Stress is basic: ABA alkalinizes both the xylem sap and the cytosol of Arabidopsis vascular bundle sheath cells by inhibiting their P-type H+-ATPase and stimulating their V-type H+-ATPase

ABSTRACTBACKGROUND AND HYPOTHESIS•Under water deprivation, in many perennial species, the stress hormone, ABA, appears in the xylem sap in the shoot (including leaf) veins and the xylem sap pH (pHEXT) increases. This study aimed to test the hypothesis that ABA is the signal for an altered proton balance of the leaf-vein-enwrapping bundle sheath cells (BSCs).METHODS•Plant Material. We used a few Arabidopsis thaliana (L.) Heynh. genotypes: wildtype (WT) of two accessions, Landsberg erecta (Ler) and Columbia (Col), and a few mutants and transformants in these backgrounds.•H+-Pumps activities. We monitored ABA effects on the H+-pump activities in the BSCs cytosol-delimiting membranes (plasma membrane and tonoplast) by monitoring the cytosol and the xylem pH, and the membrane potential (EM), by imaging the fluorescence of pH- and membrane potential (EM)-reporting probes: (a) the BSCs’ pHEXT – with the ratiometric fluorescent dye FITC-dextran petiole-fed into detached leaves in unbuffered xylem perfusion solution (XPS), (b) the BSCs’ pHCYT – with the ratiometric dye SNARF1 loaded into BSCs isolated protoplasts, and (c) the BSCs’ EM – with the ratiometric dye di- 8-ANEPPS.RESULTS•ABA increased the pHEXT; this response was abolished in an abi1-1 mutant with impaired signaling via a PP2C (ABI1) and in an aha2-4 mutant with knocked-down AHA2;•ABA depolarized the WT BSCs;•ABA increased pHCYT irrespective of AHA2 activity (i.e., whether or not AHA was inhibited by vanadate, or in the aha2-4 mutant);•The ABA-induced cytosol alkalinization was abolished in the absence of VHA activity (i.e., when VHA was inhibited by bafilomycin A1, or in the vha-a2 vha-a3 double mutant with inactive VHA);•All these results resemble the ABA effect on GCs;•In contrast to GCs, AHA2 and not AHA1 is the ABA major target in BSCs;•Blue light (BL) enabled the response of the BSCs’ VHA to ABA;•The ABA- and BL-signaling pathways acting on both BSCs’ pumps, AHA2 and VHA, are likely to be BSCs autonomous, based on (a) the presence in the BSCs of many genes of the ABA- and BL-signaling pathways and (b) ABA responses (depolarization and pHCYT elevation) demonstrated under BL in isolated protoplasts.SIGNIFICANCE STATEMENTWe reveal here an alkalinizing effect of the plant drought-stress hormone ABA on the pH on both sides of the plasmalemma of the vein-enwrapping bundle sheath cells (BSCs), due to ABA inhibition of the BSCs’ AHA2, the plasmalemma H+- ATPase and stimulation of VHA, their vacuolar H+-ATPase. Since pH affects the BSCs’ selective regulation of solute and water fluxes into the leaf, these H+- pumps may be attractive targets for manipulations aiming to improve plant drought response.

Effect of Purine Alkaloids on the Proliferation of Lettuce Cells Derived from Protoplasts

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with >250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

The influence of kinetin on regeneration of the protonema from isolated protoplasts of the moss Funaria hygrometrica L. Sibth

No stimulating effect of kinetin was noted on formation of the cell wall surrounding isolated protoplasts of the moss <em>Funaria hygrometrica</em>, although kinetin at a concentration of 1µM distinctly accelerated division of cells obtained from protoplasts and stimulated formation of the protonema and gametophores. At higher kinetin concentrations (5 µM) protoplasts, and later cells, increased their volume, whereas regeneration processes took place considerably more slowly than in the control.

Effect of nanosilver on potato plant growth and protoplast viability

Potato tissue culture is sensitive to ethylene accumulation in the culture vessel. Ag inhibits ethylene action but no information on nanosilver application in potato tissue culture has been published so far. In our study, potato cv. White Desiree was treated with nanosilver (0, 1.0, 1.5, and 2.0 ppm) in vitro. Leaf surface was increased, while stem length and root length decreased. Nanosilver caused also a decrease in the number of isolated protoplasts and in the viability of isolated protoplasts when applied either directly or indirectly.

Detection of Silver Nanoparticles Internalization into Petunia, Petunia hybrida, Isolated Protoplasts

The use of nanoparticles has spannded many divisions of biological fields including sensors, drug delivery and clinical diagnosis. These functions all based on the most important action in cells called Endocytosis. The study of endocytosis in higher order plants remained inactive for a long period of time. In this work we use Silver Nano Particles (S.N.Ps) as a endocytosic substances were incubated by isolated protoplast of petunia .Concurrently, with the study of internalization, their appear aggregation inter and intracellular were investigated by Transmissions Electron Microscopy (TEM). Results and observations showed that the S.N.Ps crossed the cell membrane and internalized in to the cells. It can be assumed that the particles with the organic functionality – PEG coated – were internalized by the petunia protoplasts.