation of some of the stress-related substances is independent of Ca+-dependent signaling when plants are challenged with stress resistance.