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A relationship between the amylose and lipid contents of starches from various mutants for amylose content in maize
Journal of Cereal Science (1991) 14 267-278
A Relationship Between the Amylose and Lipid Contents of Starches from Various Mutants for Amylose Content in Maize 1. B. SOUTH*, W. R. MORRISONt and O. E. NELSONt
* Allied Breweries Ltd,
R&D Laboratory, 107 Station Street, Burton-on-Trent, Staffs. DEl4 lBZ, UK. t University of Strathclyde, Department of Bioscience and Biotechnology, Glasgo'rl' Gl lSD, UK. and t University of Wisconsin-Madison, Laboratory of Genetics, Madison, Wisconsin 53706, US.A.
Received 1 March 1991 Starches were isolated from the endosperms of single and double mutants of maize and from endosperm gene dosage series of waxy and amylose extender mutants. In many of the starches, anomalous types of amylopectin were present, which gave false values for amylose (AM) content, measured either colorimetrically or by gel permeation chromatography (GPC) of native starches, although an accurate measure of long-chain cx-l,4-glucan (~AM) content was obtained by GPC of debranched starches. AM contents (colorimetric and by GPC) were correlated with lipid contents in the non-waxy starches and in each gene dosage series, but separate regression lines were required if the waxy starches were included. However, a single regression line described the excellent linear correlation between long-chain cx-l,4-glucan content (measured after debranching) and lipid content in the non-waxy starches (r = 0,974, n = 17) and in the full set of starches (r = 0,994, n = 22). These results demonstrate the nature of the amylose-lipid relationship in maize starches, although they provide no evidence of the possible biochemical role of lipids.
Introduction It has been known for some time that cereal starches are unusual in containing monoacyl lipids inside the granules, and that waxy (zero or low-amylose) starches have very low levels of lipids, while the high-amylose starches have more lipids than normal starches from the same species l . It has also been established that there is a good correlation between amylose and lipid contents (the so-called amylose-lipid relationship) in starches isolated from three cultivars of wheat 2 and from four genotypes of barley 3 at various stages of grain development. This relationship is also seen in F 2 progeny of crosses between waxy and high-amylose barley parents, in which 0-3 doses of the high-amylose gene were expressed in both amylose and lipid contents 4 • Since maize offers a unique selection of mutations that affect amylose contentS, it was used to provide yet another range of material to test the generality of the amylose-lipid relationship, as described in this paper.
0733-5210/91/060267 + 12 $03.00/0 II
© 1991 Academic Press Limited CEll. 14
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J. B. SOUTH ET AL.
TABLE I. Maize mutants used in this study Background W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A 0h43 0h43 0h43 Oh43
Description of mutations
Genotype n
wx/wx/wx wx/wx/Wx wx/Wx/Wx Wx/Wx/Wx ae/aejae ae/aejAe ae/Ae/Ae AejAejAe
+
wx su ae du du;wx su;wx du;su2 ae;du ae;su
+
Slt
su2 du;wx
Three doses of waxy gene Two doses of waxy gene One dose of waxy gene Normal Three doses amylose extender gene Two doses amylose extender gene One dose amylose extender gene Normal Normal (= WxjWx/Wx, and = Ae/Ae/Ae) Waxy (= wx/wx/wx) Sugary Amylose extender (= ae/ae/ae) DulI DulI-waxy Sugary-waxy Dull-sugary-2 Amylose extender--dull Amylose extender-sugary Normal dent com Sugary Sugary-2 Dull-waxy
n Since the starchy endosperm is triploid tissue, each gene occurs three times, but this is only written in full for the gene dosage series, where one, two or three of the genes are the mutant form.
Experimental
Materials Single and double mutants of maize in two genetic backgrounds, and endosperm gene dosage series of waxy and amylose-extender genes, as described in Table I, were from stocks grown at the University of Wisconsin-Madison.
Methods Prior to isolating starch, precautions were taken to minimize adsorption of surface lipid artefacts B by removing non-starch lipids from maize kernels. Thus, kernels were degermed with a scalpel, milled for 60 s in a coffee-grinder and then non-starch lipids were removed by extracting three times with diethyl ether-methanol [4: I (v/v), 1-2 mljg]. The lipid-extracted material was steeped overnight in 0'5 % (w jv) sodium metabisulphite at 37°C to soften the tissue without risk of annealing the starches. Starch was then released by gentle grinding in a pestle and mortar, and repeated centrifuging (3500 g, 10 min) of the starch slurry with removal of any protein visible on top of the starch layer. The starch was then washed in water ( x 3), acetone (x 2) and finally air-dried to give a free-flowing powder. Starch polysaccharides were analysed in three ways. Amylose (AM) content was determined calorimetrically in starch with lipids present (apparent amylose) and in lipid-free starch (total amylose), the difference (t1AM) representing a measure of amylose-lipid complexing under assay conditions'. In the second method, native starches were fractionated by gel permeation
269
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
Fraction
Fraction
n
IT
75%
25%
22%
78%
23%
77%
I
100
19%
54%
49%
21%
30%
43%
36%
21%
27%
I
200
I
100
m
I
200
Elution volume (mil
FIGURE 1. GPC elution profiles of native and debranched starches. Left-GPC of native starches on sepharose CL-2B, fraction I near void volume (c. 90 ml), fraction II from 100 ml to 200 ml approximately. Right-GPC elution profiles of debranched starches on Sepharose CL-6B, fraction I from void volume to 170 ml approximately, fraction II from 160 to 210 ml approximately, fraction III from 180 to 240 ml approximately. Top row W64A, centre row W64A ae;du, bottom row W64A ae.
270
J. B. SOUTH ET AL.
chromatography (GPC) on columns (16 mm diameter x 90 cm) of Sepharose CL-2B. Starch (I mg) dissolved in urea-dimethyl sulphoxide? was applied to the column, which was eluted with 0'25 M KOH, and a-glucan in the effluent detected using cysteine-sulphuric acid reagents. In the third method, starches were debranched with isoamylase j the debranched starches were then separated on a column of Sepharose CL-6B, and the effluent monitored as befores. Peaks in GPC chromatograms were resolved by curve-fitting (Fig. 1), photocopied and quantified by the 'cutand-weigh' method. Total lipids were extracted from starches with propanol-water [3: I (v Iv)] at 100°C, and converted to fatty acid methyl esters (FAME) for quantification by gas chromatography (GC)a-ll. A1iquots of lipid extract were also separated by thin-layer chromatography (TLC) into free fatty acid (FFA) and lysophospholipid (LPL) components, and quantified by GC using methyl heptadecanoate as internal standard 10. ll. Total phosphorus was determined directly 12 on samples of dry starch and on aliquots of lipid extract. The conversion factors used were : FFA = 0'95 x FAME: LPL = 16'3 x P; or 1'76 x FAME (FAME were prepared from FFA or LPL bands recovered from TLC plates 10 ). Coefficients of variation were < I % for colorimetric determination of AM, and < 2 % for all other analyses. Since only single samples of each maize genotype were available, more sophisticated analysis of error and variance was not appropriate.
Results
Starch polysaccharides For the purposes of this paper, maize starches are considered to consist of three major types of polysaccharides 13 • 14 • It is recognized that there can be considerable variation within each type, but it was beyond the scope of this study to investigate the polysaccharides in more detail. Most of the starches contain amylose (AM), likely to be variable in mean molecular weight (mol. wt) and polydispersity, and with different proportions of linear and branched molecules 14 • Their common features are that they have similar iodine binding capacities, their mol. wts are much lower than that of normal amylopectin (AP) so that they can be separated by GPC, and, on debranching, they give linear chains (average chain length, CL, > 270) much longer than those from AP (with the possible exception of the' superlong' chains described by Hizukuri 15 , which comprise < 10 % of the AP molecule in maize starch 18 ). All starches contain the second type of polysaccharide, high mol. wt AP, which is readily separated from AM by GPC, but some starches contain a third type of polysaccharide, low mol. wt (anomalous) AP, which appears to hl~.ve a mol, wt similar to that of AM14.17. 18. The iodine binding capacity of both types of A:R is normally quite low, but it can be enhanced considerably when external chains (typically ECL = 12-51) are lengthened by 5-15 glucosyl residues through the action of amylose extender (ae) and similar genes 1 ?, 18. Colorimetric assays (e.g. Morrison and Laignelet 7) use absorbances at Amax of the AM-polyiodide complex (630-640 nm) and a small correction for the absorbance of normal AP (A max 530-540 nm) is incorporated into the calibration. However, when anomalous types of AP with extended external chains are present (A max 560-590 nm) this correction is not sufficient and false high values for AM contents are obtained. In this study, three analytical methods were used to quantify starch polysaccharides (Table II). The iodometric (colorimetric) assay gave reliable values for AM content only
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
271
when AP had a normal low iodine-binding capacity, but this method was included because it is widely used and because flAM values give some indication of AM-lipid interactions. Low resolution GPC of native starches gave two main fractions. Fraction I was high mol. wt AP and fraction II was AM plus low mol. wt AP when present (Fig. 1, left). GPC of debranched starches gave three principal fractions-fraction I was long linear chains considered to be mainly from AM (i.e. any 'superlong' chains from AP were also in this fraction), fraction II comprised longer chains from AP and fraction III comprised shorter chains from AP (Fig. 1, right). Referring to the model of AP proposed by HizukurP5, debranched fraction II would include B 2 and B3 chains, while fraction III would include A and B1 chains. Low mol. wt (anomalous) AP was estimated as the difference between native starch fraction II and debranched starch fraction I. The results given in Table II are generally in good agreement with published information if allowance is made for genotypic and edaphic variation. W64A waxy starch (wx) contained no detectable AM, and the effect of increasing doses ofthe normal Wx gene showed that it was almost completely dominant over the wx gene, in agreement with previous reports 5. There were no anomalous types of AP in these starches. The two W64A high amylose (amylose extender, ae) starches both had about 40 % AM, 35 % low mol. wt AP and 25 % high mol. wt AP. Since the colorimetric values for AM were 53 and 63 %, it seems that both types of AP had extended chains with enhanced iodine binding capacities (cf. Baba et a/Y' 18). These results are in good agreement with the more detailed analyses of Baba et a/. 1?, 18 and Boyer et a/. 19 • In the ae/ Ae gene dosage series, the normal Ae gene showed almost complete dominance over the mutant ae gene, which was comparable with the Wx gene in the wx/Wx series. Very little starch was recovered from the sugary (su) mutants ofW64A and 0h43, and phytoglycogen was lost as water-soluble material during starch isolation. Since AM contents by colorimetric assay and by debranching were in good agreement, AP probably did not have extended chain lengths. The small amount of anomalous (low mol. wt) AP may have been due to residues of phytoglycogen, but is more probably anomalous material in view of the result for the su;wx double mutants discussed below. The 0h43 sugary-2 (su2) mutant contained more AM than the normal starch, and small differences in AM and AP contents given by the three assays were most likely due to experimental variation rather than to small increases in AP chain lengths. There was no evidence of low mol. wt AP. The AM content of W64A dull (du) starch was about 10 % higher than normal, and there was about 10 % low mol. wt AP, but no evidence of extended chains in the AP fractions. This agrees with the observations ofYeh et al. 20 in particular, and with other reports21, 22. In the double mutants homozygous for the waxy gene, the debranched starches gave three or more peaks in chromatograms, whereas only two were obtained from normal AP. In the case of du;wx, this agrees with a report by Fuwa 23 . Since no particular significance is attached to this aspect of starch structure with respect to the AM-lipid relationship, the chromatograms were divided arbitrarily into fractions II and III at points similar to those for normal AP. The wx gene is epistatic to both the du and su genes (i.e. in the double mutant endosperms-wx/wx/wx;du/du/du or wx/wx/wx; su/su/su-no amylose is produced, as is characteristic of wx/wx/wx endosperms 21 ; it 12
CER 14
IV
-J
IV
TABLE II. Percentage compositions of polysaccharides in maize starches AM, colorimetric
GPC, debranchedb
Low mol. wt
Genotype
Total
Apparent
Ii
I
II
I
II
III
APe
wxjwx/wx wxjwxjWx wxjWxjWx WxjWxjWx aejaejae aejaejAe aejAejAe AejAejAe
0 20·2 24·7 25·7 52·9 32·4 25·8 25·2 29·9 0·4 51·9 63-2 36·9 0 3'0 55·0 59·1 53-9 29'1 36·0 43·7 0
0 18·4 23-2 23·7 43·6 27·3 2I-l 20·8 27-3 1·0 40'2 59·4 32·0 0 3·0 51-8 56·5 59-6 27-8 32'5 40·4 0
0 1-8 1·5 1·5 6-6 5·2 4'7
100 75 75 75 19 23 35 66 75 100 42 23 55 100 52 47 22 27 76 60 61 100
0 25 25 25 81 77 65 34 25 0 58 77 45 0 48 53 78 73 24 40 39
0 23 25 26
25 22 23 16
75 55 52 58
0 2 0 -1
19 26 13 36 21 59 68 14 21 24 21 17 18 54
54 74 36 21 43 40 31 35 30 37 54 48 41 45
Background W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A Oh43 OM3 Oh43 Oh43
GPC, native"
+
wx SU
ae du du;wx su;wx du;su2 ae;du ae;su
+
su su2 du;wx
4-4
2·6 -0,6 11·7
3-8
4·9 0 0 3'2 2·6 5-4
1-3
3·5 3·3 0
"GPC on Sepharose CL-2B, 1= AP near void volume, II = AM + anomalous AP. bGPC on Sepharose CL-6B, I = linear AM, II and III chains from AP and anomalous AP. e Low mol. wt AP calculated from GPC, native (lI}--GPC, debranched (I).
a
-
27 0 51 43 36 I 1 51 49 49 25 35 41 1
-
-
~
-2 0 7 34 9 -I 47
(/)
-
2
29 24 -I 5 -2 -1
!=" 0
c::
>-l tI: ~ ....,
::....
f'
273
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH TABLE III. Lipid compositions of maize starches (mg/lOO g starch dry wt.) Genotype
FFA
LPL
Total lipid
Total, as FAME
wx/wx/wx wx/wx/Wx wx/Wx/Wx Wx/Wx/Wx ae/aejae ae/aejAe ae/ AejAe AejAejAe
5 285 339 336 382 361 373 392 334 6 n.d. 410 375 43 46 671 540 544 297 n.d. 635 24
4 192 229 240 588 407 329 295 297 3 n.d. 494 434 17 29 362 441 602 162 n.d. 262 19
10 477 568 576 970 768 702 687 631 9 n.d. 904 809 60 65 1033 981 1146 459 n.d. 897 43
7 475 502 489 730 603 565 591 541 9 n.d. 760 668 53 60 953 857 983 414 n.d. 861 58
Background W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A Oh43 Oh43 OM3 Oh43
+
wx su ae du du,'wx su;wx du;su2 ae;du ae;su
+
su su2 du;wx
Total starch phosphorus 3·0
In
16·0
11·7
46·8 26·3 22-3 25·1 21·0 2'5 65·2 3% 31·4 6·8 8·2 24·9 30·7 42·1 10·9 22·9 18·8 52
n.d., not determined.
is irrelevant whether at the du or su loci a non-mutant or mutant allele is present, although in these double mutant endosperms there was some evidence of interaction in the types of AP produced as shown by ope after debranching). Thus, W64A du;wx starches were exclusively AP, but with much more intermediate chain length material (debranched fraction II) than found in normal starches. This did not lead to an increase in the iodine binding capacity of the starches. Fuwa 23 has reported very similar results. W64A su ,'wx starch contained negligible AM (debranched fraction I), but there may have been some longer unbranched AP chain segments in the native AP (cf. Takeda et al. 16 ), which might account for the AM content measured colorimetrically. The most interesting feature is that nearly half the starch was low mol. wt AP. These results are quite different from previous reports, but no explanation can be offered at this time. W64A du,' su2 starch contained much more AM than W64A du, probably due to the presence of the su2 gene as shown in the Oh43 background 21 • A small increase in chain length of the debranched starch may account for the slightly higher colorimetric value for AM content. Unlike W64A du, there was negligible low mol. wt AP. W64A ae,' du and W64A ae; su starches were very similar to the single mutant W64A ae starch, indicating the major contribution of the ae gene. All contained a substantial 12·2
274
J. B. SOUTH ET AL.
proportion oflow mol. wt AP, and extended chain lengths in AP gave colorimetric AM values 10% (ae,odu) to 15% (ae,osu) above the true values (debranched fraction I), although less than the 20 % found for the single mutants. These results support the view& that the ae gene causes a reduction in branching enzyme activity during starch biosynthesis, and that there is little influence of the du or su genes, which, on their own, gave modified starches, as described above.
Starch lipids The compositions of the lipids in the various starches are given in Table III. Surface lipids adsorbed onto cereal starches tend to be FFA with less unsaturated fatty acids than the true starch Iipids 24, but great care was taken to avoid such artefacts in this study, and it is believed that the very low levels of lipids found in the zero-AM starches were true starch lipids. In the majority of the samples, FFA comprised 53-72 % of the total lipids, but only 45-47% in the high-AM lines ae,odu, and ae,osu ofW64A. In the W64A ae/Ae gene dosage series FFA contents increased in the order 39, 47, 53 and 59 % as AM contents increased, but in the wx/Wx gene dosage series FFA contents were constant at 58-60 %. In a previous study!, we found on average 62 %, FFA in starches from 20 normal and six high-AM cultivars. Starch LPL accounted for 75-85 % of the total phosphorus in the non-waxy starches and 7-15 % in the waxy starches, and non-lipid phosphorus was in the range 1,1-9,9 (mean 4,3) mg/IOO g for all starches. The fatty acid compositions of the lipids in the starches are given in Table IV. Fatty acids were mostly saturated in the wx lines, but were more unsaturated in the FFA of W64A SU,. WX, in the LPL of W64A du,. wx, and in the total lipids of Oh43 du,o wx. The lowest levels of saturation were seen in the FFA of the high-AM lines. However, there was no clear pattern in these results to which any biochemical significance could be given.
The amylose-lipid relationship In previous studies 1 • 2&, correlations were reported between AM contents measured colorimetrically and lipid contents, but it was noted that, with maize starches, better correlations were obtained using AM values from debranching and GPe, since this eliminated false values caused by extended chain lengths in some types of AP. With wheat and barley starches, debranching followed by GPe offers no advantages, since these starches contain only normal AP, but in retrospect, there could have been an advantage in using the debranching method with rice starches, since it is now known that the intermediate- and high-AM types have normal AM contents and variable levels of AP with an enhanced iodine binding capacity26. In the present study, there were excellent linear correlations between AM (colorimetric) and total lipids in the wx and in the ae gene dosage series. Taking all the non-waxy starches as a group, lipid content was linearly correlated (r = 0'906, n = 15),
275
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
TABLE IV. Fatty acid compositions of lipids in maize starches Background and genotype W64A wx/wx/wx W64A wx/wx/ Wx W64A wx/Wx/Wx W64A Wx/Wx/Wx W64A ae/ae/ae W64A ae/ae/Ae W64A ae/Ae/Ae W64A Ae/Ae/Ae W64A + W64A wx W64A ae W64A du W64A du;wx W64A su;wx W64A du;su2 W64A ae;du W64A ae;su Oh43+ Oh43 su2 OM3 du;wx
Fatty acid (wt %)A 16:0
18: 1
18:1
18:2
62,68,64 42,48,44 32, 50, 39 31,49,38 42, 33, 54 40, 30, 53 43,33,58 38,33,53 29,49,39 69,69,69 33,54,44 34,54,45 62,48,58 44, 70, 52 32,46,36 31,51,40 26,41,34 28, 55, 36 27, 37, 30 40,56,46
12,22,16 3, 3, 3 2, 1, 2 3, 3, 3 4, 4, 4 3, 3, 3 3, 3, 3 3, 3, 3 2, 3, 3 18,31,22 3, 4, 4 3, 4, 4 12, 18, 14 13, 12, 13 3, 3, 3 2, 4, 3 3, 2, 3 1, 2, I 2, I, 2 18, 9, 14
8,10, 9 4, 6, 5 5, 5, 5 5, 6, 5 7, 8, 7 6, 7, 7 6, 6, 5 6, 6, 6 6, 7, 7 1,-, 1 8, 8, 8 8, 6, 7 11,15,12 27, 5,20 8, 6, 7 9, 7, 8 7, 7, 7 9, 8, 9 14, 12, 3 18, 12, 16
18, -,11 51,54,59 58,41,52 58,42,52 44,53,33 47,56,36 51,55,38 51,55,38 59,39,48 12,-, 8 53,33,42 51,34,42 15, 19, 16 15,13,15 54,43,51 52,36,46 60,48,53 56,35,50 52,46,50 24,24,24
18:3
-,-,-,-,3,-, 3, 1, 3, 3, 3, 4, 3, 3, 2, 2, 4, 2,
2 2 1 1 1 1
3 -,-,3, 1, 2 4, 1, 2
-,-,-
-,-,3, 2, 3 3, 2, 3 4, 2, 3 6,-, 4 5, 4, 5
~,-,-
AMean values are given in the order FFA, LPL, total lipids for each starch. There was insufficient material for analysis of W64A su and Oh 43 suo
but the regression line extrapolated to a value of 280 mg lipid/IOO g starch at zero AM, which was clearly unsatisfactory. Including the waxy starches, which represented a separate population, resulted in a high overall linear correlation (r = 0'959, n = 20), which was highly significant statistically (P < 0'001). Correlations using AM contents of native starches determined by GPC (fraction II) were similar for the non-waxy starches (r = 0'894, n = 12) and for the complete set (r = 0,830, n = 16), but were obviously misleading because this fraction included low mol. wt AP in several mutants. Since W64A su;wx was an extreme outlier, it was excluded from these regression calculations. When long linear ()(-1,4-glucan chains (~AM), determined by GPC of debranched starches, were used, there was an excellent linear correlation for the non-waxy starches (r = 0'974, P < 0'001, n = 12), and the regression extrapolated to zero AM gave a lipid content of - 0·8 mg/100 g starch, close to the values found for the waxy starches. Including the waxy starches (which were strictly a separate population) improved the correlation coefficient (0'994, P < 0'001, n = 17).
276
J. B. SOUTH ET AL.
Discussion The amylose-lipid relationship that was originally postulated from data for waxy, normal and high-AM starches of maize and barleyl is open to criticism because the samples represented separate populations in terms of AM content, and there was no correlation within any population. Much better evidence was obtained using starches from three wheat cultivars 2 and from four barley genotypes 3 at consecutive stages of grain and starch granule development, and from F 2 progeny of waxy and high-AM barley parents 4 • The results presented in this paper show that the relationship does apply to waxy and amylose extender gene dosage series in maize and to various maize mutants. The linear correlations with AM content, determined colorimetrically or by GPC of native starches, were good, despite the presence of anomalous types of AP that gave false values in several single and double mutants. However, when only the long linear O:-I,4-glucan chain data were used, the experimental points gave an exceptionally high linear correlation coefficient, which is strong evidence that lipid content is in some way intimately related to long chain a-I,4-g1ucan content. In a previous paper, a selection of rice starches was examined, but no relationship between AM content (colorimetric) and lipid content was found l . It has been shown recently that intermediate and high-AM rice starches have only 15-19 % AM, and that the remainder of the iodine binding capacity and colour is attributable to long unbranched chain segments in AP 26 • It is possible, therefore, that, if these starches were re-examined using debranching and GPC to determine long linear a-I ,4-glucan content, a better relationship might be found. Recent work 27 in the authors' laboratory has revealed another important effect. Four barley genotypes were grown in controlled environment chambers at 10, 15 and 20°C. In the waxy and the two normal genotypes, AM content was relatively unaffected by temperature, but lipid content showed a strong positive relationship with ambient temperature. Thus, ambient temperature in the field may be expected to disturb the relationship in other cereals if they respond in a similar manner. There is evidence that this may, indeed, happen in rice 28 • The biochemical significance of lipids in cereal starches has been discussed, but is still obscure 24 • It would be convenient to assume that the lipids participate somehow in the biosynthesis of amylose, but several considerations cast doubt on such a simple explanation. First, the effects of temperature on barley, discussed above, show that AM-lipid stoichiometry can be substantially perturbed with no effect on AM content. Second, there is enough lipid in mature non-waxy cereal starches to form lipid-saturated complexes 29 with about 3(}-35 % of the amylose. Given a degree of polymerization of 80(}-1200 for cereal amyloses, the ratio of lipid to primer maltosaccharides (say, of DP < 6, i.e. 0·5-0·75 % of the final AM) is far too high for lipid to serve as an activator, as has been suggested 30 • 31. Third, similar considerations lead to the conclusion that there is too much lipid for it to serve as a terminator of AM chain elongation by forming a terminal AM-lipid complex32 • This concept is also incompatible with the observation that when normal cereal starches are heated in water they can leach a substantial proportion of water-soluble lipid-free amylose 33 • Fourth, the non-waxy cereal starches
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
277
have integral protein identified as granule-bound starch synthase (UDP-glucose starch glucosyltransferase)34.35, the enzyme primarily responsible for the synthesis of amylose. In the case of wheat starch, where integral proteins and lipids have been quantified separately36, there is about three-times as much lipid as protein, which is an improbable ratio if the native enzyme is a lipoprotein complex. Thus, we are no closer to understanding the precise location within the granule or the biochemical significance of lipids in cereal starches, except that it no longer seems likely that lipids have a direct role in AM synthesis. However, from the practical point of view, it can be stated with some confidence that lipid content will vary with long linear a-l,4glucan content and in response to environmental temperature in the field, and that these lipids will modify considerably the swelling and rheological properties of these starches when gelatinized 27 • This work was supported by a grant from the Agricultural and Food Research Council. Dr R. F. Tester and Mrs A. Grant gave valuable technical assistance in part of this work. Professor C. D. Boyer, Pennsylvania State University, placed mutants in the W64A background and Professor D. Glover, Purdue University, developed the Oh 43 series. These maize lines were subsequently grown for this study by O. E. Nelson.
References 1. Morrison, W. R., Milligan, T. P. and Azudin, M. N. J. Cereal Sci. 2 (1984) 257-271. 2. Morrison, W. R. and Gadan, H. J. Cereal Sci. 5 (1987) 263-275. 3. McDonald, A. M. 1., Stark, J. R., Morrison, W. R. and Ellis R. P. J. Cereal Sci. 13 (1991) 93-112. 4. Morrison, W. R. in 'Cereals in a European Context', (1 D. Morton, ed.), Ellis-Horwood, Chichester, U.K. (1987) pp 438-445. 5. Nelson, O. E. Adv. Cereal Sci. 3 (1980) 41-71. 6. Morrison, W. R., Starch 33 (1981) 408-410. 7. Morrison, W. R. and Laignelet, B., J. Cereal Sci. 1 (1983) 9-20. 8. Sargeant, J. G. Starch 34 (1982) 89-92. 9. Morrison, W. R. and Coventry, A. M., Starch 37 (1985) 83-87. 10. Morrison, W. R., Mann, D. 1., Wong, S. and Coventry, A. M., J. Sci. Food Agric. 26 (1975) 507-521. 11. Morrison, W. R., Tan, S. 1. and Hargin, K. D. J. Sci. Food Agric. 31 (1980) 329-340. 12. Morrison, W. R. Anal. Biochem. 7 (1964) 218-224. 13. Manners, D. J. Cereal Foods World 30 (1985) 461-467. 14. Morrison, W. R., Karkalas, J. in 'Methods in Plant Biochemistry', Vol. 2 (p. M. Dey and J. Harboume, eds.), Academic Press, London (1990) pp 323-352. IS. Hizukuri, S. Carbohydr. Res. 147 (1986) 342-347. 16. Takeda, Y., Suzuki, A. and Hizukuri, S. Starch 40 (1988) 132-135. 17. Baba, T., Demura, R., Hirota, M. and Arai, Y. Denpun Kagaku (1. Jpn Soc. Starch Sci.) 34 (1987) 196--202. 18. Baba, T., Uemura, R., Hirota, M. and Arai, Y. Denpun Kagaku (J. Jpn Soc. Starch Sci.) 34 (1987) 213-217. 19. Boyer, C. D., Garwood, D. 1. and Shannon, J. C. Starch 28 (1976) 405-410. 20. Yeh, J. Y., Garwood, D. 1. and Shannon, J. C. Starch 33 (1981) 222-230. 21. Ikawa, Y., Glover, D. V., Sugimoto, Y. and Fuwa, H. Starch 33 (1981) 9-13. 22. Inouchi, N., Glover, D. V. and Fuwa, H. Starch 39 (1987) 259-266. 23. Fuwa, H. D~npun Kagaku (J. Jpn Soc. Starch Sci.) 36 (1989) 25-33. 24. Morrison, W. R. J. Cereal Sci. 8 (1988) 1-15.25. Morrison, W. R. and Milligan, T. P. in 'Maize: Recent Progress in Chemistry and Technology' (G. E. Inglett, ed.), Academic Press, New York (1982) pp 1-8.
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26. Takeda, Y., Hizukuri, S. and Juliano, B. O. Carbohydr. Res. 168 (1987) 79-88. 27. Tester, R. F., South, J. B., Morrison, W. R. and Ellis, R. P. J. Cereal Sci. 13 (1991) 113-127. 28. Morrison, W. R. and Nasir Azudin, M. J. Cereal Sci. 5 (1987) 35--44. 29. Karkalas, J. and Raphaelides, S. Carbohydr. Res. 157 (1986) 215-234. 30. Vieweg, G. H. and de Fekete, M. A. R. Planta 129 (1976) 155-159. 31. Vieweg, G. H. and de Fekete, M. A. R. in 'Mechanisms of Saccharide Polymerization and Depolymer ization' (1. J. Marshall, ed.), Academic Press, New York (1980) pp 175-185. 32. Downton, W. J. S. and Hawker, J. S. Phytochemistry 14 (1975) 1259-1263. 33. Tester, R. F. and Morrison, W. R. Cereal Chern. 67 (1990) 551-557. 34. Greenwell, P. and Schofield, J. D. Cereal Chem. 63 (1986) 379-380. 35. Goldner, W. and Boyer, C. D. Starch 41 (1989) 250-254. 36. Sulaiman, B. D. and Morrison, W. R. J. Cereal Sci. 12 (1990) 53-61.
A Relationship Between the Amylose and Lipid Contents of Starches from Various Mutants for Amylose Content in Maize 1. B. SOUTH*, W. R. MORRISONt and O. E. NELSONt
* Allied Breweries Ltd,
R&D Laboratory, 107 Station Street, Burton-on-Trent, Staffs. DEl4 lBZ, UK. t University of Strathclyde, Department of Bioscience and Biotechnology, Glasgo'rl' Gl lSD, UK. and t University of Wisconsin-Madison, Laboratory of Genetics, Madison, Wisconsin 53706, US.A.
Received 1 March 1991 Starches were isolated from the endosperms of single and double mutants of maize and from endosperm gene dosage series of waxy and amylose extender mutants. In many of the starches, anomalous types of amylopectin were present, which gave false values for amylose (AM) content, measured either colorimetrically or by gel permeation chromatography (GPC) of native starches, although an accurate measure of long-chain cx-l,4-glucan (~AM) content was obtained by GPC of debranched starches. AM contents (colorimetric and by GPC) were correlated with lipid contents in the non-waxy starches and in each gene dosage series, but separate regression lines were required if the waxy starches were included. However, a single regression line described the excellent linear correlation between long-chain cx-l,4-glucan content (measured after debranching) and lipid content in the non-waxy starches (r = 0,974, n = 17) and in the full set of starches (r = 0,994, n = 22). These results demonstrate the nature of the amylose-lipid relationship in maize starches, although they provide no evidence of the possible biochemical role of lipids.
Introduction It has been known for some time that cereal starches are unusual in containing monoacyl lipids inside the granules, and that waxy (zero or low-amylose) starches have very low levels of lipids, while the high-amylose starches have more lipids than normal starches from the same species l . It has also been established that there is a good correlation between amylose and lipid contents (the so-called amylose-lipid relationship) in starches isolated from three cultivars of wheat 2 and from four genotypes of barley 3 at various stages of grain development. This relationship is also seen in F 2 progeny of crosses between waxy and high-amylose barley parents, in which 0-3 doses of the high-amylose gene were expressed in both amylose and lipid contents 4 • Since maize offers a unique selection of mutations that affect amylose contentS, it was used to provide yet another range of material to test the generality of the amylose-lipid relationship, as described in this paper.
0733-5210/91/060267 + 12 $03.00/0 II
© 1991 Academic Press Limited CEll. 14
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J. B. SOUTH ET AL.
TABLE I. Maize mutants used in this study Background W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A 0h43 0h43 0h43 Oh43
Description of mutations
Genotype n
wx/wx/wx wx/wx/Wx wx/Wx/Wx Wx/Wx/Wx ae/aejae ae/aejAe ae/Ae/Ae AejAejAe
+
wx su ae du du;wx su;wx du;su2 ae;du ae;su
+
Slt
su2 du;wx
Three doses of waxy gene Two doses of waxy gene One dose of waxy gene Normal Three doses amylose extender gene Two doses amylose extender gene One dose amylose extender gene Normal Normal (= WxjWx/Wx, and = Ae/Ae/Ae) Waxy (= wx/wx/wx) Sugary Amylose extender (= ae/ae/ae) DulI DulI-waxy Sugary-waxy Dull-sugary-2 Amylose extender--dull Amylose extender-sugary Normal dent com Sugary Sugary-2 Dull-waxy
n Since the starchy endosperm is triploid tissue, each gene occurs three times, but this is only written in full for the gene dosage series, where one, two or three of the genes are the mutant form.
Experimental
Materials Single and double mutants of maize in two genetic backgrounds, and endosperm gene dosage series of waxy and amylose-extender genes, as described in Table I, were from stocks grown at the University of Wisconsin-Madison.
Methods Prior to isolating starch, precautions were taken to minimize adsorption of surface lipid artefacts B by removing non-starch lipids from maize kernels. Thus, kernels were degermed with a scalpel, milled for 60 s in a coffee-grinder and then non-starch lipids were removed by extracting three times with diethyl ether-methanol [4: I (v/v), 1-2 mljg]. The lipid-extracted material was steeped overnight in 0'5 % (w jv) sodium metabisulphite at 37°C to soften the tissue without risk of annealing the starches. Starch was then released by gentle grinding in a pestle and mortar, and repeated centrifuging (3500 g, 10 min) of the starch slurry with removal of any protein visible on top of the starch layer. The starch was then washed in water ( x 3), acetone (x 2) and finally air-dried to give a free-flowing powder. Starch polysaccharides were analysed in three ways. Amylose (AM) content was determined calorimetrically in starch with lipids present (apparent amylose) and in lipid-free starch (total amylose), the difference (t1AM) representing a measure of amylose-lipid complexing under assay conditions'. In the second method, native starches were fractionated by gel permeation
269
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
Fraction
Fraction
n
IT
75%
25%
22%
78%
23%
77%
I
100
19%
54%
49%
21%
30%
43%
36%
21%
27%
I
200
I
100
m
I
200
Elution volume (mil
FIGURE 1. GPC elution profiles of native and debranched starches. Left-GPC of native starches on sepharose CL-2B, fraction I near void volume (c. 90 ml), fraction II from 100 ml to 200 ml approximately. Right-GPC elution profiles of debranched starches on Sepharose CL-6B, fraction I from void volume to 170 ml approximately, fraction II from 160 to 210 ml approximately, fraction III from 180 to 240 ml approximately. Top row W64A, centre row W64A ae;du, bottom row W64A ae.
270
J. B. SOUTH ET AL.
chromatography (GPC) on columns (16 mm diameter x 90 cm) of Sepharose CL-2B. Starch (I mg) dissolved in urea-dimethyl sulphoxide? was applied to the column, which was eluted with 0'25 M KOH, and a-glucan in the effluent detected using cysteine-sulphuric acid reagents. In the third method, starches were debranched with isoamylase j the debranched starches were then separated on a column of Sepharose CL-6B, and the effluent monitored as befores. Peaks in GPC chromatograms were resolved by curve-fitting (Fig. 1), photocopied and quantified by the 'cutand-weigh' method. Total lipids were extracted from starches with propanol-water [3: I (v Iv)] at 100°C, and converted to fatty acid methyl esters (FAME) for quantification by gas chromatography (GC)a-ll. A1iquots of lipid extract were also separated by thin-layer chromatography (TLC) into free fatty acid (FFA) and lysophospholipid (LPL) components, and quantified by GC using methyl heptadecanoate as internal standard 10. ll. Total phosphorus was determined directly 12 on samples of dry starch and on aliquots of lipid extract. The conversion factors used were : FFA = 0'95 x FAME: LPL = 16'3 x P; or 1'76 x FAME (FAME were prepared from FFA or LPL bands recovered from TLC plates 10 ). Coefficients of variation were < I % for colorimetric determination of AM, and < 2 % for all other analyses. Since only single samples of each maize genotype were available, more sophisticated analysis of error and variance was not appropriate.
Results
Starch polysaccharides For the purposes of this paper, maize starches are considered to consist of three major types of polysaccharides 13 • 14 • It is recognized that there can be considerable variation within each type, but it was beyond the scope of this study to investigate the polysaccharides in more detail. Most of the starches contain amylose (AM), likely to be variable in mean molecular weight (mol. wt) and polydispersity, and with different proportions of linear and branched molecules 14 • Their common features are that they have similar iodine binding capacities, their mol. wts are much lower than that of normal amylopectin (AP) so that they can be separated by GPC, and, on debranching, they give linear chains (average chain length, CL, > 270) much longer than those from AP (with the possible exception of the' superlong' chains described by Hizukuri 15 , which comprise < 10 % of the AP molecule in maize starch 18 ). All starches contain the second type of polysaccharide, high mol. wt AP, which is readily separated from AM by GPC, but some starches contain a third type of polysaccharide, low mol. wt (anomalous) AP, which appears to hl~.ve a mol, wt similar to that of AM14.17. 18. The iodine binding capacity of both types of A:R is normally quite low, but it can be enhanced considerably when external chains (typically ECL = 12-51) are lengthened by 5-15 glucosyl residues through the action of amylose extender (ae) and similar genes 1 ?, 18. Colorimetric assays (e.g. Morrison and Laignelet 7) use absorbances at Amax of the AM-polyiodide complex (630-640 nm) and a small correction for the absorbance of normal AP (A max 530-540 nm) is incorporated into the calibration. However, when anomalous types of AP with extended external chains are present (A max 560-590 nm) this correction is not sufficient and false high values for AM contents are obtained. In this study, three analytical methods were used to quantify starch polysaccharides (Table II). The iodometric (colorimetric) assay gave reliable values for AM content only
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
271
when AP had a normal low iodine-binding capacity, but this method was included because it is widely used and because flAM values give some indication of AM-lipid interactions. Low resolution GPC of native starches gave two main fractions. Fraction I was high mol. wt AP and fraction II was AM plus low mol. wt AP when present (Fig. 1, left). GPC of debranched starches gave three principal fractions-fraction I was long linear chains considered to be mainly from AM (i.e. any 'superlong' chains from AP were also in this fraction), fraction II comprised longer chains from AP and fraction III comprised shorter chains from AP (Fig. 1, right). Referring to the model of AP proposed by HizukurP5, debranched fraction II would include B 2 and B3 chains, while fraction III would include A and B1 chains. Low mol. wt (anomalous) AP was estimated as the difference between native starch fraction II and debranched starch fraction I. The results given in Table II are generally in good agreement with published information if allowance is made for genotypic and edaphic variation. W64A waxy starch (wx) contained no detectable AM, and the effect of increasing doses ofthe normal Wx gene showed that it was almost completely dominant over the wx gene, in agreement with previous reports 5. There were no anomalous types of AP in these starches. The two W64A high amylose (amylose extender, ae) starches both had about 40 % AM, 35 % low mol. wt AP and 25 % high mol. wt AP. Since the colorimetric values for AM were 53 and 63 %, it seems that both types of AP had extended chains with enhanced iodine binding capacities (cf. Baba et a/Y' 18). These results are in good agreement with the more detailed analyses of Baba et a/. 1?, 18 and Boyer et a/. 19 • In the ae/ Ae gene dosage series, the normal Ae gene showed almost complete dominance over the mutant ae gene, which was comparable with the Wx gene in the wx/Wx series. Very little starch was recovered from the sugary (su) mutants ofW64A and 0h43, and phytoglycogen was lost as water-soluble material during starch isolation. Since AM contents by colorimetric assay and by debranching were in good agreement, AP probably did not have extended chain lengths. The small amount of anomalous (low mol. wt) AP may have been due to residues of phytoglycogen, but is more probably anomalous material in view of the result for the su;wx double mutants discussed below. The 0h43 sugary-2 (su2) mutant contained more AM than the normal starch, and small differences in AM and AP contents given by the three assays were most likely due to experimental variation rather than to small increases in AP chain lengths. There was no evidence of low mol. wt AP. The AM content of W64A dull (du) starch was about 10 % higher than normal, and there was about 10 % low mol. wt AP, but no evidence of extended chains in the AP fractions. This agrees with the observations ofYeh et al. 20 in particular, and with other reports21, 22. In the double mutants homozygous for the waxy gene, the debranched starches gave three or more peaks in chromatograms, whereas only two were obtained from normal AP. In the case of du;wx, this agrees with a report by Fuwa 23 . Since no particular significance is attached to this aspect of starch structure with respect to the AM-lipid relationship, the chromatograms were divided arbitrarily into fractions II and III at points similar to those for normal AP. The wx gene is epistatic to both the du and su genes (i.e. in the double mutant endosperms-wx/wx/wx;du/du/du or wx/wx/wx; su/su/su-no amylose is produced, as is characteristic of wx/wx/wx endosperms 21 ; it 12
CER 14
IV
-J
IV
TABLE II. Percentage compositions of polysaccharides in maize starches AM, colorimetric
GPC, debranchedb
Low mol. wt
Genotype
Total
Apparent
Ii
I
II
I
II
III
APe
wxjwx/wx wxjwxjWx wxjWxjWx WxjWxjWx aejaejae aejaejAe aejAejAe AejAejAe
0 20·2 24·7 25·7 52·9 32·4 25·8 25·2 29·9 0·4 51·9 63-2 36·9 0 3'0 55·0 59·1 53-9 29'1 36·0 43·7 0
0 18·4 23-2 23·7 43·6 27·3 2I-l 20·8 27-3 1·0 40'2 59·4 32·0 0 3·0 51-8 56·5 59-6 27-8 32'5 40·4 0
0 1-8 1·5 1·5 6-6 5·2 4'7
100 75 75 75 19 23 35 66 75 100 42 23 55 100 52 47 22 27 76 60 61 100
0 25 25 25 81 77 65 34 25 0 58 77 45 0 48 53 78 73 24 40 39
0 23 25 26
25 22 23 16
75 55 52 58
0 2 0 -1
19 26 13 36 21 59 68 14 21 24 21 17 18 54
54 74 36 21 43 40 31 35 30 37 54 48 41 45
Background W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A Oh43 OM3 Oh43 Oh43
GPC, native"
+
wx SU
ae du du;wx su;wx du;su2 ae;du ae;su
+
su su2 du;wx
4-4
2·6 -0,6 11·7
3-8
4·9 0 0 3'2 2·6 5-4
1-3
3·5 3·3 0
"GPC on Sepharose CL-2B, 1= AP near void volume, II = AM + anomalous AP. bGPC on Sepharose CL-6B, I = linear AM, II and III chains from AP and anomalous AP. e Low mol. wt AP calculated from GPC, native (lI}--GPC, debranched (I).
a
-
27 0 51 43 36 I 1 51 49 49 25 35 41 1
-
-
~
-2 0 7 34 9 -I 47
(/)
-
2
29 24 -I 5 -2 -1
!=" 0
c::
>-l tI: ~ ....,
::....
f'
273
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH TABLE III. Lipid compositions of maize starches (mg/lOO g starch dry wt.) Genotype
FFA
LPL
Total lipid
Total, as FAME
wx/wx/wx wx/wx/Wx wx/Wx/Wx Wx/Wx/Wx ae/aejae ae/aejAe ae/ AejAe AejAejAe
5 285 339 336 382 361 373 392 334 6 n.d. 410 375 43 46 671 540 544 297 n.d. 635 24
4 192 229 240 588 407 329 295 297 3 n.d. 494 434 17 29 362 441 602 162 n.d. 262 19
10 477 568 576 970 768 702 687 631 9 n.d. 904 809 60 65 1033 981 1146 459 n.d. 897 43
7 475 502 489 730 603 565 591 541 9 n.d. 760 668 53 60 953 857 983 414 n.d. 861 58
Background W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A W64A Oh43 Oh43 OM3 Oh43
+
wx su ae du du,'wx su;wx du;su2 ae;du ae;su
+
su su2 du;wx
Total starch phosphorus 3·0
In
16·0
11·7
46·8 26·3 22-3 25·1 21·0 2'5 65·2 3% 31·4 6·8 8·2 24·9 30·7 42·1 10·9 22·9 18·8 52
n.d., not determined.
is irrelevant whether at the du or su loci a non-mutant or mutant allele is present, although in these double mutant endosperms there was some evidence of interaction in the types of AP produced as shown by ope after debranching). Thus, W64A du;wx starches were exclusively AP, but with much more intermediate chain length material (debranched fraction II) than found in normal starches. This did not lead to an increase in the iodine binding capacity of the starches. Fuwa 23 has reported very similar results. W64A su ,'wx starch contained negligible AM (debranched fraction I), but there may have been some longer unbranched AP chain segments in the native AP (cf. Takeda et al. 16 ), which might account for the AM content measured colorimetrically. The most interesting feature is that nearly half the starch was low mol. wt AP. These results are quite different from previous reports, but no explanation can be offered at this time. W64A du,' su2 starch contained much more AM than W64A du, probably due to the presence of the su2 gene as shown in the Oh43 background 21 • A small increase in chain length of the debranched starch may account for the slightly higher colorimetric value for AM content. Unlike W64A du, there was negligible low mol. wt AP. W64A ae,' du and W64A ae; su starches were very similar to the single mutant W64A ae starch, indicating the major contribution of the ae gene. All contained a substantial 12·2
274
J. B. SOUTH ET AL.
proportion oflow mol. wt AP, and extended chain lengths in AP gave colorimetric AM values 10% (ae,odu) to 15% (ae,osu) above the true values (debranched fraction I), although less than the 20 % found for the single mutants. These results support the view& that the ae gene causes a reduction in branching enzyme activity during starch biosynthesis, and that there is little influence of the du or su genes, which, on their own, gave modified starches, as described above.
Starch lipids The compositions of the lipids in the various starches are given in Table III. Surface lipids adsorbed onto cereal starches tend to be FFA with less unsaturated fatty acids than the true starch Iipids 24, but great care was taken to avoid such artefacts in this study, and it is believed that the very low levels of lipids found in the zero-AM starches were true starch lipids. In the majority of the samples, FFA comprised 53-72 % of the total lipids, but only 45-47% in the high-AM lines ae,odu, and ae,osu ofW64A. In the W64A ae/Ae gene dosage series FFA contents increased in the order 39, 47, 53 and 59 % as AM contents increased, but in the wx/Wx gene dosage series FFA contents were constant at 58-60 %. In a previous study!, we found on average 62 %, FFA in starches from 20 normal and six high-AM cultivars. Starch LPL accounted for 75-85 % of the total phosphorus in the non-waxy starches and 7-15 % in the waxy starches, and non-lipid phosphorus was in the range 1,1-9,9 (mean 4,3) mg/IOO g for all starches. The fatty acid compositions of the lipids in the starches are given in Table IV. Fatty acids were mostly saturated in the wx lines, but were more unsaturated in the FFA of W64A SU,. WX, in the LPL of W64A du,. wx, and in the total lipids of Oh43 du,o wx. The lowest levels of saturation were seen in the FFA of the high-AM lines. However, there was no clear pattern in these results to which any biochemical significance could be given.
The amylose-lipid relationship In previous studies 1 • 2&, correlations were reported between AM contents measured colorimetrically and lipid contents, but it was noted that, with maize starches, better correlations were obtained using AM values from debranching and GPe, since this eliminated false values caused by extended chain lengths in some types of AP. With wheat and barley starches, debranching followed by GPe offers no advantages, since these starches contain only normal AP, but in retrospect, there could have been an advantage in using the debranching method with rice starches, since it is now known that the intermediate- and high-AM types have normal AM contents and variable levels of AP with an enhanced iodine binding capacity26. In the present study, there were excellent linear correlations between AM (colorimetric) and total lipids in the wx and in the ae gene dosage series. Taking all the non-waxy starches as a group, lipid content was linearly correlated (r = 0'906, n = 15),
275
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
TABLE IV. Fatty acid compositions of lipids in maize starches Background and genotype W64A wx/wx/wx W64A wx/wx/ Wx W64A wx/Wx/Wx W64A Wx/Wx/Wx W64A ae/ae/ae W64A ae/ae/Ae W64A ae/Ae/Ae W64A Ae/Ae/Ae W64A + W64A wx W64A ae W64A du W64A du;wx W64A su;wx W64A du;su2 W64A ae;du W64A ae;su Oh43+ Oh43 su2 OM3 du;wx
Fatty acid (wt %)A 16:0
18: 1
18:1
18:2
62,68,64 42,48,44 32, 50, 39 31,49,38 42, 33, 54 40, 30, 53 43,33,58 38,33,53 29,49,39 69,69,69 33,54,44 34,54,45 62,48,58 44, 70, 52 32,46,36 31,51,40 26,41,34 28, 55, 36 27, 37, 30 40,56,46
12,22,16 3, 3, 3 2, 1, 2 3, 3, 3 4, 4, 4 3, 3, 3 3, 3, 3 3, 3, 3 2, 3, 3 18,31,22 3, 4, 4 3, 4, 4 12, 18, 14 13, 12, 13 3, 3, 3 2, 4, 3 3, 2, 3 1, 2, I 2, I, 2 18, 9, 14
8,10, 9 4, 6, 5 5, 5, 5 5, 6, 5 7, 8, 7 6, 7, 7 6, 6, 5 6, 6, 6 6, 7, 7 1,-, 1 8, 8, 8 8, 6, 7 11,15,12 27, 5,20 8, 6, 7 9, 7, 8 7, 7, 7 9, 8, 9 14, 12, 3 18, 12, 16
18, -,11 51,54,59 58,41,52 58,42,52 44,53,33 47,56,36 51,55,38 51,55,38 59,39,48 12,-, 8 53,33,42 51,34,42 15, 19, 16 15,13,15 54,43,51 52,36,46 60,48,53 56,35,50 52,46,50 24,24,24
18:3
-,-,-,-,3,-, 3, 1, 3, 3, 3, 4, 3, 3, 2, 2, 4, 2,
2 2 1 1 1 1
3 -,-,3, 1, 2 4, 1, 2
-,-,-
-,-,3, 2, 3 3, 2, 3 4, 2, 3 6,-, 4 5, 4, 5
~,-,-
AMean values are given in the order FFA, LPL, total lipids for each starch. There was insufficient material for analysis of W64A su and Oh 43 suo
but the regression line extrapolated to a value of 280 mg lipid/IOO g starch at zero AM, which was clearly unsatisfactory. Including the waxy starches, which represented a separate population, resulted in a high overall linear correlation (r = 0'959, n = 20), which was highly significant statistically (P < 0'001). Correlations using AM contents of native starches determined by GPC (fraction II) were similar for the non-waxy starches (r = 0'894, n = 12) and for the complete set (r = 0,830, n = 16), but were obviously misleading because this fraction included low mol. wt AP in several mutants. Since W64A su;wx was an extreme outlier, it was excluded from these regression calculations. When long linear ()(-1,4-glucan chains (~AM), determined by GPC of debranched starches, were used, there was an excellent linear correlation for the non-waxy starches (r = 0'974, P < 0'001, n = 12), and the regression extrapolated to zero AM gave a lipid content of - 0·8 mg/100 g starch, close to the values found for the waxy starches. Including the waxy starches (which were strictly a separate population) improved the correlation coefficient (0'994, P < 0'001, n = 17).
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Discussion The amylose-lipid relationship that was originally postulated from data for waxy, normal and high-AM starches of maize and barleyl is open to criticism because the samples represented separate populations in terms of AM content, and there was no correlation within any population. Much better evidence was obtained using starches from three wheat cultivars 2 and from four barley genotypes 3 at consecutive stages of grain and starch granule development, and from F 2 progeny of waxy and high-AM barley parents 4 • The results presented in this paper show that the relationship does apply to waxy and amylose extender gene dosage series in maize and to various maize mutants. The linear correlations with AM content, determined colorimetrically or by GPC of native starches, were good, despite the presence of anomalous types of AP that gave false values in several single and double mutants. However, when only the long linear O:-I,4-glucan chain data were used, the experimental points gave an exceptionally high linear correlation coefficient, which is strong evidence that lipid content is in some way intimately related to long chain a-I,4-g1ucan content. In a previous paper, a selection of rice starches was examined, but no relationship between AM content (colorimetric) and lipid content was found l . It has been shown recently that intermediate and high-AM rice starches have only 15-19 % AM, and that the remainder of the iodine binding capacity and colour is attributable to long unbranched chain segments in AP 26 • It is possible, therefore, that, if these starches were re-examined using debranching and GPC to determine long linear a-I ,4-glucan content, a better relationship might be found. Recent work 27 in the authors' laboratory has revealed another important effect. Four barley genotypes were grown in controlled environment chambers at 10, 15 and 20°C. In the waxy and the two normal genotypes, AM content was relatively unaffected by temperature, but lipid content showed a strong positive relationship with ambient temperature. Thus, ambient temperature in the field may be expected to disturb the relationship in other cereals if they respond in a similar manner. There is evidence that this may, indeed, happen in rice 28 • The biochemical significance of lipids in cereal starches has been discussed, but is still obscure 24 • It would be convenient to assume that the lipids participate somehow in the biosynthesis of amylose, but several considerations cast doubt on such a simple explanation. First, the effects of temperature on barley, discussed above, show that AM-lipid stoichiometry can be substantially perturbed with no effect on AM content. Second, there is enough lipid in mature non-waxy cereal starches to form lipid-saturated complexes 29 with about 3(}-35 % of the amylose. Given a degree of polymerization of 80(}-1200 for cereal amyloses, the ratio of lipid to primer maltosaccharides (say, of DP < 6, i.e. 0·5-0·75 % of the final AM) is far too high for lipid to serve as an activator, as has been suggested 30 • 31. Third, similar considerations lead to the conclusion that there is too much lipid for it to serve as a terminator of AM chain elongation by forming a terminal AM-lipid complex32 • This concept is also incompatible with the observation that when normal cereal starches are heated in water they can leach a substantial proportion of water-soluble lipid-free amylose 33 • Fourth, the non-waxy cereal starches
AMYLOSE-LIPID RELATIONSHIP IN MAIZE STARCH
277
have integral protein identified as granule-bound starch synthase (UDP-glucose starch glucosyltransferase)34.35, the enzyme primarily responsible for the synthesis of amylose. In the case of wheat starch, where integral proteins and lipids have been quantified separately36, there is about three-times as much lipid as protein, which is an improbable ratio if the native enzyme is a lipoprotein complex. Thus, we are no closer to understanding the precise location within the granule or the biochemical significance of lipids in cereal starches, except that it no longer seems likely that lipids have a direct role in AM synthesis. However, from the practical point of view, it can be stated with some confidence that lipid content will vary with long linear a-l,4glucan content and in response to environmental temperature in the field, and that these lipids will modify considerably the swelling and rheological properties of these starches when gelatinized 27 • This work was supported by a grant from the Agricultural and Food Research Council. Dr R. F. Tester and Mrs A. Grant gave valuable technical assistance in part of this work. Professor C. D. Boyer, Pennsylvania State University, placed mutants in the W64A background and Professor D. Glover, Purdue University, developed the Oh 43 series. These maize lines were subsequently grown for this study by O. E. Nelson.
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26. Takeda, Y., Hizukuri, S. and Juliano, B. O. Carbohydr. Res. 168 (1987) 79-88. 27. Tester, R. F., South, J. B., Morrison, W. R. and Ellis, R. P. J. Cereal Sci. 13 (1991) 113-127. 28. Morrison, W. R. and Nasir Azudin, M. J. Cereal Sci. 5 (1987) 35--44. 29. Karkalas, J. and Raphaelides, S. Carbohydr. Res. 157 (1986) 215-234. 30. Vieweg, G. H. and de Fekete, M. A. R. Planta 129 (1976) 155-159. 31. Vieweg, G. H. and de Fekete, M. A. R. in 'Mechanisms of Saccharide Polymerization and Depolymer ization' (1. J. Marshall, ed.), Academic Press, New York (1980) pp 175-185. 32. Downton, W. J. S. and Hawker, J. S. Phytochemistry 14 (1975) 1259-1263. 33. Tester, R. F. and Morrison, W. R. Cereal Chern. 67 (1990) 551-557. 34. Greenwell, P. and Schofield, J. D. Cereal Chem. 63 (1986) 379-380. 35. Goldner, W. and Boyer, C. D. Starch 41 (1989) 250-254. 36. Sulaiman, B. D. and Morrison, W. R. J. Cereal Sci. 12 (1990) 53-61.