DOI: 10.5061/dryad.pk0p2ngjd
title: Vacuolar sucrose homeostasis is critical for development, seed properties and survival of dark phases of Arabidopsis

description: Although we know that most of the cellular sucrose is present in the
 cytosol and vacuole, our knowledge on the impact of this sucrose
 compartmentation on plant properties is still fragmentary. Here we
 attempted to alter the intracellular sucrose compartmentation of
 Arabidopsis mesophyll cells by either, overexpression of the vacuolar
 sucrose loader BvTST2.1 or by generation of mutants with decreased
 vacuolar invertase activity (amiR vi1-2). Surprisingly, BvTST2.1
 overexpression led to increased monosaccharide levels in leaves, while
 sucrose remained constant. Latter observation allows the conclusion, that
 vacuolar invertase activity in mesophyll vacuoles exceeds sucrose uptake
 in Arabidopsis, which gained independent support by analyses on tobacco
 leaves transiently overexpressing BvTST2.1 and the invertase inhibitor
 NbVIF. However, we observed strongly increased sucrose levels in leaf
 extracts from independent amiR vi1-2 lines and non‑aqueous fractionations
 confirmed that sucrose accumulation in corresponding vacuoles. amiR vi1-2
 lines exhibited impaired early development and decreased weight of seeds.
 When germinated in the dark, mutant seedlings showed problems to convert
 sucrose into monosaccharides. Cold temperatures induced marked
 downregulation of the expression of both VI genes, while frost tolerance
 of amiR vi1-2 mutants was similar to WT indicating that increased
 vacuolar sucrose levels fully compensate for low monosaccharide
 concentrations.
description: <b>Materials and
 methods</b> <b>Plant material and growth
 conditions</b> <i>Nicotiana
 benthamiana</i>, <i>Arabidopsis thaliana</i> (ecotype
 Columbia-0) and corresponding Arabidopsis mutants were cultivated in a
 growth chamber (Weiss-Gallenkamp, Heidelberg, Gemany) on standardized soil
 (ED-73; Patzer; www.einheitserde.de), at a constant tempe­rature of 22°C
 and a light intensity of 120 µmol quanta m<sup>-2</sup>
 s<sup>-1 </sup>(µE). Plant cultivation was carried out under
 short day conditions,10 h light, 14 h darkness (standard conditions). For
 cold experiments, plants were grown for four weeks under standard
 conditions and subsequently acclimated to cold temperature for three days
 at 4°C (all other conditions were kept constant). For etiolation growth
 analyses, seeds were stratified for 24 h at 4°C, and cultivated in
 darkness for seven days on water soaked blotting paper. For seed analyses,
 plants were transferred after cultivation for four weeks at standard
 conditions to long day conditions (22°C and 200 µE, 16 h light per day).
 For dark recovery experiments, 4-week old plants grown under standard
 conditions were darkened for five days, followed by recovery for further 7
 days under standard conditions.   <b>Generation of mutants
 </b> For transient transformation of
 <i>Nicotiana benthamiana</i> leaf mesophyll cells the
 Agrobacterium infiltration method was performed according to an
 established method (Jung <i>et al</i>., 2015). For generation
 of <i>VI1-2</i> double knock down mutants, we followed an
 established protocol for gene silencing by artificial microRNA (amiRNA)
 <i>(</i>Schwab <i>et al</i>., 2006). With the web
 based amiRNA designer tool program (http://wmd3.weigelworld.org) an
 amiRNA, which targets <i>VI1</i>
 (<i>At1g62660</i>) and <i>VI2</i>
 (<i>At1g12240</i>) simultaneously, was designed. The sequence
 <i>TAAGGATGAATAAAAGCACGG </i>was used for generation of
 primers including the Gateway™ compatible sequences attP1 and attP2 to
 engineer the amiRNA fragment. The primer sequences are listed in Table S1.
 Subsequently, the fragment was sub-cloned via BP reaction into the
 Gateway™ entry vector pDONR/Zeo and via LR reaction into the destination
 vector pK2GW7, which contains a 35S-CaMV promotor. For generation of
 stably transformed Arabidopsis mutant plants, Agrobacterium-mediated
 transformation using floral dip was performed (Clough and Bent, 1998).
 <i>VI1-2</i> double knock-down mutants were selected by
 screening for the lowest remaining <i>VI1</i> and
 <i>VI2</i> transcript levels via qRT-PCR leading to the two
 independent lines no. 4 and 5.   <b>cDNA
 synthesis, qRT-PCR and RNA gel</b><b>‐</b><b>blot
 hybridization</b> RNA was isolated from 50 mg of
 frozen, fine ground plant material with the NucleoSpin RNA Plant Kit
 (Macherey-Nagel, Düren, Germany), according to the manufacturer’s
 protocol. For cDNA synthesis, RNA was transcribed into cDNA with the
 qScript cDNA Synthesis Kit (Quantabio, Beverly, MA, USA). The primers used
 for gene expression analysis by qRT-PCR are listed in Table S2.
 <i>NbAct</i>, <i>AtPP2A</i> and
 <i>AtSAND</i> were used as reference genes for transcript
 normalization. Alternatively, gene expression was analyzed by RNA gel‐blot
 hybridization.   <b>Acidic invertase activity
 assay</b> The enzyme assay was performed as
 described by (Tamoi <i>et al</i>., 2010) with slight
 modifications. 100 mg of frozen and fine ground plant material were
 homogenized with 1 ml of ice cold 200 mM Hepes/HCl (pH 5.0), 1 mM EDTA,
 1 mM PMSF on a vortex mixer for 20 sec. The samples were incubated for 20
 min on ice prior to further mixing with a vortex mixer for 20 sec.
 Subsequently, samples were centrifuged at 20.000 <i>g</i> for
 10 min at 4°C and the supernatant was transferred into a new reaction
 tube. The rate of sucrose hydrolysis was quantified spectrophotometrically
 at 22°C using a NADP-coupled enzymatic test (Stitt <i>et
 al</i>., 1989). For this, 15 µl of enzyme extract were added to
 190 µl 200 mM Hepes/HCl (pH 5.0), 10.5 mM MgCl<sub>2</sub>,
 2.1 mM ATP, 0.8 mM NADP, 0.5 U Glucose-6-Phosphate Dehydrogenase, 0.18 U
 Hexokinase and 0.48 U Phosphoglucose Isomerase. To start the enzyme
 reaction, 5 µl of 200 mM sucrose solution were added to the
 sample.   <b>Metabolite
 quantification</b> For sugar
 extraction, we added 400 µl of 80% of ethanol to 100 mg of frozen, fine
 grounded plant material, mixed and incubated for 30 min at 80°C, in a
 thermomixer at 500 rpm. After centrifugation at 16000 <i>g</i>
 (10 min at 4°C) the supernatant was used for sugar quantiﬁcation using a
 NADP-coupled enzymatic test (Stitt <i>et al</i>.,
 1989). <b> </b> <b>Non-aqueous
 fractionation</b> Subcellular sugar distribution of
 sugars in leaves was determined by non-aqueous fractionation of 4-week old
 plants. To this end, 15 mg of freeze-dried and fine grounded plant
 material was used and processed as described previously (Fürtauer
 <i>et al</i>., 2016). Acid phosphatase served as vacuolar
 marker, UGPase activity served as a cytosolic marker and alkaline
 pyrophos­phatase served as chloroplast marker. For sugar quantiﬁcation, a
 NADP-coupled enzymatic test was performed (Stitt <i>et
 al</i>., 1989) and subcellular metabolite distribution was
 calculated using an established algorithm (Fürtauer <i>et
 al</i>., 2016). <b> </b> <b>Seed
 analyses</b> Lipid quantification was performed
 according to a routine protocol (Reiser <i>et al</i>., 2004)
 with slight modifications. 0.1 g of mature, air-dried seeds were
 homogenized in a mortar and liquid nitrogen. Subsequently, 1.5 mL of
 isopropanol was added and the sample was further homo­genized. The
 suspension was transferred into a 1.5 mL-reaction tube and incubated for
 12 h at 4°C and 100 rpm. Subsequently, samples were centrifuged at 13000
 <i>g </i>for 10 min and the supernatant was transferred into a
 pre-weighed 1.5-mL reaction tube. Tubes were incubated at 60°C for 12 h to
 completely evaporate the isopropanol. Total lipid content was quantified
 gravimetrically. For determination of seed weight, 1000 mature and
 air-dried seeds were counted and their weight was quantified
 gravime­trically.   <b>Electrical
 conductivity</b> The frost
 tolerance, as measured by frost induced release of ions from leaf sample,
 of wild-type and mutant, was quantified by electrical conductivity assays,
 described earlier (Klemens <i>et al</i>., 2014).