Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein inArabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures  

Abstract

We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H + -pyrophosphatase (H + -PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H + -PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H + -PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H + -PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H + -PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.

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

“…S5). Of note, this vhp1-1 mutant retains approximately 25% of PPase activity in total membrane fractions compared with Col-0 (Segami et al, 2014). Other AVP1 mutants (fugu5-1, fugu5-2, and fugu5-3) without detectable tonoplast PPase activity have been reported (Ferjani et al, 2011).…”

“…An AVP1 mutant, Vacuolar proton pyrophosphatase1-1 (vhp1-1; Ferjani et al, 2011;Segami et al, 2014), also showed a significant growth reduction in Suc-depleted medium but no growth impairment under Suc supplementation (Supplemental Fig. S5).…”

“…The plant materials used in this work are as follows: Col-0, gain-of-function AVP1-1 line (Gaxiola et al, 2001), T-DNA promoter insertion line avp1-2 (SALK 046492), transgenic reporter line p1.7AVP1::GUS (Yang et al, 2007), pCoYMV::AVP1 and p35S::AVP1 (Col-0 background; this work), pCoYMV:: RNAi AVP1 (in an AVP1-1 background), pCoYMV::ppa (in an avp1-2 background), fugu5-1 (Ferjani et al, 2011), and vhp1-1 (Segami et al, 2014).…”

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism

“…However, highly dynamic tonoplast deformations have been reported in many different developmental contexts and environmental conditions (Saito et al, 2002(Saito et al, , 2011a(Saito et al, , 2011bReisen et al, 2005;Oda et al, 2009;Zheng and Staehelin, 2011;Segami et al, 2014;Zheng et al, 2014). Vacuolar "bulbs" are defined as complex local invaginations of the tonoplast that could serve as membrane reservoirs for rapid vacuolar volume changes.…”

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

“…AVP1 overexpression has also been reported to result in increased cell division at the onset of organ formation and increased auxin transport, which appeared to be a consequence of increased DpH PM (visualized as cytosolic alkalinization) resulting from altered distribution and abundance of the plasma membrane (PM) H + -ATPase and the PIN-FORMED1 auxin efflux facilitator . Although AVP1 clearly is an abundant tonoplast protein (Segami et al, 2014), it has been reported to be localized at the PM in sieve element companion cells and upon overexpression also in other cell types (Langhans et al, 2001;Paez-Valencia et al, 2011;Pizzio et al, 2015). The mechanistic base of the beneficial traits achieved by overexpression of AVP1 is thus unclear, and it remains to be determined if and to what extent increased vacuolar solute accumulation due to increased proton pumping by the V-PPase is involved.…”