Study On The Regulation Between The Cytoskeleton And Osmotic Pressure

Osmotic pressure is one of the most important environmental factors and plays crucial roles in plant growth and development. Alternation of osmotic pressure is also one of the most universal mechanostimuli plants have to deal with. It is involved in drought, salty stresses and can induce morphological and physiological changes to plant cells, which in turn will affects the growth direction and developmental pattern of plants. However, the pathways underlying plant osmotic perception and translate the osmotic signals into physiological responses and morphological outputs remain largely unknown. The present thesis work is a part of the grant project entitled"Functions and interactions of CW-PM-CSK continuum and Ca2+ signal in plant cell mechanotransduction". In this thesis, the correlation between osmotic pressure changes and microtubule/microfilament cytoskeleton arrangement was investigated, and the functions of microtubule/microfilament cytoskeletons in plant osmotic perception were analyzed.The rthogonal experiment was designed to find the best hormone level for GFP-FABD2 and GFP-MBD callus inducing. Then Arabidopsis suspension cells were used in this thesis to observe the morphological and cytoskeleton changes under osmotic changes which were induced by altering the concentration of mannitol in the liquid growth medium. Cytoskeleton arrangement was observed by imaging the GFP-FABD2 (for actin microfilament) and GFP-MBD (for microtubule) transgenic Arabidopsis suspension cells with laser confocal microscopy. Specific cytoskeleton-targeted agents were used to disrupt the normal functions of cytoskeletons. By comparing morphological and cytoskeleton arrangement of Arabidopsis suspension cells under different osmotic changes and drug treatments, we got the following results:①Optimized growth medium for GFP-FABD2 transgenic Arabidopsis suspension cells: MS+100mg/L inositol+150mg/L glutamine+30g/L sucrose +2.0 mg/L2,4-D +0.05 mg/L NAA+0.05 mg/L KT; optimized growth medium for GFP-MBD transgenic Arabidopsis suspension cells: MS+100mg/L inositol +150mg/L glutamine +30g/L sucrose +2.0 mg/L 2,4-D +0.5 mg/L NAA+0.5 mg/L KT.②Effects of osmotic pressure change to microtubule cytoskeleton arrangement: under hyperosmotic condition, protoplasts shrinked, microtubule cytoskeletons depolymerized, diffused in the cytosol and accompanied with aggregated dots; under hyposmotic condition, protoplast swelled, microtubule cytoskeleton bundled.③Effects of osmotic pressure change to actin microfilament cytoskeleton arrangement: under hyperosmotic condition, protoplast shrinked, actin microfilaments were compressed together and accompanied with emergence of Hechtian strands at the longitudinal ends, further compression of actin microfilaments accompanied with the breakage of these Hechtian strands; under hyposmotic condition, protoplast swelled, fine actin microfilaments disappeared, diffused in the cytosol but not aggregated into dots.④Regulation of microtubule cytoskeleton in perception of osmotic pressure change: under hyperosmotic condition, the stronger the average intensity of microtubule cytoskeletons, the stronger the protoplasts shrinked; under hyposmotic condition, the stronger the average intensity of microtubule cytoskeletons, the weaker the protoplasts swelled.⑤Regulation of actin microfilament cytoskeleton in perception of osmotic pressure change: under hyperosmotic condition, there is no regulation between the average intensity of microtubule cytoskeletons or the protoplasts shrinked; under hyposmotic condition, the stronger the average intensity of actin microfilament cytoskeletons, the strongerr the protoplasts swelled.Through the above results, we conclude that not only osmotic changes can regulate the morphology of plant cells, but also microtubule/microfilament cytoskeletons can regulate the changes of protoplasm to against the osmotic changes.