Plant Physiol. (1992) 99, 1245-12500032-0889/92/99/1245/06/$01.00/0

Received for publication November 26, 1991Accepted February 13, 1992

Expression of the Genes for the a- and ,B-Subunits ofPyrophosphate-Dependent Phosphofructokinase in

Germinating and Developing Seeds from Ricinus communis

Stephen D. Blakeley, Linda Crews, James F. Todd, and David T. Dennis*Department of Biology, Queens University, Kingston, Ontario, Canada K7L 3N6

ABSTRACT

Various tissues from both germinating and developing castorseeds (Ricinus communis L.) have been analyzed for the level ofexpression of the genes for the a- and ,-subunits of pyrophosphate-dependent phosphofructokinase (PFP). In tissues in which PFP isexpressed, there is a single mRNA species of approximately 2kilobases for each of the subunits. In germinating endosperm, thegene for the a-subunit is expressed at an earlier time after imbi-bition than that for the j-subunit, whereas in developing castorseed endosperm, both genes are highly and coordinately expressed.During seedling development, there is tissue-specific expression ofthe two genes. Tissues in which there is a high level of mRNAcorrespond with tissues in which both subunits of PFP can bedetected. The differential expression of the two subunit genes ingerminating endosperm does not result in the presence of the a-

subunit polypeptide in the absence of the ,-subunit polypeptide.Southern analysis of castor genomic DNA indicates the presence

of a single gene for both the a- and a-subunits of PFP in contrastwith potato, in which there are at least two genes for each subunit.

Two enzymes in the cytosol of plant cells catalyze thephosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. The first of these, PFK (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), utilizes ATP,catalyzes an irreversible reaction, and is found in the majorityof organisms. The second, PFP2 (EC 2.7.1.90), catalyzes a

reversible reaction with PPi as the phosphoryl donor and isfound in a wide variety of tissues from a number of plantspecies (1, 6, 7, 9, 10, 13, 16, 18, 29, 30) and a limited numberof other organisms (20-22).The kinetic properties of PFP have been studied in some

detail (4, 7, 15, 23, 28, 29), although the physiological role ofthe enzyme is still unknown. Several possible functions havebeen suggested for PFP including the regulation of the levelsof cytosolic PPi (1, 2, 5, 25), equilibration of the hexose/triosephosphate pools (12), and, more recently, as an adenylatebypass for glycolysis during periods of phosphate limitationor starvation (13).PFP has been purified from a variety of plant species. In

l This work was supported by the Natural Sciences and Engineer-ing Research Council of Canada.

2Abbreviations: PFP, PPi:D-fructose-6-phosphate 1-phospho-transferase; Fru 2,6-P2, fructose 2,6-bisphosphate.

potato tubers, PFP is a heterotetramer composed of a- and,3-subunits with Mr values of 66,000 and 60,000, respectively(16). Although the two subunits are immunologically distinct,at the amino acid level they are approximately 60% homol-ogous (8), and evidence has been presented that indicatesthat the a- and d-subunits are the regulatory and catalyticsubunits, respectively (8, 10). PFP from cucumber cotyledonsis also composed of a- and 13-subunits, although differentisoforms can be detected during seedling establishment andthe ratio of the a- to 13-subunit varies between tissues (6). Inwheat, PFP is present as two isoforms, one a homodimercontaining only the 1-subunit, the other a heterotetramer ofboth a- and 13-subunits (30). The presence of the 12-homo-dimer in wheat may be a result of the comigration of the twosubunits of PFP, as is the case with PFP from mung bean(10).The physical properties of PFP, therefore, vary among both

plant species and tissue type, suggesting that there may bedifferential expression of the genes for the two subunits. Thepresent study was initiated to determine whether the genesfor the two subunits of PFP are coordinately or differentiallyexpressed in developing and germinating castor seeds andwhether the expression of the genes could be related to theobserved subunit composition in these tissues. Clear evidencefor differential expression of PFP a- and 1-subunit genes ispresented for germinating castor seeds, as is evidence fortissue-specific differences in the level of gene expression. Incontrast, the genes for both subunits are both highly andcoordinately expressed in developing castor seed endosperm.

MATERIALS AND METHODS

Castor plants (Ricinus communis L. cv Baker 296) weregrown in a glasshouse under daylight supplemented with 16h fluorescent light. Developing endosperm was harvested atvarious times postanthesis and developmental stages wereselected according to Greenwood and Bewley (14) (see figurelegends). For collection of germinating endosperm tissue,seeds were imbibed for 24 h in running tap water and thenplanted in vermiculite and grown as described above. Differ-ent tissues were collected at various time points, beginning 1d after the seeds were first imbibed (see figure legends).Rabbit anti-(potato PFP) polyclonal antibodies were gener-ously provided by W.C. Plaxton and had been prepared asdescribed (19). Near full-length cDNA clones for the a- and1-subunits of PFP from a developing castor seed endosperm

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Plant Physiol. Vol. 99, 1992

cDNA library had been isolated previously and were similarin sequence to those described for the potato PFP a- and 1#-subunits (8). All chemicals, radiochemicals, and Biotransmembrane were from ICN Biomedicals, Inc. (St. Laurent,Quebec) and immunochemicals were from Promega (Madi-son, WI).

Northern Analysis

Relative abundances of the mRNAs for the a- and f3-subunits of PFP were determined by northern hybridizationanalysis. Total RNA was isolated from both germinating anddeveloping castor seed endosperm (see figure legends) asdescribed (17). Total RNA (15 jug per lane) was denaturedand electrophoretically separated on 1.0% formaldehyde aga-rose gels. RNA integrity was routinely assessed by staining aseparate gel with ethidium bromide and subsequent exami-nation under UV irradiation. RNA was transferred to Biotransmembrane and subjected to northern hybridization in thepresence of 50% formamide according to the manufacturer'sinstructions. After hybridization, the blots were washed twotimes in 0.1 X SSC, 0.1% (w/v) SDS at 600C. These washesare more stringent than those used in a previous report (8),in which there was no detectable cross-hybridization betweenthe a- and fl-subunits of potato PFP, which show the samenucleotide homology (49%) as the castor subunits used inthis study.The blots presented in Figures 1 and 3 represent two gels

run at the same time, from the same sample of RNA, andsubsequently probed with cDNA clones for the a- or i#-subunit of castor PFP and are representative of several in-dependent experiments. Probes were generated by labelingcDNA fragments excised from the appropriate plasmids with[a-32P]dCTP to a specific activity of 2 to 4 x 108 cpm Mg'.Probes were added to hybridization solution at a concentra-tion of 106 cpm mL-1. Autoradiography was performed at-700C for 12 to 48 h using Dupont Cronex film. An LKBUltroscan XL Enhanced Laser Densitometer was used to scanthe northern blots. Densitometric data were integrated usingthe LKB Gelscan XL software (version 2.1) and the resultsare presented in terms of the relative abundance of mRNAfor the two subunits.

Extraction of PFP and Western Analysis

Proteins were extracted from the same samples of tissue aswere used for RNA extraction, and assayed for PFP activityas described (6). Electrophoresis and immunoblotting wereperformed as in ref. 19.

Southern Analysis

Genomic DNA from castor bean and potato leaf tissue wasisolated as described (11). DNA (10 Mg) was digested over-night with EcoRI or HindIII and electrophoretically separatedon 0.8% agarose gels in the presence of ethidium bromide.DNA was transferred to Biotrans membrane and subjectedto Southern hybridization (27) according to the manufactur-er's instructions. Probes were labeled and autoradiographyperformed (for 2-4 d) as described above. Castor leaf DNA

was hybridized with the near full-length cDNA clones forthe castor PFP a- and p-subunits, and potato leaf DNA washybridized with the potato cDNA clones isolated and de-scribed previously (8).

RESULTS AND DISCUSSION

There have been many studies of the kinetic and physicalproperties of PFP from a wide variety of plant species (4, 6,7, 10, 15, 16, 23, 24, 28, 29). The primary structure of PFP,however, as deduced from cDNA clones for the a- and 1B-subunits, has only recently been established (8), and thereare no detailed reports describing patterns or levels of expres-sion of the genes for the two subunits of this enzyme. In thisstudy, the expression of the genes for the a- and a-subunitsof PFP in both developing and germinating castor seeds wasexamined and the corresponding subunit composition of PFPin these tissues was determined.

Northern blots and densitometric analysis showing thetime course of accumulation of mRNA for both the a- andA-subunits of PFP in germinating castor seed endosperm, andin various tissues as the seedling develops, are presented inFigures 1 and 2A. The gene for the a-subunit is expressedvery early in the endosperm with detectable levels of mRNAfor this subunit present 1 d after imbibition. Expression ofthis gene rises rapidly for a further 12 h and then remains

pf: i

Figure 1. Northern blot analysis of castor seed total RNA at varioustimes after imbibition. The blots were probed with cDNA clonesfor the a- and ,8-subunits of PFP. Germinating endosperm washarvested up to 5 d. Subsequently, 7 and 11 d postimbibition,specific tissues were collected: r, root; c, cotyledon; h, hypocotyl;L, leaf. The autoradiographs were exposed for 48 h at -70'C.

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EXPRESSION OF THE GENES FOR PPi-PHOSPHOFRUCTOKINASE

NOOX

c)

a;

a;)

0 1 1.5 2 2.5 3 3.5 4 5 7C

Time after imbibition (days)IV V VI vii viII Ix

Developmental Stage

Figure 2. Accumulation of mRNAs for the a- and ,8-subunits of PFPin the endosperm of (A) germinating and (B) developing castorseeds. Time after imbibition and developmental stages are as de-scribed in the legends for Figures 1 and 3. Relative RNA levelsrepresent arbitrary optical transmittance values obtained by densi-tometric scanning of northern blot autoradiograms from a singlerepresentative experiment.

endosperm parallels that of the gene for the plastid isozymeof pyruvate kinase (S.D. Blakeley, L. Crews, D.T. Dennis,unpublished data) and also the measured activity of otherglycolytic enzymes in this tissue (25). Because the glycolyticpathway in developing oilseeds is believed to be supplyingcarbon for fatty acid biosynthesis, these data are consistentwith a glycolytic role for PFP in this tissue.The presence of a single mRNA of approximately 2 kilo-

bases for each of the subunits of PFP in both developing andgerminating castor seeds is consistent with a previous reportof PFP a- and 3-expression in various tissues in potato (8).Southern blot analysis of castor genomic DNA (Fig. 4A)reveals the presence of a single band in an EcoRI or HindIIIdigest for the a-subunit and a single band in a HindIII digestfor the A-subunit of PFP. These data suggest the presence ofone gene for each of the subunits of PFP in castor plants. Incontrast, parallel blots performed with potato genomic DNA(Fig. 4B) have multiple bands for the a- and two bands forthe (3-subunit, respectively. This may indicate that potato PFP

kb

relatively constant. In contrast, detectable levels of mRNAfor the (-subunit are only present 1.5 d after the seeds arefirst imbibed and then rise slowly to a maximum level after3.5 d. The level of expression then declines up to 5 d.

In a previous study (8) using potato tuber PFP a- and (3-subunit cDNAs, each probe detected an mRNA of differentsize and no cross-hybridization was observed between them.Because the stringency of hybridization used in this study ishigher than that used in ref. 8, we believe that the datareported here reflect genuine differences in expression of thegenes for the a- and (3-subunits. Seven days after imbibition,mRNA levels for both the a- and (-subunits are higher inroot tissue than in the developing cotyledons. After 11 d,expression of both the a- and (-subunit genes is relativelylow in both the hypocotyls and roots and there is virtuallyno detectable mRNA for either subunit in the first leaf (Fig.1), indicating that expression of the PFP genes is tissuespecific. These observations are consistent with a previousreport (3) in which PFP activity was found to be much lowerin expanding leaves than in root or stem homogenates.

Northern blots and densitometric analysis showing thepattern of expression of the two subunits of PFP in devel-oping castor seed endosperm are presented in Figures 2B and3. There is a high level of expression of both subunits in thenucellus tissue (stage II in ref. 14) prior to the developmentof the endosperm. High levels of expression are also foundthroughout the development of the endosperm up to approx-imately stage VII, at which time the seed ceases oil biosyn-thesis, loses much of its metabolic activity (25), and beginsto dry and mature. Thus, in contrast with germinating seeds,the genes for the a- and (3-subunits of PFP appear to becoordinately expressed in the endosperm of developing seeds.The high level of mRNA detectable at stage III (Fig. 3) may

be the result of contamination of the endosperm tissue, whichis very small and difficult to harvest (14), with nucellus, atissue that contains high levels of mRNA for both subunitsof PFP. Expression of the two subunits of PFP in developing

3.5-

2Q -1.1.6-

3.5-

2.0-1.6-

11 III IV V VI VII ViIl Ix

11 il IV V VI VII Vll

PFPa

PFPI3

Ix

Developmental Stage

Figure 3. Northern blot analysis of PFP-a and PFP-0 in developingcastor seed endosperm. Tissue was harvested according to thestages defined in ref. 14. Stage II refers to nucellus tissue, stage IlIlto endosperm 15 d postanthesis, and stages increase in approxi-mately 5-d increments to stage Vil and IX at 40 and 50 d postan-thesis, respectively. The autoradiographs were exposed for 16 h at-70C.

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Plant Physiol. Vol. 99, 1992

Figure 4. Southern blot of (A) castor leaf and(B) potato leaf genomic DNA hybridized withcDNAs for castor and potato PFP-a and PFP-#,respectively. E, EcoRI; H, Hindll. cDNA insertswere eluted from the cloning vector after diges-tion with EcoRI and labeled as described in"Materials and Methods."

is encoded by a multigene family or it may simply result fromthe polyploid nature of this plant. The data from the Southernand northern blots of castor plant DNA and RNA indicatethat the single genes for the PFP a- and ,8-subunits, respec-

tively, are expressed in all tissues analyzed, except the firstleaf.

There are several reports describing variability in the sub-unit composition of PFP in different plant tissues (6, 10, 16,30), and also changes in PFP activity that coincide withspecific physiological conditions (e.g. 5, 13). Whether theseobservations result from differential levels of gene expressionfor the two subunits is not known. Here we have reportedthat the genes for the a- and 13-subunits of PFP in germinatingcastor endosperm are expressed differentially, whereas in thedeveloping seeds of the same plant the subunits appear to becoordinately expressed. To determine whether the physicalproperties of PFP in a given tissue can be related to theexpression of the genes for the two subunits, the same

developmental stages were analyzed immunologically for thepresence of the a- and 13-subunit polypeptides.The antibody used in this study was raised against potato

tuber PFP and has been studied in some detail (19). Quanti-fication of western blots revealed that, for purified potatotuber PFP, the 1-subunit is 1.5-fold more immunoreactivethan the a-subunit and that this figure rises to 4.8-fold usingcrude tuber extracts. The reported discrepancy in immuno-reactivity in crude extracts may reflect structural differencesbetween two subunits present in equimolar quantities. Alter-natively, the a- and (3-subunits may have similar immuno-reactivity, as is seen with purified PFP, and the differencesobserved using crude tuber extracts may result from genuinevariation in the amount of each subunit present in this tissue(e.g. 6, 10, 16, 30). The exact relative cross-reactivity of thea- and 13-subunits of PFP in the crude extracts of castor seedendosperm used in this study is not known. Based on pre-vious reports of subunit heterogeneity in germinating seeds(6), western blots were performed on both germinating and

developing castor seed endosperm extracts to determinewhether there were differences in subunit composition thatcould be correlated with the level of subunit gene expression.Western blots of total protein extracted from the same

tissue samples as those used for RNA isolation were probedwith anti-(potato PFP) polyclonal antibodies (Fig. 5, A andB). In developing castor seed endosperm (Fig. 5A), both thea- and ,3-subunits of PFP can be detected in all four of thedevelopmental stages selected for this study (II, III, V, VII),and the relative amount of each subunit remains constant.There are correspondingly high levels of mRNA for bothsubunits in the first three of these stages (Fig. 3), althoughby stage VII the abundance of message for both PFP subunitshas dropped by approximately 95% from that found at stageV (Fig. 2B). The presence of PFP at stage VII, in the absenceof high mRNA levels, indicates that the protein is relativelystable in this tissue. PFP activity, which is dependent on Fru2,6-P2, is also high in these tissues (data not shown).The results of western analysis of a developmental profile

of germinating castor seeds are presented in Figure 5B. Arecent report (6) demonstrated that 4 h after imbibition ofcucumber seeds, only the a-subunit of PFP could be detectedby western blot, whereas both subunits were present after 60h. In the present study, both the a- and 1-subunits are barelydetectable for 2.5 d after seed imbibition, although the levelof a-subunit appears to be marginally elevated over thisperiod. Because northern analysis of the same tissue revealedthe presence of both a- and 13-subunit mRNAs as early as 1d after imbibition, the discrepancy between the appearance

of mRNA and the detection of a- and 13-subunit polypeptidesimplies the presence of posttranscriptional regulation of PFPexpression. Levels of mRNA for both subunits of PFP attaina maximum level 3.5 d after imbibition and then remain highup to 5 d. The western analysis presented in Figure 5Bdemonstrates that the level of both a- and 13-subunit poly-peptides is correspondingly high in this tissue over the sameperiod. We have been unable to detect PFP activity in ger-

A

F H

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EXPRESSION OF THE GENES FOR PPi-PHOSPHOFRUCTOKINASE

Figure 5. Western blot analysis of PFP in crudeextracts of (A) developing and (B) germinatingcastor seed endosperm. Developmental stageand times after imbibition are as described inthe legends for Figures 1 and 3. All lanes con-tain 20 Atg of crude protein extract.

minating castor seed endosperm until 3.5 d after imbibition,at which time both of the subunits of PFP are clearly detect-able by western analysis. As in developing endosperm, thisactivity is dependent upon Fru 2,6-P2.Western blot analysis of the tissues harvested 7 d after

imbibition indicates that PFP is present in the root but not inthe cotyledons. Northern analysis revealed the presence ofmRNA for both subunits of PFP in cotyledon tissue, albeit atreduced levels when compared with root. Our inability todetect either subunit polypeptide in this tissue again indicatesthat there may be some form of posttranscriptional regulationcontrolling expression of PFP. The cotyledon becomes green

and photosynthetically active after 7 d and may be exportingsucrose, in which case high levels of PFP may be unnecessary

for metabolism in this tissue. It is interesting that there are

very low levels of mRNA for both subunits of PFP in thefirst leaf, another photosynthetically active tissue (Fig. 1).

CONCLUSIONS

The data presented in this report demonstrate that thegenes for the a- and /3-subunits of PFP are not coordinatelyexpressed in all tissues because there are substantial differ-ences in the expression of the genes for the two subunits incastor seed endosperm soon after imbibition. The high levelof mRNA for the a-subunit relative to the /3-subunit, 1.5 and2 d after imbibition, is not, however, reflected in elevatedlevels of protein for the a-subunit (Figs. 1 and 5B); hence,PFP gene expression cannot be directly related to subunitcomposition in this tissue. A previous report (6) demonstratedthat only the a-subunit of PFP is present 4 h after seeds wereimbibed. Although there are elevated levels of mRNA for thea-subunit for the first 2 d after castor seeds were imbibed,tissues in which this subunit is predominant have not beenfound. The presence of significant quantities of a singlesubunit only 4 h after seeds were imbibed is probably toorapid to have resulted from de novo transcription and proteinsynthesis. Our data indicate that in castor seed endosperm,transcription and the subsequent accumulation of the twosubunits of PFP occur over a period of 3 to 4 d, suggestingthat the a-subunit of PFP in cucumber may be present in

seeds before they are imbibed, as was reported for dry seedsof Phaseolus mungo (24).There are other tissues in cucumber that have been re-

ported to contain only one subunit of PFP (6). Northernanalysis of these tissues would indicate whether the observedheterogeneity in subunit composition results from differentialgene expression in these tissues or whether other processes,

such as differential mRNA stability or subunit proteolysis,give rise to the observed variety in subunit composition.The discrepancy between the appearance of mRNAs and

polypeptides for the a- and /3-subunits of PFP in germinatingcastor seed endosperm, and the presence of mRNAs in theabsence of protein in cotyledon tissue, implies a regulationof expression that is posttranscriptional. The data presentedin this report also demonstrate that there is transcriptionaland tissue-specific regulation of expression of the subunits ofPFP. If, as has been suggested, the a- and /3-subunits of PFPperform regulatory and catalytic functions, respectively (8), a

combination of mechanisms such as these could allow forthe generation, when required, of considerable subunit het-erogeneity that could ultimately affect properties such as

sensitivity to Fru 2,6-P2. We are currently isolating genomicclones with associated promoters for the a- and /-subunitsof PFP to better understand the underlying factors regulatingthe transcription of these genes. Furthermore, we are seekingtissues in which there is clear subunit heterogeneity in whichwe can subsequently investigate levels of a- and /3-subunitgene expression.

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A B

- ,(- -,.P .. .. --(3- -Rwol w 11A&

11 III V VII

Developmental Stage

-aa -(

1 1.5 2.5 3 3.5 4 5 7r 7C

Time after Imbibition ( Days)

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Plant Physiol. Vol. 99, 1992

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