Immunolocalization of plasma-membrane H + -ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L.

Abstract

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…Immunolocalization of PIN1 was performed using an affinity-purified rabbit anti-PIN1 antibody as described by Gälweiler et al (1998). Dilutions of primary anti-PIN2 guinea pig antibody (Ditengou et al, 2008) was 1:600 and for the anti-PM H + -ATPase rabbit antibody (Langhans et al, 2001; Agrisera AS07 260) 1:1000. As secondary antibodies, ALEXA Fluor 488 goat anti-guinea pig (Invitrogen) and ALEXA Fluor 555 goat anti-rabbit (Invitrogen) diluted to 1:600 were used.…”

Arabidopsis Type I Proton-Pumping Pyrophosphatase Expresses Strongly in Phloem, Where It Is Required for Pyrophosphate Metabolism and Photosynthate Partitioning

“…Interestingly, double-labeling immunolocalizations showed the H + -PPase and the well-characterized P-type H + -ATPase in proximity at the PM of companion cells in Ricinus communis (Langhans et al, 2001). Langhans et al (2001) suggested that both H + pumps generate the PMF to maintain high Suc, K + , and amino acid concentrations in the phloem.…”

“…Langhans et al (2001) suggested that both H + pumps generate the PMF to maintain high Suc, K + , and amino acid concentrations in the phloem. Coimmunogold labeling studies also showed that Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) is localized at the PM of SE-CC complexes .…”

“…Given the localization of H + -PPases at the PM of SE-CC complexes (Langhans et al, 2001;Paez-Valencia et al, 2011), the reversibility of the plant enzyme (Rocha Facanha and de Meis, 1998;Marsh et al, 2000), the thermodynamic constraints of PP i hydrolysis coupled to proton transport (Davies, 1997), and the key role of PP i in companion cells (Lerchl et al, 1995), a model was recently proposed , in which the PM-localized H + -PPase can function as a synthase in the phloem to maintain PP i homeostasis for the Suc oxidation and ATP production required for the generation of the PMF through PM H + -ATPase activity. As a corollary, increased PP i synthase activity in companion cells would augment phloem loading and long-distance transport in transgenic plants by indirectly energizing the SE-CC complex of source leaves.…”

Structural basis for the reversibility of proton pyrophosphatase

“…[5][6][7][8][9][10] However, H C -PPase also saliently localizes at the vascular tissues of plants, wherein this protein is predominantly localized at the plasma membrane (PM) of the sieve element companion cell (SE-CC) complexes. 8,[11][12][13] Thermodynamic, 14 genetic, immunohistochemical, and physiological evidence 8,15,16 is consistent with a reverse PPi synthase activity of H C -PPase at the PM in the phloem, where it plays an important role in PPi homeostasis and photosynthate partitioning. 10,[15][16][17] We contend that the magnitude of proton gradients associated with the tonoplast or PM location of this enzyme drives the protein's structurally congruent potential to work in a reversible manner -either in the synthesis or hydrolysis of its substrate, PPi.…”