The Plant Cell, Vol. 12, 535–546, April 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

Cosuppression of a Plasma Membrane H

1

-ATPase Isoform Impairs Sucrose Translocation, Stomatal Opening, Plant Growth, and Male Fertility

Rongmin Zhao,

a

Vincent Dielen,

b

Jean-Marie Kinet,

b

and Marc Boutry

a,1

a

Unité de Biochimie Physiologique, Université Catholique de Louvain, Croix du Sud, 2-20, B1348, Louvain-la-Neuve, Belgium

b

Unité de Botanique Générale, Université Catholique de Louvain, Croix du Sud, 2-20, B1348, Louvain-la-Neuve, Belgium

The plasma membrane H

1

-ATPase builds up a pH and potential gradient across the plasma membrane, thus activatinga series of secondary ion and metabolite transporters.

pma4

(for plasma membrane H

1

-ATPase 4), the most widely ex-

pressed H

1

-ATPase isogene in

Nicotiana plumbaginifolia

, was overexpressed in tobacco. Plants that overexpressedPMA4 showed no major changes in plant growth under normal conditions. However, two transformants were identifiedby their stunted growth, slow leaf initiation, delayed stem bolting and flowering, and male sterility. Protein gel blot anal-ysis showed that expression of the endogenous and transgenic

pma4

was cosuppressed. Cosuppression was develop-mentally regulated because PMA4 was still present in developing leaves but was not detected in mature leaves. Theglucose and fructose content increased threefold, whereas the sucrose content remained unchanged. The rate of su-crose exudation from mature leaves was reduced threefold and the sugar content of apical buds was reduced twofold,suggesting failure of sucrose loading and translocation to the sink tissues. Cosuppression of PMA4 also affected theguard cells, stomatal opening, and photosynthesis in mature leaves. These results show that a single H

1

-ATPase iso-form plays a major role in several transport-dependent physiological processes.

INTRODUCTION

The plasma membrane proton-translocating ATPase (H

1

-ATPase) acts as a primary transporter that pumps protonsout of the cell, thus creating a pH and electrical potentialgradient across the plasma membrane that in turn activatesmany secondary transporters involved in ion and metaboliteuptake (reviewed in Serrano, 1989; Sussman, 1994; Micheletand Boutry, 1995; Palmgren, 1998). H

1

-ATPase is encodedby a gene family of

z

10 members that are differentially ex-pressed depending on the cell type, developmental stage,and environmental factors (Ewing and Bennett, 1994; Harperet al., 1994; Michelet et al., 1994; Moriau et al., 1999; Oufattoleet al., 2000). All of the genes encoding H

1

-ATPases thathave thus far been isolated as cDNA clones belong to twosubfamilies, which probably represent the most highly ex-pressed H

1

-ATPases (Moriau et al., 1999). Possible func-tional differences between H

1

-ATPase isoforms belongingto the same or different subfamilies have been studied usingthe heterologous expression in yeast of Arabidopsis and

Nicotiana plumbaginifolia

H

1

-ATPase genes. Three Arabi-dopsis H

1

-ATPase genes (

aha1

,

aha2

, and

aha3

, for Arabi-

dopsis H

1

-ATPases 1, 2, and 3) belonging to the samesubfamily have been shown to have different kinetic param-eters (Palmgren and Christensen, 1994), whereas two

N.plumbaginifolia

genes belonging to different subfamilies(

pma2

and

pma4

, for plasma membrane H

1

-ATPases 2 and4) were shown not only to have different kinetics but also toconfer different sensitivity of yeast growth to the external pH(Luo et al., 1999).

Although these data clearly suggest that the plant H

1

-ATPases are not functionally identical, it remains to be es-tablished whether this is also the case in the plant and whatthe significance of this might be for transport activation. Forinstance, study of Arabidopsis H

1

-ATPase gene expressionsuggests that certain H

1

-ATPase isoforms might be special-ized in different major transport functions (e.g., mineral nutri-tion, phloem loading, or stomatal opening) (DeWitt et al.,1991; Harper et al., 1994; DeWitt and Sussman, 1995). Thesituation is different in

N. plumbaginifolia,

in which at leasttwo genes (

pma2

and

pma4

) are expressed in many cell typesand with a partial overlap (Moriau et al., 1999; Oufattole etal., 2000). Whatever the case may be, a functional study ofH

1

-ATPase isoforms in the plant is required to clarify the re-lationship between H

1

-ATPase expression and activity andplant physiology. Young et al. (1998) recently took a furtherstep toward this goal. They analyzed Arabidopsis transgenic

1

To whom correspondence should be addressed. E-mail boutry@fysa.ucl.ac.be; fax 32-10-473872.

536 The Plant Cell

plants expressing the AHA3 H

1

-ATPase with an altered Cterminus. When grown in vitro, the Arabidopsis transfor-mants were more resistant to acid medium, thus suggestinga role for the plasma membrane H

1

-ATPase in cytoplasmicpH homeostasis.

In

N. plumbaginifolia

, nine

pma

genes have been identified(Perez et al., 1992; Moriau et al., 1999; Oufattole et al.,2000). Studies with seven of them with the

b

-glucuronidase(

gus

A) reporter gene have clearly indicated that subfamilies I(

pma1

,

pma2

, and

pma3

) and II (

pma4

) represent the majorgenes expressed in this species.

pma2

and

pma4

were alsoshown to be expressed in several different cell types, suchas root epidermis and hairs, phloem companion cells, guardcells, and most parts of flower organs. Moreover, these twogenes appear to be coexpressed in certain cell types at thesame developmental stage (Moriau et al., 1999), thus mak-ing their individual characterization difficult. Because theproperties of their encoded products (expressed in yeast)are different (Luo et al., 1999), it is of the utmost interest todevelop tools to study the roles of a single isoform in theplant.

In this study, we report an attempt to modify the in-the-plant expression of

pma4

by introducing

pma4

under thecontrol of its own promoter but activated by the cauliflowermosaic virus (CaMV) 35S enhancer into tobacco plants.Several PMA4-overexpressing plants and three PMA4-cosuppressing plants were identified. Because the lattergrew slowly and were male sterile, they were studied inmore detail. PMA4 cosuppression was developmentally reg-ulated in the leaves and was associated with several de-fects, such as impaired sucrose translocation from themature leaves, impaired stomatal opening, and male infertil-ity. We conclude that

pma4

is a major H

1

-ATPase gene in-volved in several aspects of plant physiology.

RESULTS

Identification of

pma4

-Overexpressing or

pma4

-Cosuppressing Transgenic Tobacco

To determine the specific physiologic roles of

pma4

, weoverexpressed it under the control of a strong transcriptionpromoter. Instead of CaMV 35S, a commonly used pro-moter, we chose to retain the

pma4

promoter but reinforce itby inserting the CaMV 35S enhancer sequence (Fang et al.,1989). Transient and stable transformation with the

gus

A re-porter gene, fused to the enhanced

pma4

promoter, showedthe latter to have a much greater activity than the native

pma

promoter and to retain a broad range of tissue specificity(Zhao et al., 1999).

We designed three different constructs (Figure 1).P4pma4 was the

pma4

cDNA linked to a 2.32-kb genomicfragment containing the

pma4

upstream region, which hasbeen shown to confer

gus

A expression on many cell types

(Moriau et al., 1999). The 500pma4 construct was similar toP4pma4, except that the CaMV 35S enhancer was inserted500 nucleotides upstream of the

pma4

transcription initia-tion site. Because overexpression of the transcript might in-duce downregulation of the enzyme, we also designed500pma4

D

, a construct identical to 500pma4 except thatthe last 103 C-terminal codons were deleted. This region isthought to be an autoinhibitory regulatory domain (Palmgrenet al., 1991) such that its deletion in PMA4 expressed inyeast constitutively activates the enzyme (Luo et al., 1999).

Transgenic tobacco leaves were screened by using pro-tein gel blot analysis with antibodies raised against peptidesspecific to either subfamily I (PMA1, PMA2, and PMA3) orsubfamily II (PMA4) (Moriau et al., 1999). The P4pma4 trans-formants did not show important PMA4 overexpression (Fig-ure 2A) and were not studied further. In contrast, of the 27500pma4 and 20 500pma4

D

primary transgenic plants ana-lyzed, 15 and 10 plants, respectively, showed at least two-fold overexpression of PMA4 (examples shown in Figures2B and 2C). In 500pma4

D

plants, the size of the new prod-uct (

z

95 kD) was as expected for the C-terminal–deletedPMA4. Finally, we detected three transgenic plants, line 30from 500pma4 and lines 44 and 45 from 500pma4

D

, inwhich expression of both endogenous and transgenic

pma

4was considerably reduced (Figures 2B and 2C), suggestinga cosuppression phenomenon.

pma4

Cosuppression Results in Stunted and Delayed Growth and Male Sterility

Overexpressing 500pma4 and 500pma4

D

plants did not dis-play any major growth modification under normal green-house conditions (data not shown), whereas growth of thecosuppressing plants was markedly reduced (Figures 3A to3C). Flowering of all three cosuppressing plants was de-layed. The anther filaments were shorter, and after antherdehiscence, all of the pollen grains were found to be shriv-

Figure 1. Constructs for PMA4 Overexpression.

The three constructs bring together the transcription promoter re-gion of pma4, the pma cDNA, and the nopaline synthase polyadeny-lation site and terminator (Tnos). In 500pma4 and 500pma4D, theCaMV 35S enhancer (EN) was inserted 500 nucleotides upstream ofthe pma4 transcription start site. In 500pma4D, the last 103 C-termi-nal codons of the pma4 cDNA were truncated.

Cosuppression of Plasma Membrane H

+

-ATPase 537

eled (Figure 3D). The flowers were male sterile but could bepollinated with wild-type pollen.

Line 44 (500pma4

D

) showed a variable cosuppressionphenotype in the F

1

generation and was not studied further.Lines 30 (500pma4) and 45 (500pma4

D

) produced plantswith the same stunted phenotype up to at least the thirdgeneration. DNA gel blot analysis indicated that line 30 con-tained three independent transgenic inserts (loci

a

,

b,

and

c

;data not shown). For the F

1

generation, analysis of 16 co-suppressing offspring showed that they carried, at mini-mum, loci

b

and

c

. Other combinations of loci resulted inPMA4 overexpression instead of cosuppression (data notshown). In line 45, a single insert was identified by DNA gel

blot analysis, and cosegregation of kanamycin resistanceand cosuppression were inherited as a single locus in the F

1

and F

2

generations.The retarded development of the mutants was easily dis-

tinguishable from the normal development of the wild type(Figures 3A and 3B). Although leaf emergence was delayedin plants from lines 30 and 45, the total number of leavesproduced up to the flowering stage was higher per plant inthese lines (Figure 4A), another reflection of flower inductionbeing delayed. Because of the shorter internodes, stembolting was also reduced (Figure 4B). Although the youngexpanding leaves displayed no significant morphologicaldifferences compared with the wild type, the fully expandedleaves of the mutants were smaller during the first growthstage (Figure 4C). In addition, the first mature leaves soonstopped growing at the tip and turned yellow. As a result,

Figure 2. Immunodetection of H+-ATPase in Leaf Microsomes fromPrimary Transgenic Plants.

(A) P4pma4 transgenic plants.(B) 500pma4 transgenic plants.(C) 500pma4D transgenic plants.Microsomal proteins (5 mg) from mature leaves were electropho-resed and subjected to immunodetection (protein gel blotting) withantibodies raised against the first (PMA1, PMA2, and PMA3 [PMA1-3])or second (PMA4) H+-ATPase subfamilies. The identification numberof each transgenic plant is indicated at the top. SR1 is an untrans-formed wild-type plant. The apparent size of the proteins immuno-detected is indicated at right.

Figure 3. Phenotype of the pma4-Cosuppressing Plants.

(A) Eight-week-old plant of cosuppressing line 30 of the F2 genera-tion (left) and a wild-type SR1 (right) plant.(B) Eight-week-old plant of cosuppressing line 45 of the F2 genera-tion (left) and a wild-type SR1 (right) plant.(C) Seventh leaf from a plant of cosuppressing line 45 of the F2 gen-eration (left) and a wild-type SR1 (right) plant.(D) Flowers from a wild-type SR1 plant (left) and a plant of cosup-pressing line 45 (right).

538 The Plant Cell

the leaves were pear shaped (Figure 3C). In some seriouslystunted plants, the first four or five leaves rapidly becamenecrotic. As occurs with the primary transgenic plants, all ofthe F

1

and F

2

pma4

-cosuppressing plants were male sterile,with shorter filaments (Figure 3D) and shriveled pollen.

pma4

Cosuppression Is Developmentally Regulated

Protein gel blot analysis of microsomal fractions showedthat

pma4

cosuppression was severe in the roots, stem, andanthers. In the petals, the shorter transgenic PMA4 wasbarely detected, whereas the full-length endogenous PMA4was expressed at a lower level (Figure 5). In stunted plants,PMA4 expression was still seen in the young leaves but wasdrastically reduced in the fully expanded leaves, whereaswild-type leaves at this position expressed more PMA4 (Fig-ure 6A). To determine whether cosuppression was intrinsicto leaf position or the developmental stage of the leaf, weconducted immunodetection experiments with a leaf at thesame position for plants of differing ages. PMA4 expressionwas gradually cosuppressed with leaf maturation (Figure 6B).Finally, analysis of various regions of a single developing leafshowed more severe PMA4 cosuppression at the leaf tip,followed by the edge, thus following the maturation gradientwithin the leaf (Figure 6C). These data indicate that PMA4cosuppression is developmentally regulated. In contrast,PMA1 to PMA3 expression was not substantially altered.

pma4

Cosuppression Reduces ATPase Activity

Protein gel blot analysis showed that PMA4 expression incosuppressing plants was drastically reduced. However, theplants expressed H

1

-ATPase isoforms other than PMA4, in-cluding PMA1, PMA2, and PMA3, which were shown to bepresent in normal amounts. To determine the effect of

pma4

cosuppression on the whole H

1

-ATPase family, we mea-sured H

1

-ATPase activity in a purified plasma membranefraction. Specific ATPase activity in leaves of PMA4-cosup-pressing plants (0.24

6

0.07 and 0.33

6

0.11 mmol Pi min21

mg protein21 for F2 plants of lines 30 and 45, respectively)was reduced to 40 to 50% of that in wild-type leaves (0.66 60.17 mmol Pi min21 mg protein21).

pma4 Cosuppression Modifies Leaf Sugar Content

We then determined what the effects of altered pma4 ex-pression might be, first focusing on sugar translocation be-

Figure 4. Growth Comparison between Wild-Type and pma4-Cosuppressing Plants.(A) Number of leaves (minimal length of 5 mm).(B) Stem length.(C) Length of fully expanded leaves.Seeds from wild-type and pma4-cosuppressing lines 30 and 45 ofthe F2 generation were sown in 96-well culture plates in sterile soil.After 30 days, the plants were transferred to pots and grown at 268C(daytime at 50 mmol m22 sec21 at the stem base level for 16 hr) and208C (night). Leaf (minimum length of 5 mm) number, stem height,and fully expanded leaf length were scored until flowering for 12

plants each of the wild-type SR1 (open squares) and cosuppressinglines 30 F2 (open circles) and 45 F2 (open triangles). The error barsrepresent the 95% confidence interval.

Cosuppression of Plasma Membrane H+-ATPase 539

tween the source and sink tissues. Apoplastic phloemloading has been supported in several species by the identi-fication of H1/sucrose symporters and by the effects of anti-sense RNA expression (Riesmeier et al., 1993; Gahrtz et al.,1994; Sauer and Stolz, 1994; Kühn et al., 1996; Bürkle et al.,1998). pma4 is expressed in various cell types but predomi-nantly in phloem tissues, suggesting that PMA4 activatessugar loading (Moriau et al., 1999). The total soluble sugarcontent was threefold greater in mature leaves of the twocosuppressing plants than in wild-type leaves (Figure 7A).Analysis of individual sugars confirmed these data andshowed that the higher content of soluble sugars in cosup-pressing leaves resulted from accumulation of glucose andfructose, whereas sucrose content was not particularly al-tered (Figure 7A). In contrast, the starch content of cosup-pressing leaves was much less than that of the wild type.This was independent of the light conditions because thecontent of soluble sugars and starch measured in line 45plants grown under different light conditions (46 to 185 mmolm22 sec21; Table 1) was in each case higher (soluble sug-ars) or lower (starch) in the cosuppressing plants than in thewild type.

Plant invertase has been shown to be involved in regulat-ing sucrose metabolism (Huber, 1989). Expression of ayeast invertase in tobacco and Arabidopsis produces an ac-cumulation of carbohydrates in leaves and results in growthretardation (von Schaewen et al., 1990; Sonnewald et al.,1991). The increased amounts of hexoses seen in the co-suppressing mature leaves could therefore result from in-creased invertase activity. When soluble and wall-boundinvertases were measured, they were in all cases found tobe substantially increased in the mutants (Figure 7C).

We then focused on the sucrose content of the aerial sinktissues (Figure 7B). The apical tissue of cosuppressing linesshowed 29 to 71% less sugar content for sucrose, glucose,and fructose alike. These data suggested that sugar translo-

cation between the source and sink tissues was not operat-ing properly. When we tested this more directly bymeasuring the sucrose content of the exudate from de-tached mature leaves, threefold less sucrose was exudatedfrom the cosuppressing plants when compared with the wildtype (Figure 8). Moreover, although the cosuppressing

Figure 5. Immunodetection of H+-ATPase in Roots, Stem, Anthers,and Petals.

Microsomal proteins (5 mg) from the roots, stem, anthers, and petalsof wild-type (W) and cosuppressing lines 30 and 45 F2 (30 and 45)plants were analyzed by protein gel blotting with antibodies raisedagainst PMA4 or PMA1, PMA2, and PMA3 (PMA1-3). The apparentsize of the proteins immunodetected is indicated at right.

Figure 6. Immunodetection of H+-ATPase in Leaves at Different De-velopmental Stages.

Microsomal proteins (5 mg) from wild-type SR1 and F2 plants of co-suppressing lines 30 and 45 were analyzed by protein gel blottingwith antibodies raised against PMA4 or PMA2.(A) Microsomes were prepared from leaves at different positions on2-month-old plants. Lane 1 represents the top unexpanded leaf(z0.5 cm long); lanes 2 to 4 represent the second, third, and fourthleaves from the top.(B) Microsomes were prepared from the tenth leaf of plants at differ-ent developmental stages. Lane 1 represents the early developmen-tal stage (z2.5 cm long); lanes 2 and 3 represent the developmentstages at 3 and 6 days later.(C) Microsomes were prepared from different regions within the 10thdeveloping leaf (SR1, z12 cm total length; lines 30 and 45, z8 cmtotal length). Lanes 1 to 4 indicate the base (1) and center (2) of theleaf, the edge in the middle of the leaf (3), and the tip of the leaf (4).

540 The Plant Cell

leaves accumulated glucose and fructose, only slight tracesof glucose and fructose were found in the exudate (data notshown). Taken together, these data indicate that pma4 co-suppression in mature leaves resulted in reduced sucrosetranslocation from the source to the sink organs.

pma4 Cosuppression Alters Stomata Functioning

Because pma4 is also expressed in guard cells, we next an-alyzed these cells. In young leaves, in which pma4 was stillbeing expressed, there was no real difference between wild-type and cosuppressing plants in terms of the shape or sizeof the epidermal cells, including the guard cells (Figures 9Aand 9B). In the fully expanded leaves, however, the cells ofthe cosuppressing plants were larger than those of the wildtype. The guard cells appeared shriveled and many stomataseemed almost closed, whereas those of the wild type wereopen (Figures 9C and 9D). When we applied fusicoccin, afungal phytotoxin known to stimulate H1-ATPase and widenthe stomata (Marrè, 1979; De Boer, 1997), the aperture ofmost of the wild-type stomata increased, whereas almost50% of the cosuppressing plant stomata failed to open (Fig-ures 9E, 9F, and 9I).

Fluorescein diacetate (Widholm, 1972) was applied to testthe plasma membrane potential of the guard cells. This non-fluorescent ester readily crosses the cell membrane, whereit is cleaved by intracellular esterases; the released polar flu-orescein moiety then accumulates within the living cells(Rotman and Papermaster, 1966). In mature leaves, mostwild-type guard cells but only a few of those in cosuppress-ing plants were strongly fluorescent (Figures 9G and 9H).This suggests that most of the mutant guard cells had a re-duced membrane potential. In developing leaves, abnormalguard cells first occurred in the tip region and increased innumber after leaf maturation (data not shown). This indi-cates that irregular guard cells occurred only at a later de-velopmental stage and that this might be closely associatedwith pma4 cosuppression. Because the size of the stomataaperture controls gas exchange, we also measured CO2

consumption and calculated the net photosynthesis rate inmature leaves under normal growth conditions and foundthis to be reduced by 25 to 39% in the cosuppressing plants(Table 2).

Finally, because pma4 cosuppression might also affectmineral nutrition and therefore nitrogen supply, we mea-sured the total nitrogen content of mature leaves. No signifi-cant difference was found between the two mutants andwild-type plants (Table 3).

Figure 7. Leaf Carbohydrate Content and Invertase Activity.

(A) and (B) Carbohydrates in fully expanded leaves and in apicalbuds, including young leaves (,4 cm long), respectively.(C) Invertase activity in fully expanded leaves.The sugars were extracted from 7-week-old wild-type SR1 plants(open bars) and F2 plants of lines 30 (hatched bars) and 45 (filledbars) under a light intensity of z50 (stem base) to 200 (flower) mmolm22 sec21 at 268C for 16 hr during the day and 208C at night col-lected after 8 hr of illumination. Total soluble sugars (Soluble) andstarch were determined by using the anthrone reagent. Sucrose(Suc), glucose (Glc), and fructose (Fru) were determined by quantita-tive HPLC. Alkaline, acid, and wall-bound invertase activities weredetermined in extracts from the fully expanded leaves. The codedbars and error bars represent the mean and 95% confidence inter-

val, respectively, from four independent assays of wild-type and F2

cosuppressing plants. FW, fresh weight.

Cosuppression of Plasma Membrane H+-ATPase 541

DISCUSSION

Overexpression of pma4 was achieved using the two con-structs 500pma4 and 500pma4D. Approximately half of theprimary transgenic plants assayed showed a marked over-expression of intact or (to a lesser extent) C terminus–trun-cated PMA4. Although the PMA4-overexpressing plantsshowed no major changes under normal greenhouse condi-tions, a more detailed analysis should be performed, for in-stance, by challenging growth under stress conditions;indeed, expression of C-terminal–modified ArabidopsisAHA3 H1-ATPase resulted in an increased resistance ofseedlings grown in vitro under acid conditions, but no modi-fied phenotype was observed under normal conditions(Young et al., 1998).

Three cases of pma4 cosuppression appeared at the het-erozygous stage; in two, this was stably inherited up to atleast the third generation. pma4 cosuppression was devel-opmentally regulated in the leaves. Cosuppression was par-tial in developing leaves but almost total in mature leaves.This was observed when comparing different leaves from asingle plant at the same time, different stages of develop-ment of the same leaf, or even different parts of a single de-veloping leaf. Other cases of developmentally regulatedcosuppression have been reported in transgenic tobacco,for example, with the genes encoding nitrate reductase(Dorlhac de Borne, 1994), chitinase (Hart et al., 1992), or glu-canase (de Carvalho et al., 1995). We hypothesize thattransgene expression increases with leaf maturation and, ata certain stage, reaches a threshold necessary to induce co-suppression. In fact, a gusA reporter gene study has shownthat GUS activity, whether conferred by the native or the en-hanced pma4 promoter in transgenic plants, increases afterleaf maturation (data not shown). Our hypothesis was con-firmed by the observation that the PMA4/PMA1 to PMA3 ra-tio, determined by protein gel blot analysis (Figure 6),increased during leaf development. This modification of

pma4 expression might correspond to the sink–source tran-sition that probably correlates with the activation of the su-crose loading process.

Among the nine N. plumbaginifolia pma genes, pma4 isthe most highly expressed (Moriau et al., 1999; Oufattole etal., 2000). Expression was found in many cell types, includ-ing root epidermal cells, companion cells, and guard cells.However, the expression pattern of pma2 overlaps to a largeextent with that of pma4. The consequences of pma4 co-suppression might therefore be expected to range from noeffect whatsoever, if pma2 alone is able to sustain the trans-port function, to a major effect if pma4 plays a preponderantrole. The latter case was experimentally proven becausepma4 cosuppression resulted in alteration of several physio-logic traits, such as stomata opening, photosynthesis, sugartranslocation, stem bolting, and male fertility. This list isprobably not exhaustive. Therefore, we must analyze thesepleiotropic physiologic alterations cautiously when trying torelate a specific functional defect to PMA4 cosuppression ina particular type of cell. Long-distance and multifactorial ef-fects are possible, and regulatory systems presumably notadapted to the PMA4 absence might respond in unexpectedways. With these reservations in mind, we can now attemptto interpret some of the phenotypic modifications at the mo-lecular level.

Stomatal opening is induced by increased turgor pressureas a result of K1 and anion influx energized by H1-ATPase.In pma4-cosuppressing leaves but not in young developingleaves, most of the stomata were closed and could not be

Table 1. Total Leaf Sugar Content of Plants Grown under DIfferent Growth Conditions

Soluble Sugarb Starchb

Light Intensitya Wild Type 45 F2 Wild Type 45 F2

46 5.5 12.3 24.9 16.767 4.7 7.0 39.3 24.896 5.3 6.4 55.9 25.3185 5.7 15.0 61.4 52.1

a The indicated light intensity (in micromoles per square meter persecond) was measured at the level of the leaves used for the deter-mination.b Sugars (in milligrams per gram fresh weight of tissue) were deter-mined as given for Figure 7, except that plants were grown underdifferent light intensity.

Figure 8. Sucrose Exudation from Detached Mature Leaves.

Exudate was collected from fully expanded leaves of wild-type SR1and F2 plants from cosuppressing lines 30 and 45; the sucrose con-centrations were quantified and expressed as micromoles of su-crose per gram fresh weight (FW) of leaves. The coded bars anderror bars represent the mean and 95% confidence interval, respec-tively, of six independent assays.

542 The Plant Cell

opened by treatment with fusicoccin; however, some sto-mata still responded to fusicoccin treatment, and some evenhad a larger stomatal aperture than that seen in the wildtype. Because cosuppression is developmentally regulated,perhaps it did not occur evenly in every cell, so pma4 wasstill normally expressed in those few guard cells that stillfunctioned well. Although guard cells have active chloro-plasts, they also rely on the import of carbohydrates (Lu etal., 1997). Defective carbohydrate transfer to and uptake byguard cells might reduce ATP supply and therefore contrib-ute to the failure of stomatal opening. A major consequenceof the ineffective stomata opening seems to be a decreasedCO2 uptake, which one would expect to affect the carbohy-drate metabolism.

pma4 cosuppression caused reduced sucrose transloca-tion from source leaves and a lower sugar content in sinktissues. According to the apoplastic phloem loading hypoth-esis, photoassimilates synthesized in the source mesophyllare released into the apoplasm, taken up into the phloemcompanion cells or sieve elements by way of H1/sucrosecotransporters, and then translocated to sink organs(reviewed in Ward et al., 1998). The plasma membrane H1-ATPase creates a proton gradient across the plasma mem-brane to energize this loading process. H1-ATPases andH1/sucrose transporters are therefore the two key partnersfor phloem loading. Disruption of the latter reportedly leadsto failure of sucrose loading in Solanaceae, for instance, inthe case of antisense inhibition of H1/sucrose symportergene expression (Riesmeier et al., 1994; Kühn et al., 1996;Bürkle et al., 1998). Moreover, hydrolysis of sucrose in theapoplasm by ectopic expression of invertase (vonSchaewen et al., 1990; Sonnewald et al., 1991) or a reducedenergy supply in the companion cells as a result of ec-topic expression of pyrophosphatase (Lerchl et al., 1995;Geigenberger et al., 1996) results in reduced sucrose trans-location and produces a crinkled phenotype.

This study demonstrates that disruption of the other part-ner by pma4 cosuppression had a similar effect. AlthoughgusA expression driven by the pma4 promoter has been de-tected in companion cells (Moriau et al., 1999), characteriza-tion of the potato sucrose transporter, SUT1, supports the

Table 2. Net Photosynthesis Rate

Plant Photosynthesis Ratea

Wild type 16.6 6 1.230 F2 10.2 6 1.645 F2 12.0 6 1.2

a The net photosynthesis rate was expressed as millimoles of CO2

per square meter per hour. The values are the means 6 95% confi-dence interval from at least 25 measurements for the F2 generationof lines 30, 45, and the wild type grown in a conditioned room (seeMethods) after 4 hr of illumination.

Figure 9. Stomatal Opening in Young and Fully Expanded Leaves.

The epidermis of young and fully expanded leaves from 2-month-oldplants was peeled off and incubated in the presence or absence of10 mM fusicoccin (FC), as indicated in Methods.(A) Wild-type SR1 young leaf.(B) Young leaf from an F2 line 30 plant.(C) Wild-type SR1 fully expanded leaf not exposed to fusicoccin.(D) Fully expanded leaf from an F2 line 30 plant not exposed to fusi-coccin.(E) and (G) Wild-type SR1 fully expanded leaf treated with fusicoccinand stained with fluorescein diacetate.(F) and (H) Fully expanded leaf from an F2 line 30 plant treated withfusicoccin and stained with fluorescein diacetate. (E) and (F) were photographed in white light; (G) and (H) were pho-tographed under UV light to reveal fluorescence. Bar below (H) 5

100 mm.(I) The distribution of stomata as a function of their aperture isshown for the samples corresponding to (C) to (H). The data repre-sent the mean stoma aperture for at least 100 stomata on each ofthree leaves.

Cosuppression of Plasma Membrane H+-ATPase 543

hypothesis that the mRNA synthesized in the companioncell is transported to the sieve element, where sucrose load-ing occurs (Kühn et al., 1997). Determining whether the pro-ton gradient failure occurs in the companion cells or thesieve elements therefore requires detailed in situ H1-ATPaseRNA and protein localization, together with pH imaging.

Although sucrose translocation was defective, the su-crose content in mature leaves remained at the same levelas in the wild type, but glucose and fructose content mark-edly increased. This most probably results from the inver-tase activity induced in the cosuppressing plants. Whetherthis induction is a direct result of the failure of sucrose load-ing or is another effect of cosuppression (e.g., reduced CO2

uptake) requires additional examination to determine, suchas measuring the sugar content in the apoplasm and withinthe cells.

Transgenic plants in which sucrose loading is impaired bydownregulation of the sucrose/H1 transporter (Riesmeier etal., 1994; Kühn et al., 1996; Bürkle et al., 1998) or by ectopicexpression of yeast-derived invertase (Sonnewald et al.,1991) accumulate more sucrose and have a greater starchcontent in the mature leaves. This contrasts with the resultsobserved with the pma4-cosuppressing plants. Indeed, de-spite the reduced translocation and the increased glucoseand fructose content, if we take the starch into account, themature leaves accumulated less total carbohydrate than didthose of the wild-type plant (Figure 7A and Table 1). Abnor-mal stomatal functioning and the resulting limitation of gasexchange and photosynthesis is the most plausible expla-nation for this phenotype. However, given the wide expres-sion pattern of pma4, we cannot exclude that the importantmodification in carbohydrate metabolism and transport is asecondary effect of pma4 cosuppression in a cell type notdiscussed so far or results from a disturbed regulatorymechanism.

Floral initiation was delayed in the cosuppressing plants.This might be related to the reduced sugar input to the api-cal meristem, because sucrose has been suggested to playan essential role in floral initiation (reviewed in Bernier et al.,1993). Delayed flowering has also been observed in cases inwhich sucrose translocation was reduced (Sonnewald et al.,

1991; Kühn et al., 1996; Bürkle et al., 1998). In addition, thepma4-cosuppressing plants were completely male sterile:the anther filaments were shorter, and the pollen grains wereless abundant and shriveled. This phenotype was not ob-served in the previously described transgenic plants andthus seems to result directly from pma4 cosuppression inthe anthers. pma4 is very active in various anther tissues,notably in the tapetum and the microspores (Moriau et al.,1999); therefore, it is reasonable that pma4 cosuppression inthese rapidly developing sink tissues resulted in male sterility.

In conclusion, pma4 cosuppression has shed some lighton the important role of this isoform in plant growth andfunctioning. Because pma4 is highly expressed in almost allplant organs, its cosuppression is expected to have pleio-tropic effects. We have identified failures in the sugar trans-port, stomatal opening, and the development of maletissues. Additional effects might still be expected. Cosup-pression, for instance, was also observed in root tissues, butit does not seem to affect greatly the mineral nutrition of theplant grown in soil because ions such as potassium andmagnesium were not modified in the leaves of the cosup-pressing plants (data not shown). The nitrogen supply alsowas not altered—the nitrogen content of leaves was notsubstantially changed. The situation might be different ifthese plants were grown under conditions of mineral deficitor salt stress. Further investigations would greatly benefitfrom tools that make it possible to address the role of PMA4or other H1-ATPase isoforms in specific organ or cell types,by using, for example, grafting experiments or cell type–specific promoters to drive antisense constructs or cosup-pression.

METHODS

Constructs and Plant Transformation

The XhoI-HindIII fragment containing the plant plasma membraneH1-ATPase pma4 cDNA (cpma4) sequence (Luo et al., 1999) wascloned into pBluescript SK+ (Stratagene, La Jolla, CA). The pma4 39

AflII-HindIII fragment was replaced by the synthetic fragment 59-ATCCTTAAGTGAGCAAAAGCTGATCAGTGAGGAAGACTTGTAAT-GAAGCTTCG, which encodes a c-myc 10–amino acid epitope(Kolodziej and Young, 1991), resulting in the deletion of the last 103C-terminal PMA4 residues. c-myc has been shown to be a conve-nient marker for plant H1-ATPase (DeWitt and Sussman, 1995) butwas not used in the experiments reported in this study. The BamHI-blunted XhoI fragment, containing the pma4 promoter reinforcedwith the cauliflower mosaic virus (CaMV) 35S enhancer introduced atposition 2500 upstream of the transcription start site (Zhao et al.,1999), was inserted into pBI101 (Jefferson et al., 1987) betweenBamHI and SmaI. After ligation, the XhoI site was recovered. Intactcpma4 and 39-deleted cpma4 were digested by HindIII (filled in byusing the Klenow fragment of DNA polymerase) and XhoI. The XhoI-blunted HindIII fragments were then cloned into XhoI and bluntedSacI sites that are downstream of the activated pma4 promoter inpBI101, resulting in 500pma4 and 500pma4D, respectively. The

Table 3. Total Nitrogen Content

Plant Nitrogena

Wild type 1.36 6 0.2830 F2 1.38 6 0.2545 F2 1.69 6 0.51

a Total nitrogen is expressed as percentage of fresh weight of tis-sues. The values are the means 6 95% confidence interval frommeasurements performed with mature leaves from four (30 F2 and45 F2) or eight (wild-type) plants.

544 The Plant Cell

activated pma4 promoter in 500pma4 was replaced by the nativepma4 from pPRP4XB (Zhao et al., 1999) by using BamHI and XhoI;this generated P4pma4.

Nicotiana tabacum cv SR1 was transformed as described by An et al.(1988). The regenerated kanamycin-resistant plants were transferred tothe greenhouse or conditioned room (258C during the day for 16 hr of il-lumination with 45 to 185 mmol m22 sec21 and 208C at night).

Microsomal and Plasma Membrane Fraction Preparation

Fresh leaf tissues (2 g) were homogenized at 28C in a Waring blenderwith 10 mL of homogenization buffer (50 mM Tris-HCl, pH 8.0, 250mM sorbitol, 2 mM EDTA, 0.7% polyvinylpyrrolidone [Polyclar T;Serva, Boehringer Ingelheim Bioproducts, Heldelberg, Germany], 5mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg mL21 leupep-tin, and 1 mg mL21 each pepstatin, chymostatin, antipain, and apro-tinin), and filtered through two layers of Miracloth (Calbiochem, LaJolla, CA). Aliquots (2 mL) of the homogenate were centrifuged twiceat 10,000g for 5 min, after which the supernatant was centrifuged at20,800g for 60 min. The pellet was suspended in 50 mL of 10 mMTris-Mes, pH 6.8, and 330 mM sucrose. For minipreparations of a mi-crosomal fraction, fresh leaf tissues (50 mg in 1.2 mL of homogeniza-tion buffer) were homogenized in a 1.5-mL Eppendorf tube by usinga pestle and centrifuged as described earlier. Plasma membraneswere prepared by phase partition (Larsson et al., 1987). Protein con-centrations were measured as described by Bradford (1976).

Immunodetection

Microsomal proteins (5 mg) were electrophoresed using SDS-PAGE,transferred to Immobilon-P (polyvinylidene difluoride membrane; Mil-lipore, Bedford, MA), incubated with rabbit antiserum raised againstPMA4 or PMA1 to PMA3 (Moriau et al., 1999), and revealed bychemiluminescence.

ATPase Assays

ATPase assays (Goffeau and Dufour, 1988) were performed at 308Cfor 10 min in 50 mL of medium containing 4 mM MgATP, 1 mM freeMg21 (MgCl2), 50 mM Mes-NaOH, pH 6.8, 10 mM NaN3, 20 mMKNO3, 0.2 mM sodium molybdate, and 5 mg of proteins.

Carbohydrate and Nitrogen Analyses

Fresh leaf tissues (1 g) were frozen in liquid nitrogen, ground to a finepowder, and then extracted three times with 8 mL of 80% ethanol.After centrifugation (9500g for 10 min), the supernatants were pooledand filtered through a 0.45-mm pore-size filter to obtain the total sol-uble sugar extract. Starch was then extracted from the pellet withperchloric acid, as described by McCready et al. (1950). Total solublesugars and starch were determined by using the anthrone reagent,as described by Yemm and Willis (1954) and McCready et al. (1950),respectively.

To measure sucrose, glucose, and fructose in the soluble sugarextract, we dried and then dissolved the samples in demineralizedH2O at one-thirtieth of the initial volume. Sucrose, glucose, and fruc-tose were measured using HPLC with a Bio-Rad Aminex HPX-87C

column (300 3 7.8 mm [i.d.]), as described by Houssa et al. (1991).Nitrogen content was determined by the Kjeldahl method.

Sugar Analysis of Leaf Exudate

Phloem exudate was collected using the EDTA method (King andZeevaart, 1974). Fully expanded leaves (after 4 hr of illumination)were cut at the base of the petioles. The tip of the petiole was againcut under water and then immediately immersed in 20 mM EDTA, pH7.0, after which the leaves were placed in the dark for 8 hr in a sealedbag to prevent transpiration. The sugars in the EDTA solution weremeasured using HPLC, as previously described, and were expressedas micromoles of sucrose per gram (fresh weight) of leaves.

Invertase Assays

The extraction of invertase from leaves was performed as describedby Tang et al. (1996). Briefly, a leaf disc (50 mg) was homogenized in1 mL of homogenization buffer (50 mM Hepes-KOH, pH 7.5, 1 mMEDTA, 1 mM EGTA, 2 mM benzamidine, 5 mM DTT, and 0.1 mM phe-nylmethylsufonyl fluoride) in an Eppendorf tube by using a pestle.The homogenate was centrifuged for 30 min at 20,800g, and the su-pernatant was collected for assay of alkaline invertase and solubleacid invertase. To assay cell wall–bound invertase, the pellet waswashed four times with demineralized H2O and then incubated over-night in 1 mL of homogenization buffer containing 1 M NaCl. The re-leased invertase was recovered by centrifugation.

Soluble and cell wall–bound acid invertase activity was assayed at378C in 250 mM sodium acetate, pH 5.5, and 100 mM sucrose. Alka-line invertase was assayed at 378C in 27 mM Hepes-KOH, pH 7.5,and 100 mM sucrose. 3,5-Dinitrosalicylic acid reagent was added toreaction samples at 0, 30, 60, and 90 min to terminate the reaction;the glucose concentration was then determined as described byArnold (1965).

Stomatal Opening Assay and Measurement of Photosynthesis

The lower epidermis of developing or fully expanded leaves from2-month-old plants was peeled off, washed by sonication (Poffenrothet al., 1992), and incubated for 5 hr in the dark in a solution contain-ing 10 mM Mes-KOH, pH 6.1, 30 mM KCl, and 0.1 mM CaCl2. Fusicoc-cin (final concentration of 10 mM) was then added, and incubationwas continued for an additional 5 hr. Stomatal apertures were mea-sured with an optical microscope. To test the viability of the guardcells, we added 0.05% fluorescein diacetate (Sigma) and observedguard cells with a fluorescence microscope. The net photosyntheticrate of the fully expanded leaves was measured by infrared gas anal-ysis with a portable analyzer (model LCA2; Analytical Development,Hoddeston, UK) under growth conditions.

ACKNOWLEDGMENTS

This work was supported by grants from the Belgian National Fundfor Scientific Research, the European Commission (BIOTECH pro-gram), and the Interuniversity Poles of Attraction program of theBelgian Government Office for Scientific, Technical, and Cultural Af-

Cosuppression of Plasma Membrane H+-ATPase 545

fairs. We thank Pierre Gosselin and Anne-Marie Faber for theirexcellent technical help.

Received December 27, 1999; accepted February 18, 2000.

REFERENCES

An, G., Ebert, P.R., Mitra, A., and Ha, S.B. (1988). Binary vectors.In Plant Molecular Biology Manual, S.B. Gelvin, R.A. Schilperoort,and D.P.S. Verma, eds (Dordrecht, The Netherlands: Kluwer Aca-demic Publishers), pp. A3/1–A3/19.

Arnold, W.N. (1965). b-Fructofuranosidase from grape berries. Bio-chim. Biophys. Acta 110, 134–147.

Bernier, G., Havelange, A., Houssa, C., Petitjean, A., and Lejeune,P. (1993). Physiological signals that induce flowering. Plant Cell 5,1147–1155.

Bradford, M.M. (1976). A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle ofprotein-dye binding. Anal. Biochem. 72, 248–254.

Bürkle, L., Hibberd, J.M., Quick, W.P., Kühn, C., Hirner, B., andFrommer, W.B. (1998). The H1-sucrose cotransporter NtSUT1 isessential for sugar export from tobacco leaves. Plant Physiol. 118,59–68.

De Boer, B. (1997). Fusicoccin—A key to multiple 14-3-3 locks?Trends Plant Sci. 2, 60–66.

de Carvalho Niebel, F., Frendo, P., Van Montagu, M., andCornelissen, M. (1995). Post-transcriptional cosuppression ofb-1,3-glucanase genes does not affect accumulation of transgenenuclear mRNA. Plant Cell 7, 347–358.

DeWitt, N.D., and Sussman, M.R. (1995). Immunocytological local-ization of an epitope-tagged plasma membrane proton pump (H1-ATPase) in phloem companion cells. Plant Cell 7, 2053–2067.

DeWitt, N.D., Harper, J.F., and Sussman, M.R. (1991). Evidencefor a plasma membrane proton pump in phloem cells of higherplants. Plant J. 1, 121–128.

Dorlhac de Borne, F., Vincentz, M., Chupeau, Y., and Vaucheret,H. (1994). Co-suppression of nitrate reductase host genes andtransgenes in transgenic tobacco plants. Mol. Gen. Genet. 243,613–621.

Ewing, N.N., and Bennett, A.B. (1994). Assessment of the numberand expression of P-type H1-ATPase genes in tomato. PlantPhysiol. 106, 547–557.

Fang, R.X., Nagy, F., Sivasubramaniam, S., and Chua, N.-H.(1989). Multiple cis regulatory elements for maximal expression ofthe cauliflower mosaic virus 35S promoter in transgenic plants.Plant Cell 1, 141–150.

Gahrtz, M., Stolz, J., and Sauer, N.A. (1994). Phloem-specificsucrose-H1 symporter from Plantago major L. supports the modelof apoplastic phloem loading. Plant J. 6, 697–706.

Geigenberger, P., Lerchl, J., Stitt, M., and Sonnewald, U. (1996).Phloem-specific expression of pyrophosphatase inhibits long dis-tance transport of carbohydrates and amino acids in tobaccoplants. Plant Cell Environ. 19, 43–55.

Goffeau, A., and Dufour, J.P. (1988). Plasma membrane ATPase

from the yeast Saccharomyces cerevisiae. Methods Enzymol.157, 528–533.

Harper, J.F., Manney, L., and Sussman, M.R. (1994). The plasmamembrane H1-ATPase gene family in Arabidopsis: Genomicsequence of AHA10 which is expressed primarily in developingseeds. Mol. Gen. Genet. 244, 572–587.

Hart, C.M., Fischer, B., Neuhaus, J.M., and Meins, F., Jr. (1992).Regulated inactivation of homologous gene expression in trans-genic Nicotiana sylvestris plants containing a defense-relatedtobacco chitinase gene. Mol. Gen. Genet. 235, 179–188.

Houssa, P., Bernier, G., and Kinet, J.M. (1991). Qualitative andquantitative analysis of carbohydrates in leaf exudate of the short-day plant, Xanthium strumarium L. during floral transition. J. PlantPhysiol. 138, 24–28.

Huber, S.C. (1989). Biochemical mechanism for regulation ofsucrose accumulation in leaves during photosynthesis. PlantPhysiol. 91, 656–662.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUSfusions: b-Glucuronidase as a sensitive and versatile gene fusionmarker in higher plants. EMBO J. 6, 3901–3907.

King, R.W., and Zeevaart, J.A.D. (1974). Enhancement of phloemexudation from cut petioles by chelating agents. Plant Physiol. 53,96–103.

Kolodziej, P.A., and Young, R.A. (1991). Epitope tagging and pro-tein surveillance. Methods Enzymol. 194, 508–519.

Kühn, C., Quick, W.P., Schulz, A., Riesmeier, J.W., Sonnewald,U., and Frommer, W.B. (1996). Companion cell–specific inhibi-tion of the potato sucrose transporter SUT1. Plant Cell Environ.19, 1115–1123.

Kühn, C., Franceschi, V.R., Schulz, A., Lemoine, R., and Frommer,W.B. (1997). Macromolecular trafficking indicated by localizationand turnover of sucrose transporters in enucleate sieve elements.Science 275, 1298–1300.

Larsson, C., Widell, S. and Kjellborn, P. (1987). Preparation ofhigh-purity plasma membranes. Methods Enzymol. 148, 558–568.

Lerchl, J., Geigenberger, P., Stitt, M., and Sonnewald, U. (1995).Impaired photoassimilate partitioning caused by phloem-specificremoval of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants.Plant Cell 7, 259–270.

Lu, P., Outlaw, W.H., Jr., Smith, B.G., and Freed, G.A. (1997). Anew mechanism for the regulation of stomatal aperture size inintact leaves. Plant Physiol. 114, 109–118.

Luo, H., Morsomme, P., and Boutry, M. (1999). The two majortypes of plant plasma membrane H1-ATPases show differentenzymatic properties and confer differential pH sensitivity of yeastgrowth. Plant Physiol. 119, 627–634.

Marrè, E. (1979). Fusicoccin, a tool in plant physiology. Annu. Rev.Plant Physiol. 30, 273–288.

McCready, R.M., Guggolz, J., Silviera, V., and Owens, H.S.(1950). Determination of starch and amylose in vegetables: Appli-cation to peas. Anal. Chem. 22, 1156–1158.

Michelet, B., and Boutry, M. (1995). The plasma membrane H1-ATPase: A highly regulated enzyme with multiple physiologicalfunctions. Plant Physiol. 108, 1–6.

Michelet, B., Lukaszewicz, M., Dupriez, V., and Boutry, M. (1994).

546 The Plant Cell

A plant plasma membrane proton-ATPase gene is regulated bydevelopment and environment and shows signs of a translationalregulation. Plant Cell 6, 1375–1389.

Moriau, L., Michelet, B., Bogaerts, P., Lambert, L., Michel, A.,Oufattole, M., and Boutry, M. (1999). Expression analysis of twogene subfamilies encoding the plasma membrane H1-ATPase inNicotiana plumbaginifolia reveals the major transport functions ofthis enzyme. Plant J. 19, 31–41.

Oufattole, M., Arango, M., and Boutry, M. (2000). Identificationand expression analysis of three new Nicotiana plumbaginifoliagenes which encode isoforms of a plasma membrane H1-ATPaseand one of which is induced by mechanical stress. Planta 210,715–722.

Palmgren, M.G. (1998). Proton gradients and plant growth: Role ofthe plasma membrane H1-ATPase. Adv. Bot. Res. 28, 1–70.

Palmgren, M.G., and Christensen, G. (1994). Functional compari-sons between plant plasma membrane H1-ATPase isoformsexpressed in yeast. J. Biol. Chem. 269, 3027–3033.

Palmgren, M.G., Sommarin, M., Serrano, R., and Larsson, C.(1991). Identification of an autoinhibitory domain in the C-terminalregion of the plant plasma membrane H1-ATPase. J. Biol. Chem.266, 20470–20475.

Perez, C., Michelet, B., Ferrant, V., Bogaerts, P., and Boutry, M.(1992). Differential expression within a three-gene subfamilyencoding a plasma membrane H1-ATPase in Nicotiana plum-baginifolia. J. Biol. Chem. 267, 1204–1211.

Poffenroth, M., Green, D.B., and Tallman, G. (1992). Sugar con-centrations in guard cells of Vicia faba illuminated with red or bluelight. Plant Physiol. 98, 1460–1471.

Riesmeier, J.W., Hirner, B., and Frommer, W.B. (1993). Expres-sion of the sucrose transporter from potato correlates with thesink–source transition in leaves. Plant Cell 5, 1591–1598.

Riesmeier, J.W., Willmitzer, L., and Frommer, W.B. (1994). Evi-dence for an essential role of the sucrose transporter in phloemloading and assimilate partitioning. EMBO J. 13, 1–7.

Rotman, B., and Papermaster, J. (1966). Membrane properties ofliving mammalian cells as studied by enzymatic hydrolysis of fluo-rogenic esters. Proc. Natl. Acad. Sci. USA 55, 134–141.

Sauer, N., and Stolz, J. (1994). SUC1 and SUC2: Two sucrosetransporters from Arabidopsis thaliana: Expression and character-ization in baker’s yeast and identification of the histidine-taggedprotein. Plant J. 6, 67–77.

Serrano, R. (1989). Structure and function of plasma membraneATPase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 61–94.

Sonnewald, U., Brauer, M., von Schaewen, A., Stitt, M., andWillmitzer, L. (1991). Transgenic tobacco plants expressingyeast-derived invertase in either the cytosol, vacuole or apoplast:A powerful tool for studying sucrose metabolism and sink/sourceinteractions. Plant J. 1, 95–106.

Sussman, M.R. (1994). Molecular analysis of proteins in plantplasma membrane. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45,211–234.

Tang, X., Ruffner, H.P., Scholes, J.D., and Rolfe, S.A. (1996). Puri-fication and characterisation of soluble invertases from leaves ofArabidopsis thaliana. Planta 198, 17–23.

von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U., andWillmitzer, L. (1990). Expression of a yeast-derived invertase inthe cell wall of tobacco and Arabidopsis plants leads to accumu-lation of carbohydrate and inhibition of photosynthesis andstrongly influences growth and phenotype of transgenic tobaccoplants. EMBO J. 9, 3033–3044.

Ward, J.M., Kühn, C., Tegeder, M., and Frommer, W.B. (1998).Sucrose transport in higher plants. Int. Rev. Cytol. 178, 41–71.

Widholm, J.M. (1972). The use of fluorescein diacetate and pheno-safranine for determining the viability of cultured plant cells. StainTechnol. 47, 189–194.

Yemm, E.W., and Willis, A.J. (1954). The estimation of carbohy-drates in plant extracts by anthrone. Biochem. J. 57, 508–514.

Young, J.C., DeWitt, N.D., and Sussman, M.R.A. (1998). A trans-gene encoding a plasma membrane H+-ATPase that confers acidresistance in Arabidopsis thaliana seedlings. Genetics 149, 501–507.

Zhao, R., Moriau, L., and Boutry, M. (1999). Expression analysis ofthe plasma membrane H+-ATPase pma4 transcription promoterfrom Nicotiana plumbaginifolia activated by the CaMV 35S pro-moter enhancer. Plant Sci. 149, 157–165.

DOI 10.1105/tpc.12.4.535 2000;12;535-546Plant Cell

Rongmin Zhao, Vincent Dielen, Jean-Marie Kinet and Marc BoutryStomatal Opening, Plant Growth, and Male Fertility

-ATPase Isoform Impairs Sucrose Translocation,+Cosuppression of a Plasma Membrane H

 This information is current as of January 31, 2018

 

References /content/12/4/535.full.html#ref-list-1

This article cites 50 articles, 21 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists