Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity

Starch granule initiation and growth are altered in barleymutants that lack isoamylase activity

Rachel A. Burton1, Helen Jenner2, Luke Carrangis1 Brendan Fahy2, Geoffrey B. Fincher1, Chris Hylton2, David A. Laurie2,

Mary Parker2, Darren Waite2, Sonja van Wegen1, Tamara Verhoeven2 and Kay Denyer2,*1Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia2John Innes Centre, Norwich Research Park, Colney, Norfolk NR4 7UH, UK3Institute of Food Research, Norwich Research Park, Colney, Norfolk NR4 7UA, UK

Received 31 October 2001; revised 18 March 2002; accepted 27 March 2002.*For correspondence (fax +44 1603 450045; e-mail [email protected]).

Summary

Two mutant lines of barley, Risù 17 and Notch-2, were found to accumulate phytoglycogen in the grain.

Like the sugary mutants of maize and rice, these phytoglycogen-accumulating mutants of barley lack

isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have

lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a

reduction in endosperm starch content, the mutations have a profound effect on the structure, number

and timing of initiation of starch granules. There are no normal A-type or B-type granules in the

mutants. The mutants have a greater number of starch granules per plastid than the wild-type and,

particularly in Risù 17, this leads to the appearance of compound starch granules. These results suggest

that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild-type endosperm plays a

role in determining the number, and hence the form, of starch granules.

Keywords: starch, barley, granules, initiation, isoamylase, phytoglycogen.

Introduction

Normal starch synthesis in plants requires, in addition to

starch synthases and starch-branching enzymes, a deb-

ranching enzyme (DBE) that cleaves (1,6) a-linkages within

amylopectin and related polysaccharides or oligosacchar-

ides. Evidence that DBE is necessary for normal starch

synthesis comes from the study of mutant plants and

algae lacking enzymes belonging to the isoamylase class

of DBE (maize, James et al., 1995; rice, Kubo et al., 1999;

Arabidopsis, Zeeman et al., 1998; Chlamydomonas,

Mouille et al., 1996). In these mutants, the starch content

is lower than normal and there is an accumulation of a

soluble (1®4 : 1®6) a-glucan, phytoglycogen. Phytogly-

cogen does not accumulate in wild-type plants and algae,

or in other low-starch mutants.

In the isoamylase (sugary1) mutants of maize and rice,

there is also a decrease in the activity of limit dextrinase

(LD), another type of DBE (Nakamura et al., 1996; Pan and

Nelson, 1984). It has been argued that this decrease, rather

than the loss of isoamylase, may be the direct cause of

phytoglycogen accumulation. There is an inverse correl-

ation between LD activity and phytoglycogen accumula-

tion in the endosperm of rice mutants carrying sugary1

alleles of different severity (Nakamura et al., 1997).

However, LD remains at wild-type levels in the phytogly-

cogen-accumulating isoamylase mutant of Arabidopsis

(dbe1; Zeeman et al., 1998). Thus, it is likely that the

isoamylase-type of DBE plays a speci®c role in the

synthesis of starch that cannot be assumed by the LD-type.

Two models have been put forward to explain the role of

isoamylase in starch synthesis. The ®rst proposes that

isoamylase plays a direct role in the synthesis of

amylopectin, the major component of starch granules

(Ball et al., 1996; Myers et al., 2000). It is suggested that

DBE is required for the synthesis of an amylopectin

molecule capable of crystallization from a soluble pre-

amylopectin precursor. In the absence of isoamylase, pre-

amylopectin is further elaborated by starch synthase and

starch-branching enzymes in the stroma to form phytogly-

cogen. The second model (Zeeman et al., 1998) proposes

that isoamylase does not play a direct role in the synthesis

of amylopectin. Instead, together with other degradative

enzymes, isoamylase degrades soluble a-glucans. In the

The Plant Journal (2002) 31(1), 97±112

ã 2002 Blackwell Science Ltd 97

absence of isoamylase, these accumulate in the form of

phytoglycogen along with reduced amounts of normal

amylopectin.

The phenotypes of the isoamylase mutants so far

described do not provide suf®cient information to allow

these models to be further evaluated. It is not clear which,

if either is correct. To investigate further the role of

isoamylase in starch synthesis, we identi®ed two allelic

isoamylase mutants of barley, Risù 17 and Notch-2, during

a screen for altered starch synthesis in the endosperm of

high-lysine mutants of barley. Detailed characterization of

starch granule structure and the sequence of the isoamy-

lase genes of these mutants have shed light on the

possible role of isoamylase during starch synthesis.

Results

Two low-starch barley mutants accumulate large

amounts of soluble a-glucan

The starch and soluble a-glucan contents of mature grains

of two wild-type barley cultivars (Bomi and Carlsberg II)

and several previously identi®ed low-starch mutants

(Balaravi et al., 1976; Bansal, 1970; Doll, 1983) were meas-

ured (Figure 1). Soluble a-glucan is material that is soluble

in aqueous extraction medium but insoluble in > 60%

aqueous methanol. Two of the low-starch mutants, Risù 17

and Notch-2, had much higher soluble a-glucan contents

than the wild-type lines and the other mutants.

The degree of branching of the a-glucans in the mature

grains of wild-type (Bomi) and mutants (Risù 17 and

Notch-2) was compared by measuring the wavelengths of

maximum absorbance (lmax) of the a-glucan-iodine

complexes. All of the starches had lmax of 550±600 nm,

which is typical of starches generally. The lmax of the

soluble a-glucan from Bomi was also 550±600 nm sug-

gesting that it had a degree of branching similar to starch.

The soluble a-glucans from the mutants had lmax values

of 420±430 nm, which indicated that they were more

highly branched than starch. These values are in the

same range as those of phytoglycogen from maize and

Arabidopsis (Zeeman et al., 1998), suggesting strongly that

the barley mutants accumulate phytoglycogen.

The soluble a-glucan in the mutants is phytoglycogen

To examine the structure of the soluble a-glucan in more

detail and to compare the starches from wild-type and

mutants, we determined the relative abundance of chains

of different lengths in these a-glucans using ¯uorophore-

assisted gel electrophoresis (O'Shea and Morell, 1996).

Starch and soluble a-glucan were extracted from endo-

sperms at ®ve different stages of development. For starch,

the pro®les obtained re¯ect the distributions of the short-

to medium-length chains of the amylopectin component of

starch.

There was little alteration in the chain-length pro®les of

the starches with developmental age, for either the wild-

type or the mutants (Figure 2a, i-iii). The mutant starches

had chain-length pro®les similar to those of the wild-type

starches except that mutant starches from older and

mature endosperms had relatively more chains of dp 10±

13 (Figure 2b, i-ii).

The chain-length pro®les of the soluble a-glucan from

the barley mutants were similar to those of phytoglycogen

from other species (e.g. maize, Dinges et al., 2001; rice,

Nakamura et al., 1997; Arabidopsis, Zeeman et al., 1998).

There was an increase in the proportion of short chains (dp

6±9) with age (Figure 2a, v-vi and Figure 2b, iii-iv). At all

stages of development, the soluble a-glucans from the

mutants had a greater proportion of chains of dp 6±8

(Figure 2b, v-vi) and, particularly in Notch-2, fewer chains

of dp 10±15 than their respective starches (Figure 2b, v-vi).

Risù 17 and Notch-2 are allelic variants

To determine whether the recessive mutations in Risù 17

and Notch-2 were in the same gene, we crossed them and

examined the F1 progeny. All of the F1 grains were

shrivelled, indicating that they had a lower than normal

starch content, and the starch granules were irregularly

shaped and small. Risù 17 and Notch-2 were crossed with

other low-starch cultivars (Risù 13, Risù 16, Risù 527, Risù

1508, Notch-1; Doll, 1983). These crosses gave F1 grains

that were all, or mostly all, normal in shape. The starch

Figure 1. Starch and soluble a-glucan contents of mature grains.a-Glucans were extracted from mature grains by homogenization inwater. Starch was puri®ed from the water-insoluble fraction and solublea-glucan by alcohol precipitation of the water-soluble fraction. Soluble a-glucan is material which is soluble in aqueous extraction medium butinsoluble in > 60% aqueous methanol. Data are means 6 SD of valuesfrom 3 to 4 separate extracts. Bomi and Carlsberg II are wild-type withrespect to their starch contents. The other cultivars were previouslyidenti®ed as low-starch mutants.

98 Rachel A. Burton et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 97±112

granules in grains resulting from the cross between Risù

17 and Notch-1 were normal in shape. These results

indicate ®rstly, that the mutations in Risù 17 and Notch-2

lie in the same gene and secondly, that none of the other

low-starch cultivars carry mutations that are allelic to those

in Risù 17 or Notch-2.

Risù 17 and Notch-2 lack isoamylase activity

Isoamylase activity cannot be quanti®ed in crude extracts

due to the absence of a unique substrate for this enzyme.

However, isoamylase activity in extracts of several plant

species is revealed on native, non-denaturing polyacryla-

mide gels containing amylopectin. The enzyme appears as

a blue-staining band with low mobility when the gels are

stained with Lugol solution (Dinges et al., 2001; Kubo et al.,

1999; Zeeman et al., 1998). Such a band was observed with

extracts of developing wild-type barley endosperms of

different developmental ages (Figure 3 and data not

shown). A second, faint blue-staining band was some-

times observed in wild-type extracts (not visible in

Figure 3). Neither blue band was observed with extracts

of endosperms of any age for either Risù 17 or Notch-2. To

estimate the minimum isoamylase activity that could be

detected using this native gel method, we compared a

series of dilutions of the wild-type extract on native gels

(not shown). From this, we estimated that the isoamylase

activity in the mutants was < 4% of that in the wild-type.

The activities of several other enzymes are affected in the

mutants

The activities of many of the enzymes involved in the

conversion of sucrose to starch were determined in

endosperms from Risù 17 and its parent variety, Bomi

and in Notch-2 and the variety from which it was derived,

NP113. When the activities in grains of 45±55 mg FW in the

mutants and their corresponding wild-types were com-

pared, the activities of many enzymes were not statistically

signi®cantly different (Table 1). Some enzymes (Table 1)

did show differences but these were not consistently

different in both mutants. For example, the activity of

soluble starch synthase was higher in Risù 17 than in Bomi

but there was no signi®cant difference in the activity of this

enzyme between NP113 and Notch-2. Most of the enzymes

that differed in activity showed a higher activity in the

mutant than in the wild-type. These increases in activities

are unlikely to cause or contribute to the decrease in total

a-glucan synthesis observed in the mutants. Two

enzymes, SBE and alkaline pyrophosphatase, showed

lower activity in Risù 17 than in Bomi. These data suggest

that the mutations cause pleiotropic effects on the activ-

ities of other enzymes in the pathway of starch synthesis.

Such pleiotropic effects are common in starch mutants of

cereals (maize, Singletary et al., 1997; barley, Schulman

and Ahokas, 1990).

Attempts to assay limit dextrinase (LD) activity in crude

extracts of developing barley endosperm were unsuccess-

ful due to the presence of protein inhibitors (Macri et al.,

1993). The activity of LD measured in mixed extracts of

developing pea embryo and developing barley endo-

sperms was much less than that expected from measure-

ments of the LD activity in these tissues extracted

separately (data not shown). Inclusion of a chemical

modi®er of the LD inhibitor, phenyl glyoxal (MacGregor

et al., 2000) did not increase the measurable LD activity in

extracts of barley. As an alternative approach to estimate

the LD activity, we used native gels similar to those

described above to identify isoamylase but containing red-

pullulan rather than amylopectin. Red-pullulan is a unique

substrate for LD, and is not degraded by isoamylase or any

other enzymes. Two bands of LD activity were observed

(data not shown). Comparison of the LD activity in extracts

of mutant and wild-type developing endosperms did not

reveal any consistent differences. This suggested that in

the barley isoamylase mutants, unlike those of rice and

maize, the LD activity is not decreased relative to that in the

wild-type. However, this conclusion must be treated with

some caution as we do not know to what extent the LD

inhibitors affect the LD activity revealed in these gels.

Cloning and sequencing the isoamylase cDNA

To discover whether the lack of isoamylase activity was

due to mutations within the isoamylase gene, PCR was

used to amplify the cDNAs encoding isoamylase in the

wild-types and mutants. A nested PCR strategy, using the

high-®delity Taq polymerase Elongase, was used to obtain

full-length isoamylase cDNAs. The cDNA sequences have

been submitted to GenBank under accession numbers

AF490375 (Bomi), AF490376 (Notch-2) and AF490377 (Risù

17), and the predicted amino acid sequences are shown in

Figure 4a. This is the ®rst published report of the full-

length sequence of barley isoamylase cDNA; the sequence

previously reported by Sun et al. (1999) is truncated at both

the 5¢ and 3¢ ends. We will refer to this cDNA as isa1.

The isa1 cDNAs from the wild-types, Bomi and NP113,

are almost identical and very similar to isoamylase cDNAs

from other cereals (data not shown). The isa1 cDNAs from

both mutants differ from the wild-type isa1 cDNAs. That

from Risù 17 contains an 872-bp deletion, starting at amino

acid 338 (Figure 4a), whilst that from Notch-2 contains a

72-bp insertion. The insertion in the Notch-2 cDNA

includes two in-frame stop codons (Figure 4a). A

BLASTN search with the 72 bp sequence reveals that it is

in the same position and has 90% sequence identity with

intron 9 of a DBE gene from Aegilops tauchii (Figure 4b).

This suggests that the insertion in Notch-2 is an intron that

Isoamylase mutants of barley 99


is either not removed or is incorrectly spliced out of the

mRNA, possibly due to a single base change (G to A) at the

5¢ intron splice junction (Figure 4b). A thorough investiga-

tion of the Notch-2 cDNA population revealed a number of

other isoamylase cDNAs, for example, Notch-2A and

Notch-2B (Figure 4a) in which the intron appears to have

been mis-spliced. The mis-splicing in both Notch-2A and B

results in the removal of two amino acids and causes

frameshifts that result in the introduction of downstream

stop codons. No full-length, wild-type cDNAs were found

in Notch-2 or Risù 17.

The relative amounts of isa1 transcript in developing

endosperms of the wild-types and mutants were com-

pared by Northern analysis (Figure 4c). Using this tech-

nique, the transcripts in the mutants were apparently

absent. However, as transcripts were detected in the

mutants using the more sensitive PCR technique, we

assume that a very low level of isa1 transcript is present in

Figure 2a.



the mutants but that it is below the level of detection in the

Northern analysis.

The isoamylase gene is located on chromosome 7H

The mutation responsible for the low-starch phenotype of

Risù 17 was previously assigned to barley chromosome 7H

using a set of translocation lines (Jensen, 1979).

We mapped the isoamylase gene, isa1 by RFLP in a

population of 86 doubled haploid (DH) lines from a

Chebec 3 Harrington cross. A single polymorphism

between restriction enzyme digests of the parental lines

was identi®ed. Scoring this polymorphism in the mapping

population con®rmed the chromosome 7H location

Figure 2b.

Figure 2. Analysis by gel electrophoresis of the short chains of a-glucans.a-Glucans were extracted from grains of different developmental ages. The stages of development were de®ned by the FWs of the grains as follows: s,> 20 mg; h, 20±40 mg; n, 40±70 mg; ,, 70±100 mg; e, mature grains. a-Glucan samples were debranched with isoamylase, derivatized with the¯uorophore APTS and subjected to electrophoresis in an Applied Biosystems DNA sequencer. Data were analysed using Genescan software.(a) Analysis of chain-length pro®les. The sum of the areas of peaks corresponding to individual chain lengths between 6 and 28 glucose units wascalculated and each peak area was expressed as a percentage of the total peak area. Each value is the mean of two replicate measurements. (i-iii) starch;(iv-vi) soluble a-glucan.(b) Comparison of chain-length pro®les. To compare different a-glucans, for each chain length, the difference between values for the percentage total peakarea was calculated.



(Figure 4d) and reference to markers listed in Langridge

et al. (1995) placed the gene genetically close to the

centromere on the short arm. The gene was also mapped

in a population of 90 DH lines from a Galleon 3 Haruna

Nijo cross (Langridge et al., 1995) which gave an equiva-

lent map location (data not shown). The limit dextrinase

gene is also located on chromosome 7H (Figure 4d; R.A.

Burton and G.B. Fincher, unpublished data).

The mutants have radically altered starch-granule

architecture

Scanning electron microscopy of mature grains (Figure 5a)

showed that the endosperm of the wild-type barley, Bomi,

contained the A- and B-type starch granules typical of the

Triticeae family (Jane et al., 1994). As observed previously

(Burgess et al., 1982; Gautam et al., 1994; Sood et al., 1992;

Tester et al., 1993), the granules in the mutants were

completely different in appearance to those in the wild-

type. They were irregular in shape and intermediate in size

compared with the A- and B-type granules in Bomi.

Some of the granules in Risù 17 resembled the com-

pound granules of rice and oat endosperm. In these Risù

17 granules, a number of irregular granulae were closely

oppressed to form a smooth compound granule

(Figure 5a). Compound granules were previously

observed in the developing endosperms of Notch-2

(Sood et al., 1992). These observations and our own of

the young developing endosperm of Risù 17 (Figure 5b),

suggested that the individual components of the com-

pound granules in these mutants were initiated separately

within the plastid. Later, these component granules grew

together to ®ll the available space and therefore became

irregular in shape. The granules in these mutants do not

appear to arise as single granules which later fracture into

Figure 3. Isoforms of debranching enzymes in developing endosperms.Crude, soluble extracts of developing endosperms were loaded onto gelscontaining amylopectin. Each track contained extract (from 1 mg FW oftissue) and loading medium in a ratio of 5 : 1. After electrophoresis, gelswere incubated at 37°C for 16 h at pH 6.0 and stained with iodinesolution. The position of the blue-staining band that is due to isoamylaseactivity is indicated with an arrow. Tracks 1 and 2: Bomi, tracks 3 and 4:Risù 17, tracks 5 and 6: NP113, tracks 7 and 8: Notch-2.

Table 1. Comparison of the maximum catalytic activities of enzymes in crude extracts of developing endosperms

Enzyme

Activity (mmol min±1 g±1FW)

Bomi Risù 17 NP113 Notch-2

Sucrose synthase 3.39 6 0.16 [8] 3.79 6 0.53 [5] 2.81 6 0.72 [4] 3.80 6 0.59 [5]UDPG pyrophosphorylase 54.13 6 4.68 [7] 68.87 6 8.51 [5] 41.04 6 5.10 [5] 55.53 6 11.91 [5]Fructokinase 0.245 6 0.022 [8] 0.311 6 0.039 [5] 0.182 6 0.021 [5] 0.291 6 0.026 [5]Glucokinase 0.435 6 0.050 [8] 0.523 6 0.056 [5] 0.386 6 0.034 [5] 0.612 6 0.054 [5]Phosphoglucomutase 19.02 6 3.92 [8] 38.35 6 10.40 [6] 27.51 6 7.87 [6] 28.37 6 6.94 [6]Phosphoglucose isomerase 19.61 6 2.26 [8] 21.40 6 1.71 [4] 18.68 6 0.79 [5] 22.83 6 2.88 [5]ADPG pyrophosphorylase 4.44 6 0.37 [8] 6.88 6 0.97 [6] 3.72 6 0.28 [5] 5.25 6 0.78 [6]Soluble starch synthase 0.216 6 0.020 [8] 0.399 6 0.026 [4] 0.131 6 0.025 [5] 0.118 6 0.037 [4]Granule-bound starch synthase 0.387 6 0.048 [8] 0.373 6 0.069 [4] 0.252 6 0.051 [4] 0.222 6 0.074 [3]Alkaline pyrophosphatase 7.34 6 0.94 [8] 4.52 6 0.64 [5] 3.68 6 0.57 [5] 3.58 6 0.64 [5]Starch-branching enzyme 42.93 6 2.62 [6] 31.54 6 0.78 [5] ND ND

Developing endosperms from grains of 45±55 mg FW were extracted as described in Experimental procedures and assayed for enzymeactivities. Values are means 6 SE of measurements made on a number of independent extracts (shown in parentheses). Comparison of theactivities of each enzyme in Bomi and Risù 17 and in NP113 and Notch-2 was done using Microsoft Excel software (t-test, 2-taileddistribution, 2-sample equal variance). This statistical analysis showed that the activities of soluble starch synthase and ADPGpyrophosphorylase were higher in Risù 17 than in Bomi (P < 0.05), the activities of fructokinase and glucokinase were higher in Notch-2than in NP113 (P < 0.05) and the activities of SBE and alkaline pyrophosphatase were lower in Risù 17 than in Bomi (P = 0.05). All other pair-wise comparisons showed no statistically signi®cant difference (P > 0.05). ND = not determined.



multiple, irregular pieces as in developing embryos of the

starch-branching enzyme mutant of pea (rr, Lloyd, 1995).

Endosperm cells contain different amounts of

phytoglycogen

To investigate the distribution of starch and phytoglyco-

gen within endosperms of barley, slices of endosperm

were cut from developing grains, ®xed and embedded in

resin. Thin sections were cut, stained and examined with

either a light microscope (Figure 6, left panels) or a

transmission electron microscope (TEM; Figure 6, right

panels).

As well as the small, irregular granules in the mutants

(Figure 6b-f), some plastids in cells of the mutant endo-

sperms contained a diffuse material that stained lightly

with toluidine blue (Figure 6b-d). The TEM pictures

showed that this material was particulate (Figure 6e,g,h).

This is likely to be phytoglycogen as it resembled similar

material seen in the leaves of isoamylase mutants of

Arabidopsis (Zeeman et al., 1998) and in sections of

developing maize endosperm from sugary1 mutant plants

prepared using the same procedures used for the barley

endosperm sections shown in Figure 6 (M. James and M.

Parker, Iowa State University and IFR Norwich, respect-

ively, personal communication).

In the endosperm of older Risù 17 grains and in both

young and older Notch-2 endosperm, most cells contained

phytoglycogen (not shown). In many plastids, the starch

granules had sharp, well-de®ned edges (e.g. Figure 6f).

However, plastids containing large amounts of phytogly-

cogen as well as starch often had granules with irregular,

diffuse edges (Figure 6c,g). At high magni®cation

(Figure 6h), it was evident that the boundary between the

granules and phytoglycogen was not well de®ned.

The phytoglycogen content of adjacent cells was vari-

able, particularly in young endosperm of Risù 17

(Figure 6b,c). Many cells contained little or no phytoglyco-

gen. The phytoglycogen-containing cells were randomly

dispersed throughout the endosperm. Within a cell, there

was variation between plastids in phytoglycogen content.

Some plastids contained large amounts of phytoglycogen

and small amounts of starch whilst other plastids con-

tained more starch and relatively little phytoglycogen

(Figure 6e). To investigate whether this could have been

due to loss of phytoglycogen during ®xation or artefacts

due to the staining procedure, we compared endosperm

sections of the maize mutant sugary1 (kindly provided by

M. James and M. Parker) with barley sections that were

prepared and stained in the same way. In the maize

sections, there was more phytoglycogen and less starch

than in the barley sections. This is consistent with meas-

urements of the phytoglycogen and starch content of

sugary1 maize endosperm (55% and 14% (w/w), respect-

ively; Dinges et al., 2001). There was also less heterogen-

eity between cells and between plastids within cells in the

amounts of these materials in maize compared to barley.

This suggested that the lack of phytoglycogen in some of

the cells and plastids of the barley endosperm was not

likely to be due to loss during preparation but to real cell-

to-cell variation in phytoglycogen and starch content. The

underlying cause of this variation is not understood.

There is a single wave of granule initiation in the

mutants

To discover whether the total number of granules in the

endosperm as a whole was altered in the isoamylase

mutants, starch was extracted from endosperms of differ-

ent ages and the numbers of granules in samples of these

were estimated using a haemocytometer (Shannon et al.,

1996). In wild-type barley endosperms, A-type granules

initiate at approximately 5±10 days after anthesis (DAA)

followed approximately 10 days later by a second wave of

granule initiation that gives rise to the B-type granules.

Our measurements of granule number in Bomi show these

two waves of initiation (Figure 7). Before 20 DAA, almost

all of the granules were large and disc-shaped, which is

typical of A-type granules. After 20 DAA, there was a

sudden increase in granule number and we observed

small, spherical B-type granules as well as the larger, A-

type granules. In the mutant Risù 17, the number of

granules per endosperm was higher than in wild-type

endosperms before 20 DAA, showing that many more

granules initiate in the young endosperm of the mutant

than in the wild-type. The mean of the values shown in

Figure 7 for endosperms less than 20 DAA was

109.6 6 15.3 million (mean 6 SE, n = 11) for Risù 17 and

19.4 6 3.8 million (mean 6 SE, n = 11) for Bomi. However,

in the mutant, there was no second wave of initiation.

Thus, the total number of granules per endosperm in the

mutant in the later half of development (after 20 DAA) was

similar to that in the endosperm of the wild-type. A

second, independent experiment on a separately grown

batch of plants gave results that showed the same trends

as those shown in Figure 7.

Discussion

In many respects, the Risù 17 and Notch-2 mutants are

similar to other cereal isoamylase mutants. They lack

isoamylase activity and have a reduced starch content, an

increased sugar content, an altered storage-protein com-

position and shrivelled grains at maturity, and they

accumulate phytoglycogen (our data and those of

Bansal, 1970; Doll, 1983; Sood et al., 1992).

The cDNA sequences con®rmed that Risù 17 and Notch-

2 have mutations in the isoamylase gene, isa1 that abolish



function and are very likely to be responsible for the

phenotype. The gene from Risù 17 has a 872-bp deletion in

the coding region. In Notch-2, there is a single-base

substitution in a 5¢-intron splice consensus sequence

(converting GT to AT) that appears to interfere with the

normal splicing of an intron from the primary mRNA

transcript. As a result, the cDNA from Notch-2 has either a

72-bp insertion containing two in-frame stop codons or a

downstream stop codon introduced by a mis-splicing

event. In addition, the abundance of isa1 transcripts is

severely reduced in the mutants compared to the wild-

types. This could be due to the fact that the mutant

transcripts are unstable or are recognized by the plant as

aberrant and are therefore rapidly turned over.

As well as causing a decrease in starch content and an

accumulation of phytoglycogen, the mutations have a

profound effect on the structure, number and timing of

initiation of starch granules. As observed previously, in

both mutants, granules are smaller than A-type granules

from the wild-type and are irregularly shaped (Burgess

et al., 1982; Gautam et al., 1994; Sood et al., 1992; Tester

et al., 1993). In Risù 17, a single wave of granule initiation

occurs at the time in endosperm development when A-

type granules initiate in the wild-type. More than one

granule initiates per plastid in the mutant and these pack

together to form compound granules resembling those

found in normal rice and oat grains. In the early stages of

endosperm development (< 20 DAA), the total number of

Figure 4a.

Figure 4b.



granules in the endosperm of Risù 17 is more than ®ve

times greater than in the wild-type, suggesting that the

mutation conditions an increase in granule initiations.

However, a second wave of granule initiations, giving rise

to the B-type granules, occurs in the wild-type. Mature

grains of Risù 17 thus contain a similar (our experiments)

or slightly reduced (71%; Tester et al., 1993) total number

of granules compared with Bomi. The lack of a second

wave of granule initiations in the mutants may be an

indirect effect due to the disruption to starch metabolism

in early endosperm development.

Phytoglycogen synthesis was previously thought to be

an inevitable consequence of the lack of isoamylase and

models to explain the function of isoamylase during starch

Figure 4c. Figure 4d.

Figure 4. Mutations in the isoamylase genes of Risù 17 and Notch-2.(a) Predicted amino acid sequences of cDNAs isolated from developing barley endosperms of wild-type (wtype), Bomi, Notch-2 and Risù 17. The putativetranslation start point has not been determined experimentally and may be any of the methionines located at positions 1, 3, 4 or 6. The deletion in Risù 17 isindicated by the dotted line. All deviations from the wild-type sequence are underlined and in bold. The sequence for Notch-2 shows the entire intronsequence although the protein would be expected to terminate at the ®rst stop codon shown. Notch-2 A and Notch-2B represent additional cDNAs resultingfrom mis-splicing of the intron beyond the normal 5¢ boundary. Amino acids numbers are indicated on the right hand margin and amino acids encoded bythe mutant transcripts that are different from those in the wild-type are indicated in bold. * = stop codon.(b) Partial cDNA sequence of isoamylase from Notch-2 compared with the genomic and cDNA sequences from Aegilops tauchii (At; GenBank AX031278 andAX031277, respectively). The intron sequence is shown in lower case, bold. The putative single base change in Notch-2 giving rise to mis-splicing of theintron is shown in white on black. Predicted amino acid sequences are shown below the cDNA sequences. Stop codons are indicated by stars.(c) Comparison of transcript abundance by Northern blot. Tracks are: B = Bomi, R = Riso17, NP = NP113 and N2 = Notch 2. RNA was extracted fromendosperms of grains of 12±20 mg FW and separated on a denaturing agarose gel. Upper panels: Northern blot hybridized with a full-length isoamylasecDNA. The approximate size of the wild-type isoamylase transcript is indicated. Lower panels: ethidium-bromide-stained gel photographed before blotting.(d )Genetic map location of the isoamylase gene in a Chebec 3 Harrington cross in relation to RFLP markers (ABC, ABG, BCD, CDO, KSU, pTAG and PSRpre®x), AFLP markers (AA/CAC) and SSR markers (HV and AWB). The full-length wild-type cDNA was used as a probe. Figures to the left of the map aregenetic distances between markers (cM). Loci were positioned using the `®nd best location' function of Map Manager QT (version b29ppc; Manly andCudmore, 1997).



synthesis have, at their core, ideas to explain the produc-

tion of this polymer. However, the present study of the

barley isoamylase mutants reveals a different picture.

Firstly, the fact that phytoglycogen does not accumulate in

all endosperm cells shows that loss of isoamylase activity

does not necessarily result in phytoglycogen synthesis.

Secondly, the initiation of abnormally large numbers of

granules early in endosperm development in all cells of

the mutants suggests that isoamylase plays a fundamental

role in granule initiation.

There is other evidence to suggest that the lack of

isoamylase affects starch synthesis without necessarily

leading to the production of phytoglycogen. Starch and

phytoglycogen accumulation was studied in developing

endosperms of maize containing 0±3 doses of the sugary1

mutant allele (Singletary et al., 1997). A statistically

signi®cant decrease in starch content was measured in

endosperms with 2 and 3 doses of the mutant allele. At

maturity, these endosperms had 71% and 57%, respect-

ively, of the starch content per endosperm of the wild-type.

However, phytoglycogen accumulation was only observed

in endosperms containing three doses of the mutant allele.

An effect of the lack of isoamylase activity on starch

granule number and/or shape has been reported for

sugary1 mutants of maize and rice. Unlike normal maize

endosperm which has simple granules, the granules in the

sugary1 mutant of maize are compound (Boyer et al.,

1977). In the sugary1 mutant of rice, numerous small

granules not observed in the wild-type are present in

addition to the normal compound granules (Kubo et al.,

1999). This implies that in maize and rice, as in barley,

there is an increase in the number of granule initiations per

plastid in mutants that lack isoamylase.

We have two suggestions for possible mechanisms

through which isoamylase activity might suppress granule

initiation. Firstly, the number of granules that are initiated

in a plastid might be determined by the concentration and/

or structure of soluble a-glucan in the stroma at the onset

of starch synthesis. High concentrations of soluble a-

glucans with a chain-length pro®le conducive to crystal-

lization may favour more nucleation events leading to a

greater number of granules. Zeeman et al. (1998) sug-

gested that isoamylase, together with other starch-degrad-

ing enzymes, might act to reduce the synthesis of soluble

a-glucans during the growth of the granule. Similarly,

isoamylase might contribute to the destruction of soluble

a-glucans at the time of granule initiation, thus limiting the

number of nucleation events leading to granules as well as

later, inhibiting the synthesis of phytoglycogen. In the

Figure 5. Scanning electron microscopy of barley grains.Mature (a) and frozen immature (b) grains were fractured to reveal thestarch granules within endosperm cells and viewed in a SEM. Themagni®cation is indicated by the scale bar. Note that panels are atdifferent magni®cations. Labels are A-type starch granules (A), B-typestarch granules (B), compound starch granule (cs), mutant starchgranules (s), plastid stroma (ps).



absence of isoamylase, more soluble a-glucans capable of

crystallization may accumulate in the stroma and this may

favour more nucleation events and hence, initiation of

more than the usual number of granules.

Secondly, isoamylase may destroy a speci®c primer

required for granule initiation. In animals and yeast, a self-

glucosylated protein primer, glycogenin, is required for the

synthesis of glycogen particles. Glycogen synthase cannot

elongate small oligosaccharides unless these are attached

to glycogenin (Alonso et al., 1995). Isoamylase might act

on some glycogenin-like protein required for granule or

polymer initiation in plants, preventing the initiation of

large numbers of granules and phytoglycogen particles by

cleaving off the associated a-glucan chains. Isoamylase

from Pseudomonas is able, in vitro, to cleave the a-glucan

chain from a primed glycogenin molecule (Lomarko et al.,

1992). Limit dextrinase, the other form of starch debranch-

ing enzyme found in plants, cannot cleave this protein-

glucan complex (Lomarko et al., 1992).

The role of glycogenin-like proteins in starch synthesis is

not established. Starch synthases can elongate small

malto-oligosaccharides and so it is not necessary to

postulate the presence of a priming protein for polymer

synthesis. However, plant genes encoding glycogenin-like

proteins have been reported (e.g. Roach and Skurat, 1997)

and various self-glycosylating proteins have been identi-

Figure 6. Light microscopy and TEM of thinof sections of developing endosperm.Developing grains (40±50 mg FW) were®xed in glutaraldehyde and osmiumtetroxide, embedded in epoxy resin andsectioned. Sections for light microscopy(left panels) and TEM (right panels) werestained with toluidine blue. Labels areprotein bodies (P), starch granules (S) andphytoglycogen (PG). The magni®cation isindicated by the scale bars.



®ed biochemically (Dhugga et al., 1997). The possibility

that granule initiation is controlled via the action of

isoamylase on a protein-glucan primer requires further

investigation.

Experimental procedures

Plants

Grains of NP113 were obtained from the National Small GrainsResearch Facility, Idaho, USA, Risù 17 from the Nordic Gene Bank,Alnarp, Sweden and all other grains from the John Innes CentreGermplasm Collection. Plants were grown in a greenhouse inindividual pots at a minimum temperature of 12°C, with supple-mentary lighting in winter.

Extraction of starch and soluble a-glucan

Glucans were extracted from individual mature grains by grindingto a ®ne powder in a pestle and mortar. The powder wassuspended in 5 ml ice-cold H2O, ground further and centrifuged at2500 g for 5 min at 4°C. The supernatant was removed and thepellet was resuspended in 2 ml ice-cold H2O, centrifuged againand the supernatant pooled with the previous supernatant. Thepellet was resuspended in 1 ml ice-cold H2O followed by 4 mlethanol and incubated on ice for 15±30 min. After centrifugationas before, the supernatant was discarded and the pellet wasresuspended in 12 ml H2O. Duplicate aliquots of the suspendedstarch were diluted 5-fold with H2O, autoclaved and assayed for a-glucan. The pooled supernatants containing the soluble a-glucanwere diluted 6-fold with aqueous methanol/KCl (75% (v/v)methanol, 1% (w/v) (KCl), incubated at 4°C for 12 h andcentrifuged as before. The supernatant was discarded and thepellet was resuspended in 12 ml H2O. Duplicate aliquots of thesupernatant were diluted 6-fold with H2O, autoclaved and assayedfor a-glucan.

The wavelength of maximum absorption of the a-glucan-iodinecomplex was determined as follows. Puri®ed a-glucans weredissolved by suspension at 20 mg ml±1 in 1 M NaOH and dilutedwith an equal volume of water. An aliquot (20±50 ml) of thedissolved a-glucan was added to a cuvette containing, in a ®nalvolume of 1 ml, 100 mM NaOH, 100 mM acetic acid and 100 mlLugol solution. The maximum absorbance (400±800 nm) wasdetermined relative to that of a sample identical except that itcontained no a-glucan.

For a-glucan chain-length analysis, a-glucans were extractedfrom grains (0.5±3.0 g FW) ground to a ®ne powder in liquidnitrogen in a pestle and mortar. The powder was suspendedin 2 ml 10% (w/v) perchloric acid, transferred to a 50-ml tubeand shaken on ice for 30 min. After centrifugation at 18000 g for30 min at 4°C, the supernatant (containing the soluble a-glucan)was retained and the pellet (containing the starch) was resus-pended in 10 ml H2O, ®ltered through muslin (washing throughwith additional H2O) and centrifuged at 2500 g for 10 min. Thegrey layer on top of the starch was removed and the starchwas washed successively in 20 g l±1 SDS (thrice) and H2O (twice).After proteinase K treatment to remove surface proteins (Rahmanet al., 1995), the starch was washed successively in 20 g l±1

SDS (once), H2O (twice) and ice-cold acetone (twice), and freeze-dried.

The soluble a-glucans were precipitated by the addition of 3.5vols methanol to the perchloric acid-soluble material, incubatedon ice overnight and the precipitate was collected by centrifuga-tion at 2500 g for 15 min at 4°C. The supernatant was discardedand the pellet was resuspended in 4 ml H2O, and subjected to re-precipitation with methanol as above. The supernatant wasdiscarded and the soluble-glucan pellets were freeze-dried.

Glucan assays

Glucans were assayed as glucose released after digestion withspeci®c glucosylases. Control reactions, in which the glucosy-lases were omitted, were also performed. Duplicate samples(0.5 ml) were each incubated with 0.5 ml 50 mM Na acetatepH 5.2, 2 U a-amylase and 10 U amyloglucosidase (enzymes fromRoche Diagnostics, Lewes, UK) at 25°C for 12±24 h. The sampleswere heated to 100°C for 2 min, centrifuged at 14000 g for 2 minand the supernatants were assayed spectrophotometrically forglucose according to Lowry (1972).

Determination of chain-length distribution of a-glucans

The methods used were based on those of O'Shea and Morell(1996) as described in Edwards et al. (1999).

Scanning electron microscopy

Mature barley grains were fractured transversely using a razorblade to initiate the fracture. The pieces were mounted onaluminium stubs using silver conducting paint. Samples weresputter-coated (Emitech Ltd, Ashford, UK) with a layer of goldapproximately 25 nm thick and examined and photographed in aLeica Stereoscan 360 SEM (LEO, Cambridge, UK).

Light microscopy and transmission electron microscopy

Tissue slices approximately 1.5 mm thick were cut from theendosperm of developing barley and ®xed for at least 4 h in 3%

Figure 7. The number of granules in developing endosperm.Starch granules were puri®ed from developing endosperms of Bomi (r)and Risù 17 (e) and suspended in water. The number of granules inreplicate aliquots of the suspension was estimated using ahaemacytometer. The age of the grains from which the endospermswere dissected is expressed as days after anthesis (DAA). Values are themeans of measurements from three replicate aliquots of a suspension.Each aliquot was sampled 10 times. Each sample containedapproximately 50±250 starch granules per 0.00625 mm3.



glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. The slices werewashed 3 times in buffer, cut into smaller pieces and ®xedovernight in 1% aqueous osmium tetroxide. The pieces weredehydrated in an ethanol series with 3 changes in 100% ethanoland transferred to acetone. Tissue was in®ltrated and embeddedin epoxy resin (Spurr's, Agar Scienti®c, Stansted, UK).

Sections 1 or 1.5 mm thick were stained with 1% (w/v) toluidineblue in 1% (w/v) borax, pH 11 and examined with an OlympusBX60 microscope (Olympus Optical, Japan) and recorded digitallywith AcQuis Bio software (Synoptics, Cambridge, UK).

Sections showing silver-gold interference colours were cutfrom embedded material with a diamond knife and collected oncopper grids. They were stained sequentially in uranyl acetate andReynold's lead citrate and examined and photographed in anelectron microscope (JEOL 1200EX/B).

Native gels

All procedures were carried out at 4±6°C. Developing endospermswere homogenized in approximately 10 vols extraction medium(100 mM MOPS pH 7.2, 10 mM EDTA, 50 ml l±1 ethanediol, 1 mM

DTT) in a pestle and mortar. The extract was centrifuged at28000 g for 5 min at 4°C. The resulting supernatant was added tosample loading medium (600 ml l±1 glycerol, 2 mg ml±1 bromo-phenol blue, 20 mM DTT) in a volume ratio of 1 : 5 (loading buffer:to extract).

Native gel electrophoresis was carried out according toLaemmli (1970), except that SDS was omitted from all solutionsand the separating gel contained 0.1% (w/v) acarbose (Glucobay100, Bayer plc, Berkshire, UK) to inhibit a-amylase activity. Theseparating gels consisted of 7.5% acrylamide, were 1 mm thickand contained potato amylopectin (1 mg ml±1, Sigma, Poole, UK)or red pullulan (10 mg ml±1, Megazyme International, CountyWicklow, Ireland).

After electrophoresis at 15 mA per gel and 4°C, native gelscontaining amylopectin were rinsed in medium A (100 mM MESpH 6.0, 5 mM DTT, 50 ml l±1 ethanediol, 0.1% (w/v) acarbose),incubated in this medium for 16 h at 37°C, rinsed brie¯y in waterand then stained with Lugol's solution. Native gels containing redpullulan were rinsed in medium A and then incubated in thismedium for 6±16 h at 37°C until bands were visible. Afterincubation, gels were soaked in 50 ml l±1 aqueous ethanol toenhance the contrast between the bands and the background.

Enzyme activities

All procedures were carried out at 4±6°C. Developing endospermswere dissected from grains of 45±55 mg FW and homogenized inapproximately 5 vols extraction medium (100 mM MOPS pH 7.2,5 mM DTT, 5 mM MgCl2, 5% (v/v) glycerol, 1% (w/v) BSA, 1% (w/v)(PVP) in a pestle and mortar followed by an all-glass homo-genizer. The extract was centrifuged at 28000 g for 10 min at 4°Cand the resulting supernatant was assayed. For total starchsynthase assays, the extract was not centrifuged.

For each enzyme, the activity reported was dependent upon thepresence in the assay of all of the appropriate substrates andcofactors and also upon extract concentration within the rangeused to make the measurements. The concentrations ofcomponents of each of the assays and their pH values wereoptimized to give the maximum rate using extracts of Bomi. Therate of the reaction was linear with respect to time for at least4 min in spectrophotometric assays and for at least 10 and 30 minin assays of sucrose synthase and starch synthase, respectively.

For starch-branching enzyme, activity was calculated from therate of reaction during the phase of the assay in which it waslinear with respect to time. Reaction mixtures were as follows:

Sucrose synthase. As in Craig et al. (1999) except that the bufferwas 82 mM AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulphonic acid) pH 9.0.

UDPG pyrophosphorylase. The assay contained, in 1 ml,100 mM HEPES pH 8.1, 2 mM MgCl2, 0.8 mM NAD, 0.8 mM UDP-glucose, 1 mM NaPPi, 2 U phosphoglucomutase (PGM), 5 UG6PDH and 10±50 ml of a 10-fold dilution of extract in extractionmedium. The reaction was initiated with sodium pyrophosphate(NaPPi) and monitored spectrophotometrically at 340 nm.

Fructokinase. As in Craig et al. (1999) except that 2.5 mM NAD,3 mM MgCl2 and 100 ml extract were used.

Glucokinase. As in Craig et al. (1999) except that 100 mM BicinepH 8.5, 2 mM NAD, 2.5 U G6PDH, 100 ml extract were used andPGM was omitted.

Phosphoglucomutase. The assay contained, in 1 ml, 50 mM

Bicine pH 8.0, 0.6 mM NAD, 6 mM glucose-1-phosphate, 2 UG6PDH, 5±10 ml extract.

Phosphoglucose isomerase. The assay contained, in 1 ml,100 mM glycyl glycine pH 8.4, 1 mM NAD, 10 mM fructose-6-phosphate, 4 U G6PDH, 5±10 ml extract.

ADPG pyrophosphorylase. As in Smith et al. (1989; assay 2b),except that 100 mM HEPES pH 7.9, 0.4 mM NAD, 1 mM ADP-glucose, 1.5 mM NaPPi and 5 U PGM were used.

Soluble starch synthase. As in Jenner et al. (1994), the resinmethod except that 0.5 mg potato amylopectin, 2 mM ADP[U-14C]glucose at 2.3 GBq mol±1 and 10 ml of extract were used.

Granule-bound starch synthase. As above for soluble starchsynthase except that extracts which had not been centrifuged toremove insoluble material (total extracts) as well as solubleextracts were assayed. The granule-bound activity was calculatedas the difference between the activities in the total and solubleextracts.

Alkaline inorganic pyrophosphatase. As in Gross and ap Rees(1986) except that 50 mM Bicine pH 8.9, 20 mM MgCl2, 1.25 mM

NaPPi and 50 ml extract were used.Starch-branching enzyme. As in Smith (1990), the phosphor-

ylase-stimulation assay using MES buffer except that assays wereprocessed using DOWEX rather than methanol/KCl essentiallyaccording to Jenner et al. (1994), the resin method. 0±10 ml of a 10-fold dilution of extract was used and activity was expressed ineach case relative to that in assays with no extract.

RNA extraction and cDNA synthesis

Total RNA was extracted from barley tissues using a commer-cially available phenol-guanidine isothiocyanate preparation(Trizol; Gibco BRL, Gaithersburg, MD, USA). Single strandedcDNA was prepared from 1 mg of total RNA with Thermoscriptreverse transcriptase (Gibco BRL) and either an oligo(dT)20 or theTRACE (Frohman et al., 1988) primer according to the manufac-turers instructions. The cDNA was treated with RNase H for20 min at 37°C prior to the PCR reaction.

Isolation of the 5¢ and 3¢ ends of the barley isoamylase

cDNA

A cDNA sequence for barley isoamylase was published by Sunet al. (1999) but comparison with published sequences from othercereals (Zea mays, Beatty et al., 1997; AF030882 and Triticumaestivum, Luetticke et al., 2000; AX010486) indicated that it was



not full length. The missing 5¢ end of the isoamylase transcriptwas obtained using nested PCR on cDNA prepared from total RNAfrom developing grain (Bomi) 10±13 days after fertilization. Theprimer sequences were 5¢-CCGATAAATAATCCCACCTCGC-3¢ and5¢-ATCACTGCCTTAGCATAAGGATCC-3¢ for the ®rst round of PCRand 5¢-GGCTGCAGGGCATGAAGATGATGGCCAT-3¢ with 5¢-GAACCTCCTCGCTCACCCTAT-3¢ for the second, nested round,with PCR conditions of 4 min at 94°C, followed by 35 cycles of1 min at 94°C, 1 min at 53°C, 1 min at 72°C and a ®nal extensiontime of 10 min at 72°C. The 3¢ end of the cDNA sequence wasobtained using a 3¢ RACE protocol where the ®rst round of PCRwas carried out with a gene-speci®c isoamylase primer 5¢-CCACTTATTGACATGATCAGC-3¢ and the RACE 3¢ primer 5¢-GACTC GAGTCGACATCG-3¢ (Frohman et al., 1988). A secondPCR reaction was carried out with a nested gene-speci®c primer5¢-CGTCAAGCTCATTGCTGAAGC-3¢ and RACE 3¢ with PCR condi-tions of 4 min at 94°C, followed by 35 cycles of 30 sec at 94°C,30 sec at 50°C, 2 min at 72°C and a ®nal extension time of 10 minat 72°C. PCR products to be analysed were cloned into the pGEM-T Easy vector (Promega, Madison WI, USA) and sequenced usingan ABI 3700 capillary sequencer.

Isolation of full-length isoamylase cDNAs

Primers for the isolation of full-length isa1 cDNAs from wild typeand mutant barley lines were designed to the 5¢ untranslatedregion upstream from the predicted translation start site and tothe 3¢ untranslated region at the 3¢ end of the transcript. A ®rst-round PCR reaction was carried out with the primers 5¢-CCGATAAATATCCCACCTCGC-3¢ and 5¢-CCGCCGAACGACTACA-TATAC-3¢ using the high-®delity Taq polymerase Elongase(Gibco, BRL) and PCR conditions of 45 sec at 94°C, followed by35 cycles of 45 sec at 94°C, 45 sec at 54°C, 3 min 30 sec at 72°Cand a ®nal extension time of 7 min at 72°C. A fully nested, secondround of PCR was carried out with 5¢-GGCTGCAGGGCATGAA-GATGATGGCCATGG-3¢ and 5¢-TCAAACATCAGGGCGTGATAC-AA-3¢ under the same PCR conditions. Putative full-length tran-scripts were cloned into the pGEM-T Easy vector (Promega). ThecDNA transcripts were fully sequenced in both directions using aset of overlapping primers on an ABI 3700 capillary sequencer.The resultant chromatograms were edited using Chromas(Technelysium, Helensvale, Queensland, Australia) software andanalysed with Genetic Computer Group (Madison, WI, USA)software in the ANGIS suite of programs at the University ofSydney.

Northern analysis

Approximately 10 mg of total RNA was separated on a 1% agarosedenaturing gel with size standards (Promega, Madison, USA).RNA was visualized with ethidium bromide under ultraviolet lightto ensure equal loading. RNA was transferred to Duralon nylonmembrane (Stratagene, La Jolla, USA) by capillary transfer andcrosslinked using UV light. A [32P]-labelled full-length isoamylasecDNA was synthesized by random priming, essentially asdescribed by Feinberg and Vogelstein (1983) and the membranewas probed as described by Banik et al., 1996). Autoradiographywas performed for 5 days at ± 80°C with X-ray ®lm and anintensifying screen.

Mapping the isoamylase gene, isa1

The probe DNA for Southern hybridization was radioactivelylabelled using standard methods. Hybridization methods were asdescribed in Rogowsky et al., 1991), except that both pre-hybridization and hybridization were carried out in the samesolution (0.9 M NaCl, 30 mM Pipes pH 6.8, 0.75 mM EDTA, 7.5%(w/v) dextran sulphate, 0.6% (w/v) BSA, 0.6% (w/v) Ficoll 400, 0.6%(w/v) polyvinyl-pyrollidone, 250 mg ml±1 denatured salmonsperm).

Counting starch granules

All procedures were carried out at 4±6°C. Developing endospermswere dissected from immature grains and homogenized inapproximately 50 vols extraction medium (50 mM HEPESpH 7.8, 10 mM EDTA, 10 mM DTT, 0.1 mg ml±1 and 0.1 mg ml±1

Proteinase K) in a pestle and mortar. The homogenate wasincubated at 37°C for 1 h, centrifuged at 28000 g for 5 min and thesupernatant discarded. The pellet was washed successively in 1 mlaliquots of 20 g l±1 SDS (twice), water (twice), 0.5 M NaCl, water,and the resulting starch preparation was resuspended in 1±10 mlof water. Three 200-ml or 1-ml aliquots of the suspension wereremoved for analysis. For each aliquot, the number of granulesper ml was estimated using a haemocytometer slide with a unitvolume of 0.00625 mm3. Starch granules stained with Lugolsolution, were diluted to approximately 50 granules per unitvolume and viewed under a light microscope. The precisenumber of granules per unit volume was determined 5 timeseach for two replicate dilutions of each sample of resuspendedstarch. These results were used to calculate the number ofgranules per endosperm.

Acknowledgements

The John Innes Centre is supported by a competitive strategicgrant from the Biotechnology and Biological Sciences ResearchCouncil (BBSRC), UK. The authors are extremely grateful to AlisonM. Smith for support, encouragement and useful discussionsthroughout the course of this work and for constructive criticismof the manuscript. Nicola Patron and Margaret Pallota are thankedfor DNA and RNA preparation, and chromosome mapping,respectively. Tamara Verhoeven thanks the BBSRC for a researchstudentship and Syngenta for additional ®nancial support. Workat the University of Adelaide was supported by grants (to GeoffFincher) from the Grains Research and Development Corporationof Australia and (to Kay Denyer) from the JIC/CSIRO/Waite fund.

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Accession numbers for GenBank database: AF490375, AF490376, AF490377



Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity

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