Apoplast acidification in growing barley (Hordeum vulgare L.) leaves

Visnovitz, Tamás (2011) Apoplast acidification in growing barley (Hordeum vulgare L.) leaves. PhD thesis, University College Dublin.

Text (Apoplast acidification in growing barley (Hordeum vulgare L.) leaves (Thesis; Visnovitz Tamás UCD, Írország))

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Supervisor name: Fricke, Wieland
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Apoplast acidification associated with growth is well-documented in roots, coleoptiles and internodes but not in leaves. In the present project on barley (Hordeum vulgare L.) advantage was taken of the high cuticle permeability in the elongation zone of leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H+-ATPase (PM-H+-ATPase) and K+ in this process was of particular interest. An in vitro gel system with bromocresol purple as pH indicator, pH microelectrodes and pH-sensitive fluorescence dye combined with confocal microscopy were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer (LVDT). Test reagents which blocked (vanadate) or stimulated (fusicoccin) PM-H+-ATPase, or which reduced (NH4 +, Cs+, tetraethylammonium) K+ uptake were applied. Plasma membranes were isolated from growing and mature leaf tissue and used to determine the activity (ATPase assay) and abundance (Western blotting) of PM-H+-ATPase protein. Protein localisation was studied by immunohistochemistry and expression of mRNA quantified using real time PCR (qPCR). Apoplast pH was by up to 1.0 pH unit lower in growing compared to nongrowing leaf tissue. Depending on the K+ concentration in the bathing medium used during electrophysiological analyses, apoplast pH in the elongation zone ranged from pH 4.8 (0.1 mM K+) to pH 5.8 (10 mM K+). In the emerged blade, apoplast pH remained at about pH 5.8 irrespective of the K+ concentration in the bathing medium Growth was more responsive to test reagents than to changes in apoplast pH. Expression of PM-H+- ATPase was comparable between growing and non-growing leaf regions when expression was related to per unit extracted RNA or cell number. However, when expression was related to per unit surface area of plasma membrane, expression of PM-H+-ATPase was about twice as high in growing compared to non-growing leaf tissue. The same applied to the protein level and activity of PM-H+-ATPase. Immunohistochemical analyses showed that PM-H+-ATPase was present in all living leaf tissues, particular in those (guard cells, phloem, and xylem parenchyma) associated with high rates of trans-membrane solute transport. It is concluded that leaf cell expansion in barley depends on the activity of the PM-H+-ATPase and K+ transport processes. The higher surface density of PM-H+-ATPase activity in growing barley leaf tissue aids apoplast acidification and growth. A H+ / K+ co-transport system may play a key role in linking growth with apoplast pH, H+ pump activity and K+-uptake.

Item Type: Thesis (PhD)
Subjects: Q Science / természettudomány > QK Botany / növénytan
Depositing User: Erika Bilicsi
Date Deposited: 23 Aug 2013 07:19
Last Modified: 22 Jan 2014 08:40

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