evidence that the rug3 locus of pea (pisum sativum l.) encodes plastidial phosphoglucomutase...

The Plant Journal (1998) 13(6), 753–762

Evidence that the rug3 locus of pea (Pisum sativum L.)encodes plastidial phosphoglucomutase confirms that theimported substrate for starch synthesis in pea amyloplastsis glucose-6-phosphate

C. J. Harrison*, C. L. Hedley and T. L. Wang

John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Summary

Mutants at the rug3 locus give rise to seeds that, unlike

those of the wild-type pea, are severely wrinkled at matur-

ity. In the present analysis, the starch contents of the

mature rug3rug3 seeds were found to be greatly reduced

relative to near-isogenic wild-type peas, with the most

severe phenotypes having only 1% of the seed dry weight

as starch, compared with 50% in the wild type. At the

microscopic level, the severely reduced starch content of

mutant embryos was reflected by the presence of

starchless plastids. The leaves of the rug3rug3 plants also

had dramatically reduced starch contents that could be

distinguished readily from the wild type by iodine staining.

Biochemical investigations showed that the activity of the

enzyme plastidial phosphoglucomutase (PGM) was not

detectable in mutant tissues. In wild-type pea embryos,

this enzyme catalyses the conversion of glucose-6-phos-

phate into glucose-1-phosphate in the plastids and there-

fore provides the substrate for the committed pathway of

starch biosynthesis. The requirement for the plastidial

PGM activity in order that starch synthesis can occur in pea

embryos and leaves indicates that glucose-1-phosphate

cannot be imported into the plastids and provides further

evidence that the imported substrate in pea embryos is

glucose-6-phosphate. Genetic analysis showed that the

rug3 locus is tightly linked to the flower morphology locus

k. This finding is consistent with the known genetic map

location of the Pgm-p isozyme locus and supports the

hypothesis that the rug3 mutations affect the plastidial

PGM structural gene.

Introduction

The r and rb loci of the pea are known to affect the starch

content and composition of the seed. Plants bearing mutant

alleles at these loci have been well characterized biochem-

ically and it has been established that the loci encode

Received 22 August 1997; revised 25 November 1997; accepted 26

November 1997.

*For correspondence (fax 01603 456844;

e-mail [email protected]).

© 1998 Blackwell Science Ltd 753

enzymes of the starch biosynthetic pathway (reviewed by

Martin and Smith, 1995; Wang et al., 1997). Collectively

these loci are called ‘rugosus’ (the Latin word for wrinkled),

since the mature seeds of homozygous recessive plants

for either of these loci are wrinkled in appearance. This

characteristic wrinkling is considered to be linked to a

reduced starch content in the seed by a scheme proposed

by Wang and Hedley (1991). The scheme predicts that a

reduction in starch synthesis in the developing embryo

gives rise to an increased pool of substrates, notably

sucrose, and this in turn generates an increased osmotic

pressure in the pea embryo. The high osmotic pressure

causes more water to be drawn into the embryo and

increases the fresh weight of the embryo relative to that of

the wild type. Subsequently, during the natural desiccation

process of the seed, the mutant embryo has more water

to lose, resulting in the wrinkling of the seed.

In 1987, a mutagenesis programme was initiated with

the specific aim of generating more genetic variation for

wrinkled seeds (Wang et al., 1990). The hypothesis pro-

posed by Wang and Hedley (1991) predicts that any mutant

with a decrease in starch content would have the wrinkled-

seeded phenotype. This phenotype therefore provided an

initial screen by which potential starch mutants could be

selected. The mutagenesis produced a number of wrinkled-

seeded lines which, following genetic analysis, fell into

five complementation groups. Of these, two contained new

mutants allelic to the original r and rb mutants and three

represented new rugosus loci. These have been named

rug3, rug4 and rug5. Five mutants at the rug3 locus were

generated and their alleles have been subsequently named

rug3-a to rug3-e (Wang and Hedley, 1993). Preliminary

analysis of the storage product content of the seeds of

these mutants revealed a starch content of between 1%

and 20% of the dry weight (Wang and Hedley, 1991),

representing a dramatic reduction from the 50% starch

found in the seed of the parental line.

Extensive studies of starch biosynthesis in pea embryos

have identified the major steps of the pathway (Martin and

Smith, 1995; Figure 1), beginning in the cytosol with a series

of reversible reactions and resulting in the production of

hexose phosphates from sucrose. In pea embryos and

roots, glucose-6-phosphate is thought to be the substrate

most likely to be imported into the plastids from the

cytosol. Studies have shown that when isolated plastids are

supplied with this substrate, they are capable of producing

754 C. J. Harrison et al.

Figure 1. Summary of the starch biosynthetic pathway in pea embryos.

Enzyme activities affected by mutations at the rugosus loci (r and rb)

are shown.

starch at a rate comparable to that seen in vivo (Borchert

et al., 1993; Hill and Smith, 1991). However, it has been

shown that glucose-1-phosphate can be translocated into

the amyloplasts of wheat endosperm (Tyson and apRees,

1988) and it has been argued that one translocator may be

capable of transporting different hexose-phosphates and

indeed triose-phosphates according to the type of plastid

(Borchert et al., 1993).

Inside the amyloplast, glucose-6-phosphate is recon-

verted to glucose-1-phosphate by the action of the plastidial

isoform of phosphoglucomutase (PGM). In the plastids of

wild-type pea embryos, the high levels of this enzyme

activity maintain a state of equilibrium between incoming

glucose-6-phosphate and glucose-1-phosphate (Smith and

Denyer, 1992), the substrate for the committed pathway of

starch synthesis. In mutants of Nicotiana sylvestris and

Arabidopsis thaliana, gross reductions in the activity of

this enzyme result in the almost complete absence of

starch in the leaves (Caspar et al., 1985; Hanson and

McHale, 1988). The action of the enzyme adenosine diphos-

phoglucose pyrophophorylase (ADPGPPase) produces

adenosine diphosphoglucose (ADPglucose) and pyro-

phosphate from glucose-1-phosphate and ATP, the pyro-

phosphate being removed by the action of inorganic

alkaline pyrophosphatase (Weiner et al., 1987) in favour of

starch synthesis. In maize, mutants exist that affect the

large and small subunits of the ADPglucose enzyme separ-

ately (shrunken-2 and brittle-2). In both of these mutants,

the effect on one subunit of the enzyme reduces the activity

of the enzyme to approximately 10% of that in the wild

type and cause a 75% reduction in starch content (Dickinson

© Blackwell Science Ltd, The Plant Journal, (1998), 13, 753–762

and Preiss, 1969; Tsai and Nelson, 1966). In A. thaliana, the

mutation adg1 is thought to affect overall regulation of

ADPGPPase production and results in the absence of both

enzyme subunits from leaves and roots (Lin et al., 1988)

and a leaf starch content of 2% of that of the wild-type. In

pea, the rb locus, mentioned above, corresponds to the

shrunken-2 locus of maize in that a mutation affects the

large subunit of the enzyme (Hylton and Smith, 1992). The

resultant phenotype is a reduction in the starch content of

mature pea seed to 35% of the dry weight, similar to that

of the original rugosus mutant (r). ADPglucose is combined

into the growing glucan chains by the action of starch

synthase enzymes which, in pea embryos, exist as both

soluble and granule-bound forms. The interaction of starch

synthases and starch branching enzymes results in the

formation of the two components of the starch granule,

amylose and amylopectin. These components differ with

respect to their degree of branching within the molecule,

amylose being essentially linear and amylopectin being

highly branched.

The studies of the biochemical pathway of starch syn-

thesis in peas coupled with information from the starchless

mutants of other plant species, mentioned above, indicated

possible biochemical lesions that may occur in the rug3

mutants and provided appropriate starting points for a

biochemical analysis. This paper describes a more detailed

analysis of the rug3 mutants and presents evidence that

the primary effect of the mutation is on the activity of the

plastidial PGM enzyme. The severe phenotype resulting

from the mutations has implications for our current know-

ledge of the starch biosynthetic pathway in peas and raises

questions about the importance of starch in plants that

normally store large amounts in their seeds.

Results and discussion

Plant phenotype

Plants of all five mutant genotypes grew normally in

the glasshouse and were indistinguishable from wild-type

plants. Figure 2 shows 3-week-old seedlings of the rug3-a

and rug3-b lines, which represent the extremes of pheno-

type with regard to starch content of the mature seed. Also

shown are wild-type plants of the same age. Germination

trials carried out on 1200 seeds each of the rug3-a, rug3-b

and rug3-c lines, showed rates of germination in excess of

96% with no significant difference between the rates of

germination of any of the lines and the wild-type, round-

seeded line. At maturity, the seeds of mutant plants were

severely wrinkled in appearance (Figure 3).

Starch content

The analysis of starch content of the seeds (Figure 4)

confirmed the initial analyses carried out by Wang et al.

The rug3 locus of pea 755

Figure 2. Four-week-old seedlings of (a) wild-type, (b) rug3-a and (c) rug3-b lines grown for leaf starch analysis.

The vertical bar represents 10 cm.

Figure 3. Mature pea seeds.

Seeds labelled (a–e) have the genotypes rug3-arug3-a to rug3-erug3-e,

respectively. Also shown is a wrinkled seed with the mutant genotype (rr),

i.e. homozygous recessive with respect to the original rugosus locus, and

a typical wild-type, round seed (RR). Note that the rug3 mutant seeds often

appeared more severely wrinkled than typical rr seeds.

(1990) except for the rug3-a line, in which the starch content

of the seeds appeared to be slightly lower than that

originally reported (12% rather than 20% of the dry weight).

In seeds of the rug3-b, rug3-d and rug3-e lines, the starch

content was approximately 1% of the dry weight. The

remaining mutant line (rug3-c) had a seed starch content

of 5%. In the rug3-a line, the amylose content of the starch

was reduced from 30% in wild-type starch to

approximately 12–15%. The second method of amylose

analysis (using perchloric acid) proved more sensitive for

measuring the amylose content of low starch samples,

such as the flours of rug3rug3 seeds, than did the DMSO

method, which relies on the extraction and quantification

of total starch, prior to the amylose measurement.

The starch of the rug3-a line was extracted for further

analysis by Sepharose CL-2B chromatography. Figure 5

shows the results of this analysis. The peak eluting at

around fraction 15 corresponds to amylopectin and the

peak at around fraction 40 corresponds to amylose, based

on the elution of pure fractions isolated from pea starch

(MacLeod, 1994). It can be seen that in both wild-type and


rug3-arug3-a samples, the amylose and amylopectin peaks

eluted in corresponding fractions. The amylose peak in the

rug3 sample was reduced relative to that of the wild-type,

however, the λmax values for this fraction in the two samples

was very similar, at approximately 600–620 nm. In contrast,

the λmax value for the amylopectin peak in the rug3-arug3-

a sample was increased relative to the wild-type, being

approximately 580 nm as opposed to 560 nm. The use of

size exclusion chromatography served to confirm the

results of the chemical analyses of the starch of rug3-

arug3-a seeds. In addition, the results demonstrate that

the amylose in the mutant is apparently chemically similar

to that of the wild type.

The effect of the rug3 mutations on leaf starch was

demonstrated visually by iodine staining (Figure 6). The

five rug3 alleles gave rise to leaves that were indistinguish-

able and all appeared to have grossly reduced starch

contents. A quantitative analysis carried out on samples

of leaves from 4-week-old seedlings confirmed the reduc-

tion in leaf starch content in rug3rug3 plants and gave

values of 1.5 6 0.14%, 1.0 6 0.08%, 1.0 6 0.04%,

1.2 6 0.02% and 1.1 6 0.1% for the mutant lines in allelic

order, respectively. Leaves from wild-type seedlings had

6.5 6 0.17% starch. The detection of some starch in the

mutant leaves at a level of approximately 1% of the dry

weight may reflect the ability of the mutants to synthesis

a small amount of starch, as would appear to be the case

in the seeds. However, measurements in this very low

range are likely to be beyond the true sensitivity of the

assay.

Microscopic investigations of sections of embryos from

one of the most severe mutants revealed distinct changes

at the cellular level by comparison with a wild-type embryo.

In rug3-brug3-b embryo sections there was an apparent

absence of starch when viewed with the light microscope

(Figure 7). In addition, at the light microscope level, the

cell walls in this mutant appeared to be thicker than those

of the wild type. With electron microscopy, structures that

were probably starchless plastids could be seen, but the

presence of very small starch grains in some plastids

supported the findings of the chemical analyses in that

this line is not entirely lacking in starch (Figure 7). Other

differences between wild-type and mutant embryos were


Figure 4. Starch and amylose content of mature rug3 and wild-type seeds.

Amylose 1 and 2 show amylose measurements made by the DMSO/iodine methods and perchloric acid methods, respectively. The DMSO/iodine method

was generally less reliable for measuring amylose content of low-starch pea flours. However, the apparent presence of significant quantities of amylose in

the lowest starch seeds, as determined by the perchloric acid method, may have been an artifact of the method. Error bars represent SEM of five replicates.

Figure 5. Separation of amylose and amylopectin by Sepharose CL-2B size

exclusion chromatography from (a) wild-type seed starch and (b) starch

from rug3-arug3-a seeds.

apparent; for example, the mutant had both larger vacuoles

and an altered storage protein deposition. It may be that

such differences resulted from the lowered starch content


Figure 6. Iodine stained leaves of the five rug3rug3 lines (a–e) with their

corresponding wild-type (Rug3Rug3) isolines.

of the embryos and subsequent pleiotropic effects such as

increased osmotic pressure and water uptake (Wang and

Hedley, 1991).


Figure 7. Cotyledon cells from 200–300 mg embryos of the rug3-b line and its wild-type isoline.

The light micrographs (a and b) show the difference between the lines with respect to starch content. The cells of the wild-type (b) contained large starch

grains (s), while the mutant cells (a) appeared starchless. Cell walls (w) appeared thicker in the mutant cells (a) and large vacuoles (v) were apparent. When

viewed with the electron microscope (c), the rug3-brug3-b cells can be seen to contain the occasional, extremely small starch grain (s). Size bars (a and b)

15 µm; (c) 1.3 µm.

Table 1. Enzyme-specific activities measured in fractions resulting from plastid preparation. Activities shown are µmol min–1g–1 fresh

weight or percentages 6 standard errors for two independent preparations

Plant type Total activity Activity in Activity % activity % activity in % recovery % activity attributable

supernatant in plastids in supernatant plastids to plastids

ADPglucose pyrophosphorylase

Wild type 0.184 6 0.048 0.176 6 0.058 0.012 6 0.0005 93.6 6 6.8 7.0 6 1.5 100.6 6 5.3 100*

Mutant 0.578 6 0.029 0.481 6 0.033 0.065 6 0.0051 83.2 6 1.4 11.2 6 1.3 94.4 6 1.7 100*

Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase

Wild type 0.711 6 0.324 0.728 6 0.353 0.0007 6 0 100.55 6 3.9 0.12 6 0.06 100.7 6 3.8 0*

Mutant 1.11 6 0.09 0.988 6 0.043 0.002 6 0.001 89.7 6 3.7 0.2 6 0.1 89.9 6 3.8 0*

Phosphoglucomutase

Wild type 6.595 6 2.77 6.375 6 2.6 0.053 6 0.017 97.2 6 1.2 0.84 6 0.1 98.0 6 1.34 10.6 6 1.0†

Mutant 7.532 6 0.5 7.322 6 0.47 0.007 6 0.001 97.2 6 0.2 0.09 6 0.02 97.3 6 0.02 –0.8 6 0.5†

*The percentage activity assumed to be present in the plastids for the cytosolic and plastidial marker enzymes.

†The activities of phosphoglucomutase in the plastids calculated as described in the text.

Enzyme activities

By analogy to the mutants of A. thaliana and N. sylvestris,

ADPGPPase and plastidial PGM enzyme activities were

investigated in rug3 lines. In crude extracts of developing

pea embryos, 200–300 mg fresh weight, ADPGPPase activ-

ities were 0.69 6 0.07 and 1.15 6 0.31 µmol min–1 g–1 for

wild-type and rug3-b mutant, respectively, which were not

significantly different (P . 0.2). Table 1 shows the data for

PGM from plastid preparations made from wild-type and

mutant embryos of approximately 200 mg fresh weight.

Calculations indicated that, of the total PGM activity in

rug3-b mutant embryos, none could be attributed to the

plastids, while values of 10.6% 6 1.0% were found in ident-


ical experiments carried out on wild-type embryos. To

confirm this finding, starch gel electrophoresis was

employed. It is known that two distinct isoforms of PGM

exist in the pea plant, corresponding to the cytosolic and

plastidial forms (Przybylska et al., 1989; Weeden and Marx,

1984; Weeden et al., 1984), the plastidial isoform being

the more anodal (Weeden et al., 1984). The zymograms

obtained from rug3 mutant leaves indicated that plastidial

PGM activity was undetectable (Figure 8).

Genetic analysis

Both PGM isozymes have been assigned to linkage groups

on the pea genetic map, the cytosolic marker (designated


Pgm-c) being located on linkage group VII and the plastidial

marker (Pgm-p) on linkage group II, closely linked to the

mo (bean yellow mosaic virus resistance) marker and the

k locus (Weeden and Marx, 1984; Weeden et al., 1984). As

a preliminary investigation of the position of the rug3 locus

on the pea genetic map and to support the evidence that

the mutation affects the PGM (p) structural gene, a linkage

analysis was carried out using classical genetic markers,

including k. The kk plant has a distinctive flower phenotype,

the wing petals being reduced and similar to the keel in

shape and coloration. The phenotype is easily distingu-

ished from that of the wild-type (KK) flower. The F2 popula-

tion from a cross between the multiply-marked, round-

seeded JI 791 line (Ambrose, 1996) with the kk genotype,

and the rug3-b isoline (KK), was scored for the classical

markers, including the phenotype associated with kk. When

F3 seed was produced by the plants, the F2 plants could

then be scored according to their genotype with respect

to rug3, i.e. they were designated rug3rug3, Rug3Rug3 or

Rug3rug3. Scores of the three F2 genotypes with respect

to the k phenotype are shown in Table 2. Statistical analysis

revealed that the rug3 and k loci are linked (χ2 5 78,

Figure 8. Starch gel electrophoresis of leaf extracts from wild-type and

rug3-brug3-b plants.

The ‘zymogram’ was produced by staining for PGM activity using thiazoyl

blue (MTT) and 8-dimethylamino-2,3-benzophenoxazine (Meldola’s blue).

The vertical arrow shows the direction of migration during the

electrophoresis (towards the anode). Note that the fainter, less anodal

isoform of PGM, known to correspond to the plastidial isoform, was absent

from the mutant extract.

Table 2. Numbers of F2 plants of the cross JI17913SIM32 (rug3-

brug3-b) in each category according to their genotypes with

respect to the rug 3 and k loci. Plants heterozygous for k (Kk) were

indistinguishable from the homozygous dominant (KK) type, and

therefore plants with these two genotypes were counted together

Genotypes KK or Kk kk

Rug2Rug3 0 13

Rug3Rug3 32 5

rug3rug3 45 0


P , 0.001). Applying Allard’s equation 5 (Allard, 1956) to

these results showed a map distance of approximately 7

centimorgans. This is in agreement with the map distance

calculated for the Pgm-p isozyme locus and the k locus

(Weeden et al., 1984).

Discussion

The rug3 locus of pea affects plastidial PGM activity

The initial analysis of the rug3 mutants carried out by

Wang and Hedley (1991) indicated a very severe reduction

in starch content of the seeds. The investigations described

here confirm this phenotype and demonstrate that the

mutations also affect the leaves of the plant. The effects

are similar to those seen in mutants of both A. thaliana

and N. sylvestris that affect plastidial PGM activity (Caspar

et al., 1985; Hanson and McHale, 1988) and the mutant of

A. thaliana lacking ADPGPPase activity (Lin et al., 1988).

Preliminary investigations of root tissues (Harrison, 1996)

indicated that these tissues were also affected. The rug3

starch phenotype together with the measurements of

enzyme activities and the genetic analysis provide strong

evidence that the rug3 mutations affect the plastidial PGM

structural gene.

Glucose-6-phosphate is the major substrate imported by

pea plastids

A gross reduction in the activity of plastidial PGM in

conjunction with no reduction in the activity of cytosolic

PGM has significance for the mechanism involved in the

transport of a substrate for starch synthesis into the plastids

of pea. Hill and Smith (1991) used the technique of incubat-

ing intact plastids with a variety of radioactively labelled

substrates in order to determine which could be imported

by the plastids and incorporated into starch. This work

provided evidence that glucose-6-phosphate was the major

substrate imported by the plastids of pea embryos in vitro.

The demonstration that a virtually starchless mutant of

pea has reduced (or no) activity of plastidial PGM provides

substantial evidence that the substrate imported into pea

amyloplasts is glucose-6-phosphate rather than one later

in the starch biosynthetic pathway. The two substrates that

are unequivocally excluded as being imported into the

amyloplasts are those that could enter the starch biosyn-

thetic pathway inside the amyloplast bypassing the step

carried out by plastidial PGM. These substrates are ADPglu-

cose and glucose-1-phosphate. The work of Hill and Smith

(1991) indicated that some substrates, such as fructose,

could be imported into isolated plastids to a lesser extent

than glucose-6-phosphate and at a rate not capable of

sustaining the rate of starch synthesis in vivo. Considering

the phenotype of the rug3 mutants, it now seems likely


that these observations were artifacts of the in vitro analysis

since the levels observed would probably result in more

starch synthesis than is seen in the rug3 mutants. It is

unlikely therefore that any substrate other than glucose-6-

phosphate is imported into the amyloplasts. It is possible

that in the rug3-b mutant there is a complete absence of

plastidial PGM activity, for example as a result of a promoter

mutation. In this case, the extremely small amount of

starch being synthesized in this mutant, as indicated by

the electron microscopy, must be due to the import of an

alternative substrate into the amyloplasts. The level of

import of such a substrate may reflect the level of affinity

of the glucose-6-phosphate translocator for glucose-1-

phosphate, or perhaps (as suggested by Pozueta-Romero

et al., 1991) the ability of an ADP/ATP translocator to

import ADPglucose. It has recently been suggested that

the apparent absence of starchless mutants from starch-

storing crops is as a result of a general feature of amylo-

plasts, that is the ability to import hexose phosphates

in the form of both glucose-6-phosphate and glucose-1-

phosphate (Ball, 1995). Clearly, such generalizations about

amyloplasts in different plant species cannot be made. In

starch-storing crops it is now apparent that different hex-

ose-phosphates are imported into the amyloplasts,

dependent on the plant species. In wheat, the ability of the

amyloplasts to import glucose-1-phosphate (Tyson and

apRees, 1988) means that a mutation affecting plastidial

PGM would not result in a starchless phenotype, whereas

the inability of the pea amyloplasts to import hexose

phosphates other than glucose-6-phosphate made the

isolation of the rug3 mutants possible. The implications of

the effects of rug3 mutations for the transport of substrates

into the amyloplasts of pea are also true for the chloro-

plasts, since these also produce very little starch in the

mutant plants. Furthermore, the very low starch content

of the seeds, leaves and roots of the rug3rug3 plants

indicates that in the cells of all of these tissues, the same

single isoform of plastidial PGM is present.

Implications for starch composition

The reduction in amylose content in the rug3-arug3-a

seeds has implications for the mechanisms of amylose

and amylopectin formation within the starch grain. The rb

and rug3 mutations of pea both affect enzyme activities

that are required for the supply of ADPglucose to the

growing glucan chains. In the rb mutant, there is a 37%

reduction in the amylose content of the starch. In the rug3-

a mutant described here, a greater reduction in total starch

content is also accompanied by a 68% reduction in amylose

content of the residual starch. Several mechanisms have

been proposed by which amylose content of starch may

be reduced in mutants where the biochemical lesion lies

in the stages of the starch biosynthetic pathway affecting


ADPglucose supply (Martin and Smith, 1995; Van den

Koornhuyse et al., 1996). However, the effects of a reduction

in ADPglucose supply on starch composition may not be

the same across different species, since a PGM mutant

of Chlamydomonas reinhardtii, for which a preliminary

investigation has been described (Van den Koornhuyse

et al., 1996), has between 4% and 12% of wild-type starch

levels and has no amylose.

An approximately linear relationship exists for the pro-

portion of the starch that is amylose in mature seeds of

wild-type, rb and rug3 peas and the proportion during

development of a wild-type pea seed. Therefore, in the rb

mutant, the proportions of amylose in the starch is equal

to that in the wild-type for the same starch content of the

embryo (Lloyd et al., 1996). It can be concluded that for

pea mutations affecting substrate supply, the factor that

determines the proportion of amylose in the starch is the

amount of starch present. Furthermore, the rate of starch

synthesis does not affect the amount of amylose being

synthesized, since a mature rug3-arug3-a seed and an

immature wild-type seed both having the same amount of

starch will have the same proportion of amylose in the

starch despite a large difference in the time period over

which the starch was synthesized. These observations are

in agreement with theories that it is the physical trapping

of GBSS1 inside the starch grain that leads to amylose

synthesis (Martin and Smith, 1995) – as the starch grain

size increases so the amount of trapped enzyme increases

and therefore the amount of amylose being synthesized

increases. Such theories may explain alterations in amyl-

opectin seen in the Chlamydomonas PGM mutant (Van

den Koornhuyse et al., 1996) and the rug3-a mutant.

Starch as a storage material?

Despite some similarities with near-starchless mutants of

other species, mentioned above, the rug3 mutants of pea

are unique. Caspar et al. (1985) stated that, since A. thaliana

is an oilseed, it probably does not require starch as a seed

carbohydrate reserve. It was argued that it is for this reason

that it was possible to isolate a near-starchless mutant of

A. thaliana and also that it would be unlikely that a

starchless mutant could be isolated from a plant species

that does rely on starch as storage carbohydrate in the

seed, since any such mutant would be seedling lethal. This

hypothesis is clearly abrogated by the isolation of the

rug3 mutants.

The isolation of a near-starchless mutant of pea, a species

that normally stores large amounts of starch as storage

carbohydrate in the seeds, poses the obvious question:

Why store starch when it is apparently not necessary

for germination or initial growth of the plant? In some

circumstances, and especially in a natural environment,

there would be a distinct advantage in having a large store


of a high energy-yielding compound in the seed. This store

could be utilized in the initial growth of the seedling, to

extend the shoot rapidly out of deep earth, or to enable

the roots to reach soft earth from a dry surface onto which

seeds may have fallen from the plant. Starch provides an

ideal storage compound as it is osmotically inactive, is

synthesized in specialized organelles and can be rapidly

mobilized by a small number of lytic enzymes. In the

evolutionary history of the pea, the presence of starch in

the seed as a storage compound probably gave a selective

advantage to the plant. True wild-type varieties of pea

contain a large proportion of starch in the mature seed.

However, it is important to note that the large, modern pea

seed has been selected by humans through centuries of

cultivation and breeding that has undoubtably resulted in

quantities of storage carbohydrate that are surplus with

regard to the survival and germination of the seed.

The rug3 mutants represent the first near-starchless

mutants to be isolated from a starch-storing crop. They

comprise an allelic series, demonstrating that alterations

in plastidial PGM activity can produce a variety of starch

contents of the pea seed. In addition, in the rug3 mutant

with the highest starch content, the proportion of amylose

in the starch is affected. The rug3 mutants therefore aug-

ment the existing starch-synthesis mutants of pea in provid-

ing tools by which the mechanisms involved in the starch

biosynthetic pathway may be more fully understood.

Experimental procedures

Plant material and growth conditions

Comparisons between mutant and wild-type material were made

using near-isogenic pea lines that were obtained by selfing and

reselecting lines heterozygous at the rug3 locus for six generations,

so that each wrinkled-seeded (rug3rug3) line had its own corres-

ponding round-seeded (Rug3Rug3) line. For the purpose of linkage

analysis the rug3-brug3-b line was crossed to a round-seeded

line carrying multiple morphological genetic markers (JI 791;

Ambrose, 1996).

Seeds were sown in the glasshouse in 2-inch ‘Jiffy’ pots con-

taining John Innes no. 1 compost to which 30% chick grit had

been added. Once established, the plants were transferred to 5-

inch pots and fed weekly with low nitrogen fertilizer. Glasshouses

were maintained in a 15/10°C minimum day/night cycle with

supplementary lighting as required to provide a minimum photo-

period of 16 h.

Starch content and composition in mature seeds

Total starch content of mature seeds and the amylose contents of

the starch were determined according to methods modified from

Carpita and Kanabus (1987) and Knutson (1986) as described in

Wang et al. (1990). Pea amylose was used as the material for the

construction of standard curves showing the relationship between

amylose concentration and OD600 (Knutson, 1986), since it has

been found to be the only suitable amylose (MacLeod, 1994). To


confirm amylose measurements made on low-starch pea flour a

second method was employed that did not rely on the measure-

ment of total starch. This method was adapted from Hovenkamp-

Hermelink et al. (1988) and Haase and Kempf (1990). Firstly,

a standard curve was produced by dissolving separate known

amounts of pea amylose and amylopectin in 1 ml 45% perchloric

acid for 5 min with gentle stirring. Standards were diluted to

10 ml with water giving a range of amylose and amylopectin

concentrations of between 0 and 8 mg 100 ml–1. One millilitre of

each standard was added to 1 ml Lugol’s iodine solution (0.04 M

potassium iodide 1 0.013 M iodine) in disposable plastic cuvettes.

Absorption spectra were produced for both amylose and amyl-

opectin and the wavelengths giving rise to the maximum

absorbance by each were noted. The optical density of each

standard was measured at each of the two peak wavelength

values. Optical densities were plotted against concentration of

amylose and amylopectin producing four standard curves.

Samples were treated in the same way as the standards;

approximately 1 mg of each sample was dissolved in perchloric

acid, diluted to 10 ml, and 1-ml aliquots mixed with 1 ml Lugol’s

iodine. An OD value at each of the peak wavelengths determined

for pure amylose and amylopectin was obtained and these values

were used in conjunction with the standard curves to calculate

the amylose:amylopectin ratio using the formula of Hovenkamp-

Hermelink et al. (1988). Absorption spectra and optical density

measurements were carried out in a Hitachi U-2000 spectropho-

tometer.

Sepharose CL-2B chromatography was carried out according to

Denyer et al. (1995).

Starch content of leaves

Fully expanded leaves were removed from 4-week old, glasshouse-

grown seedlings, 8 h into the photoperiod. The leaves were freeze-

dried and subjected to ball-milling in the same way as mature

seeds. Duplicate samples in the region of 5 mg of the resulting

powder were accurately weighed and boiled in 5 ml of 70% ethanol

for 30 min with stirring in capped tubes. The solid material in

the tubes was pelleted by centrifugation and the supernatant

discarded. This process was repeated and, after centrifugation

and removal of the supernatant, the pellet was dried in a rotary

evaporator. The ethanol extractions were designed to remove

soluble sugars that may have been present in the samples. To

check for sample loss during these extractions, a sample of wild-

type seed flour was treated in the same manner as the leaf

material and analysed for starch content. The solid material

remaining after the ethanol extractions was subjected to total

starch analysis in exactly the same manner as used for pea flour

from mature seeds.

Visualization of starch in leaves was carried out by first soaking

mature leaves in 100% ethanol overnight in order to remove

pigmentation. They were then rinsed with 100% ethanol and

immersed in 50% Lugol’s iodine solution for approximately 1 h.

The iodine solution was then removed and the leaves rinsed in

water before being photographed.

Determination of ADPglucose pyrophosphorylase activity

Preparation of crude extracts of pea embryos and the determina-

tion of ADPGPPase specific activity were carried out according to

Smith (1988). Results given are the means and standard errors

of three separate extractions from embryos in the 200–300 mg

weight range.


Determination of plastidial PGM activity

Preparation of plastids and the determination of PGM activity in

both crude extracts of embryos and plastid preparations was

carried out according to Foster and Smith (1993). Plastidial PGM

activity was calculated from the activities of marker enzymes, i.e.

those known to be present only in the cytosol (in the case of PFP)

or in the plastid (in the case of ADPGPPase) as follows:

P 1 C 5 T (1)

Where P is plastidial PGM activity, C is cytosolic PGM activity and

T is total PGM activity in an embryo extract.

(Mc% 3 C) 1 (Mp% 3 P) 5 Pa

Where Mc is cytosolic marker activity, Mp is plastidial marker

activity and Pp is the PGM activity assayed in the plastid frac-

tion, i.e.

McC 1 MpP 5 100 Pa (2)

Substituting the values for Mc, Mp and Pa obtained from the

assays into the equation and solving equations (1) and (2) simultan-

eously gives the values for P and C.

Starch gel electrophoresis

Buffers for starch gel electrophoresis were prepared according to

Selander et al. (1971). The electrode buffer was lithium-borate,

pH 8.1 (0.03 M lithium hydroxide, 0.19 M boric acid). The gel buffer

comprised one-part electrode buffer and nine-parts Tris–citrate

buffer, pH 8.4 (0.05 M Tris, 0.008 M citric acid). Extraction buffer

was prepared according to N. Weeden (personal communication)

and consisted of 0.05 M Tris–HCl, pH 8, 2 mM DTT. The starch gels

consisted of 7.5% potato starch (hydrolysed for electrophoresis;

Sigma-Aldrich Co. Ltd, Dorset, UK) in 150 ml gel buffer. Samples

for electrophoresis were prepared by grinding leaves and embryos

from wild-type and rug3-brug3-b plants in a minimum amount of

extraction buffer so that a thick slurry was produced. The extract

was then allowed to soak into filter paper wicks (Whatman no.

182) for approximately 10 min at 4°C. When fully impregnated,

the wicks were inserted into a slit made 2 cm from the cathodal

edge of the gel. Electrophoresis of the starch gel was carried out

at 300 V and 4°C. After approximately 30 min, the current was

switched off and the paper wicks were removed from the gel.

Electrophoresis was then continued for a further 6 h. Horizontal

slices were made through the gel and stained for PGM activity by

a method adapted from Thorpe et al. (1987) and N. Weeden

(personal communication). The stain consisted of 50 ml 0.1 M

Tris–histidine buffer (pH 8; 0.1 M Tris titrated to correct pH with

histidine–HCl) containing 100 mg MgCl2, 120 mg glucose-1-phos-

phate, 10 mg NADP, 20 mg thiazoyl blue (MTT), a trace of 8-

dimethylamino-2,3-benzophenoxazine (Meldola’s blue) and 50

units of glucose-6-phosphate dehydrogenase (from Leuconostoc

mesenteroides). The starch gel was immersed in stain for 60 min

at 37°C in darkness. Violet bands representing enzyme activity

were easily visible after this time.

Light and electron microscopy of embryo sections

Embryos of 200–300 mg fresh weight from wild-type and

rug3-arug3-a and rug3-brug3-b plants were harvested and

approximately 2mm3 pieces cut from the cotyledons. Preparation

and sectioning of samples for light and electron microscopy were

carried out according to Liu (1995) and Liu et al. (1995). The


samples of cotyledons were fixed overnight at room temperature

in a solution containing 4% (V/V) formaldehyde, 2.5% (W/V)

gluteraldehyde, 0.75% (V/V) acrolein and 0.1 M sodium cacodylate

(pH 7.2). Fixed samples were treated with 1% osmium tetroxide

for 1 h at room temperature followed by dehydration in an ethanol

series. The samples were infiltrated with LR White resin (London

Resin Co. Ltd, Hampshire, UK) and polymerized overnight at 60°C

in gelatin capsules. Processing of sections for electron microscopy

was carried out according to Liu et al. (1995). Sections were

prepared for light microscopy from the same resin-embedded

samples by taking 0.5 µm sections, mounting them on glass

microscope slides and staining with filtered 0.5% (w/v) toluidine

blue for 2 min followed by rinsing with water. These sections were

observed with a Zeiss Axiophot microscope.

Acknowledgements

This research was funded by Unilever plc whose support we

gratefully acknowledge. We wish to thank Alison Smith for help

with biochemistry, Noel Ellis for assistance with the genetic

analysis and Lorraine Barber for the Sepharose chromatography.

The John Innes Centre is supported by a grant-in-aid from the

BBSRC.

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evidence that the rug3 locus of pea (pisum sativum l.) encodes plastidial phosphoglucomutase...

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