Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis

Abstract

Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H(+)-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1(Pro):IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

Figure 1.

Morphological and Cellular Phenotypes of the fugu5 Mutant. (A) Effect of growth media composition on the gross phenotype of the fugu5 mutant. Wild-type (WT) and fugu5-1 mutant seedlings were grown for 8 DAS either on rockwool (left panels) or on standard MS medium (right panels). Bars = 2 mm. (B) Cell proliferation is compromised in fugu5 cotyledons after germination. Number of palisade mesophyll tissue cells along proximodistal and mediolateral axes of either embryonic cotyledons (dissected from imbibed dry seeds) or mature cotyledons was determined after growth on rockwool for 25 DAS. Data are means and sd (n = 8). Cot, cotyledons; PD, proximodistal; ML, mediolateral. (C) Effect of various carbohydrates on the gross phenotype of fugu5 cotyledons. The effect of exogenously supplied Suc, Glc, and Fru (58 mM each) on cotyledon phenotype is shown. The effect of simultaneous addition of Glc and Fru was also tested. Eight-day-old seedlings of the wild type (left panels) and fugu5-1 (right panels) are shown. Bar = 2 mm. (D) Effect of different kinds of carbohydrates on fugu5-1 cotyledon cell number. Palisade mesophyll tissue cell numbers in seedlings of either the wild type or fugu5-1 mutants were determined 8 DAS. Data are means and sd (n = 8). NS, no significant difference between the two genotypes (the wild type and fugu5-1) under the indicated growth conditions. (E) Effect of exogenously supplied Suc on compensated cell enlargement. Cotyledons were collected from the wild type and fugu5-1 mutant grown on MS media with or without 2% Suc for 21 DAS. Data are means and sd (n = 8). NS, no significant difference between the two genotypes (the wild type and fugu5-1). Asterisk indicates significant difference at P < 0.01.

Figure 2.

V-PPase and V-ATPase Activities in fugu5 Mutant Alleles. (A) Schematic representation of the AVP1/FUGU5 gene. Exons are shown as filled rectangles. The molecular lesion in each of the three loss-of-function fugu5 alleles is indicated by an asterisk. In fugu5-1, the Ala-709 residue is replaced by Thr. In fugu5-2, the Glu-272 residue is replaced by Lys. In fugu5-3, the Ala-553 residue is replaced by Thr, and the five residues from Leu-554 to Ala-558 are deleted. (B) Substrate hydrolysis activity of V-PPase (top) and V-ATPase (bottom) in crude membranes prepared from wild-type (WT) and fugu5 mutants. Plants were grown in culture medium without Suc for 17 DAS. Crude membranes were prepared from shoots of more than 240 plants and used for enzyme assays as described in Methods. (C) to (F) Protein levels of vacuolar membrane proton pumps, BIP, and aquaporin. Crude membrane aliquots (2 μg protein) of the membrane fractions were separated by SDS-PAGE and subsequently immunoblotted with anti-V-PPase (C), anti-subunit A and anti-subunit a of V-ATPase (D), anti-BIP (E), and anti-TIP1s (F). Apparent molecular masses of the immunostained bands are shown in each panel. The relative signal intensity of immunostained bands was quantitatively measured and calculated as the ratio to that of the wild type of each protein. V-PPase protein was not detected in the fugu5-3 mutant line.

Figure 3.

Expression of DR5:GUS and Effects of Exogenous Auxins. (A) to (F) Expression pattern of the DR5:GUS reporter gene in young seedlings. Expression of DR5:GUS in the seedlings of the wild type (A) and fugu5-1 mutant (B) at 6 DAS. Expression of DR5:GUS in wild-type root and lateral root primordium at 6 DAS, respectively ([C] and [D]). Expression of DR5:GUS in fugu5-1 mutant root and lateral root primordium at 6 DAS, respectively ([E] and [F]). DR5:GUS signals around root apical meristem (black arrowheads), in the vascular system (open arrowheads), and in the emerging lateral root primordia (asterisk). Bars = 2 mm in (A), 1 mm in (B), and 100 μm in (C) to (F). (G) Effect of exogenous auxin on the growth of fugu5 mutant. Seedlings of the wild type (WT) and fugu5-1 grown on MS alone, MS + 2% Suc, MS + indole-3-acetic acid (5 μM), and MS + 1-naphthaleneacetic acid (5 μM) 8 at DAS. Bar = 2 mm. (H) Effect of exogenous auxin on cell number in the cotyledons. Average cell numbers of samples described in (G) were determined. Data are means and sd (n = 8). NS, no significant difference between the two genotypes (the wild type and fugu5-1). Asterisk indicates significant difference at P < 0.01.

Figure 4.

Degradation of Protein Bodies in fugu5 and the Morphological and Cellular Phenotype of AVP1_Pro:IPP1 Transgenic Lines. (A) Effect of V-PPase dysfunction on mobilization of seed storage proteins. Protein samples were collected at 0, 48, 72, and 96 h (time after transfer to growth temperature) from either 30 seeds or seedlings of the wild type and fugu5-1 mutants. Proteins were separated using SDS-PAGE and subsequently stained with Coomassie blue. Lanes 1, 3, 5, and 7 are wild-type samples. Lanes 2, 4, 6, and 8 are the fugu5-1 mutant samples. Results were reproducible in three independent experiments. (B) The heterologous expression of IPP1 in the fugu5-1 mutant background. RT-PCR analyses of IPP1 were performed in six independent AVP1_Pro:IPP1 transgenic lines that we constructed. RT(−) = no reverse transcriptase. WT, wild type. (C) The heterologous expression of IPP1 rescues fugu5 gross phenotypes. Gross morphology of seedlings of the wild type, fugu5-1, and two representative lines of AVP1_Pro:IPP1 transgenic plants at 7 DAS. Bar = 2 mm. (D) to (G) The heterologous expression of IPP1 rescues delayed growth of fugu5. Gross morphology of the wild type, fugu5-1, and AVP1_Pro:IPP1#4-4 and AVP1_Pro:IPP1#8-3 transgenic plants, respectively, at 28 DAS. Bar = 2 cm. (H) The heterologous expression of IPP1 gene totally rescues fugu5 cellular phenotypes. Average area, cell number, and cell size of cotyledons of the wild type, fugu5-1, and two representative lines of AVP1_Pro:IPP1 grown on rockwool for 25 DAS. Data are means and SD (n = 8). Asterisk indicates significant difference at P < 0.01 compared with the wild-type.

Figure 5.

Germination Rate, Postgerminative Growth, and Seedling Growth Phenotype of fugu5 Mutant in the Dark. (A) The germination rates are not affected in three fugu5 mutant alleles. One hundred seeds of each genotype (three sets) were sown on rockwool, and plants were grown in a 16/8-h light/dark cycle. The germination rates are relative mean values and sd from three independent experiments. WT, wild type. (B) fugu5 mutants exhibit a slight postgerminative growth delay. The germination rate and postgerminative growth phenotype of plants were scored at 12 and 21 DAS, respectively. Data are relative mean values and sd from three independent experiments. Asterisk indicates significant difference at P < 0.05 compared with the wild type. Normal, plants without significant growth delay; semi-dwarf, plant size reduced by ~50% compared with normal plants; dwarf, plant size reduced by ~75% compared with normal plants. (C) and (D) Hypocotyl elongation is severely inhibited in fugu5-1 in the dark in the absence of Suc. (C) Photographs of hypocotyls of etiolated seedlings of the wild type, fugu5-1, and AVP1_Pro:IPP1#8-3 grown for 4 DAS in the dark on either MS alone (top panel) or MS + Suc media (bottom panel). Bars = 5 mm. (D) Hypocotyl length of etiolated seedlings was determined at 4 DAS. Data are means and sd (n = 20). Asterisk indicates significant difference at P < 0.001 compared with the wild type.

Figure 6.

Effects of the Loss of V-PPase Activity on Suc and PPi Contents and the Vacuolar pH. (A) The amounts of Suc are significantly decreased in the fugu5-3 mutant. The amounts of Suc in the wild type (WT) and the fugu5-3 mutant were determined during postgerminative growth in 3-d-old etiolated seedlings grown on MS-only medium (four hundred etiolated seedlings per experiment) as described in Methods. Data are means and sd from three independent experiments. Asterisk indicates significant difference at P < 0.05 compared with the wild type. (B) The amounts of PPi are significantly increased in the fugu5-3 mutant. Seedlings grown under the same conditions and collected at the same stage as described in (A) were used for the quantification of PPi. Data are means and sd from three independent experiments. Asterisk indicates significant difference at P < 0.05 compared with the wild type. (C) Loss of the V-PPase activity causes a slight alkalinization of vacuolar pH in the fugu5-1 mutant. The vacuolar pH of 10-d-old seedlings grown on MS medium without Suc was determined using the fluorescent cell-permeant dye BCECF-AM as described (Krebs et al., 2010). The loss of V-PPase activity increases the vacuolar pH in root cells by 0.25 pH units. Data are means and sd of 23 measurements from 10 seedlings (the wild type), 25 measurements from nine seedlings (fugu5-1), and 26 measurements from 10 seedlings (AVP1pro:IPP1#8-3). Asterisk indicates significant difference at P < 0.005 compared with the wild type. NS, no significant difference between the two genotypes (fugu5-1 and AVP1pro:IPP1#8-3). [See online article for color version of this figure.]

Figure 7.

Suc Synthesis and PPi Generation Processes during Germination in Oilseeds. The metabolic pathways shown here were deduced from Taiz and Zeiger (2010). Fatty acids from TAG are converted to acetyl-CoA by β-oxidation and then to succinate by the glyoxylate cycle operating in glyoxysomes. PPi is generated by the reaction of fatty acyl-CoA synthase (a). Succinate is transferred into mitochondria and converted to malate by the citric acid cycle reactions. Malate exported into the cytosol is oxidized to oxaloacetate and converted to phosphoenolpyruvate. During gluconeogenesis from phosphoenolpyruvate, PPi is generated by the reaction of PPi-dependent phosphofructokinase (b). PPi is also produced by the reaction of UDP-Glc pyrophosphorylase (c), which provides UDP-Glc. Syntheses of macromolecules, such as cellulose, also generate PPi as a by-product (d). PPi in the cytosol is consumed by V-PPase/AVP1/FUGU5.