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Non-cellulosic cell wall polysaccharides are subject to genotype × environment effects in sorghum (Sorghum bicolor) grain
Journal of Cereal Science 63 (2015) 64e71
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Non-cellulosic cell wall polysaccharides are subject to genotype environment effects in sorghum (Sorghum bicolor) grain Natalie S. Betts a, Glen P. Fox b, Alison M. Kelly c, Alan W. Cruickshank d, Jelle Lahnstein a, Marilyn Henderson a, David R. Jordan b, Rachel A. Burton a, * a
ARC Centre of Excellence in Plant Cell Walls, Waite Campus, University of Adelaide, Glen Osmond 5064, Australia Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Centre for Nutrition and Food Science, Queensland 4350, Australia Department of Agriculture, Fisheries and Forestry Queensland, Leslie Research Facility, Queensland 4350, Australia d Department of Agriculture, Fisheries and Forestry Queensland, Hermitage Research Facility, Queensland 4072, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2014 Received in revised form 20 February 2015 Accepted 23 February 2015 Available online 1 April 2015
Sorghum is a staple food for half a billion people and, through growth on marginal land with minimal inputs, is an important source of feed, forage and increasingly, biofuel feedstock. Here we present information about non-cellulosic cell wall polysaccharides in a diverse set of cultivated and wild Sorghum bicolor grains. Sorghum grain contains predominantly starch (64e76%) but is relatively deficient in other polysaccharides present in wheat, oats and barley. Despite overall low quantities, sorghum germplasm exhibited a remarkable range in polysaccharide amount and structure. Total (1,3;1,4)-b-glucan ranged from 0.06 to 0.43% (w/w) whilst internal cellotriose:cellotetraose ratios ranged from 1.8 to 2.9:1. Arabinoxylan amounts fell between 1.5 and 3.6% (w/w) and the arabinose:xylose ratio, denoting arabinoxylan structure, ranged from 0.95 to 1.35. The distribution of these and other cell wall polysaccharides varied across grain tissues as assessed by electron microscopy. When ten genotypes were tested across five environmental sites, genotype (G) was the dominant source of variation for both (1,3;1,4)-b-glucan and arabinoxylan content (69e74%), with environment (E) responsible for 5e14%. There was a small G E effect for both polysaccharides. This study defines the amount and spatial distribution of polysaccharides and reveals a significant genetic influence on cell wall composition in sorghum grain. Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.
Keywords: Sorghum Grain GE Cell wall
1. Introduction An increasing global population and our changing climate have led to a concomitant decrease in arable land area. Sorghum bicolor (L.) Moench has long been used as a food and feed staple in Africa and India due to its ratoon habit and ability to grow in marginal areas with minimal inputs (Rooney, 2004), but it is also an
Abbreviations: A, arabinose; DP3, degree of polymerisation of 3 (cellotriose); DP4, degree of polymerisation of 4 (cellotetraose); E, environment; G, genotype; GAX, glucuronoarabinoxylan; G E, genotype environment; HPLC, high performance liquid chromatography; PAD, pulsed amperometric detection; X, xylose. * Corresponding author. Level 4 WIC Building, Waite Campus, The University of Adelaide, Urrbrae, SA 5064, Australia. Tel.: þ61 8 8313 1057; fax: þ61 8 8303 7116. E-mail addresses: [email protected] (N.S. Betts), [email protected] (G.P. Fox), [email protected] (A.M. Kelly), [email protected]. au (A.W. Cruickshank), [email protected] (J. Lahnstein), [email protected] (M. Henderson), [email protected] (D.R. Jordan), [email protected] (R.A. Burton). http://dx.doi.org/10.1016/j.jcs.2015.02.007 0733-5210/Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.
attractive crop for other purposes. It is commonly used to make European lager-style beers in areas where more traditional malting cereals are unavailable (Agu and Palmer, 1998). Sorghum's popularity among coeliac patients and health-conscious consumers is rising due to its gluten-free nature and 'ancient grains' appeal (Gelski, 2014); and it is a cheap replacement for rice in the pet food industry, worth over US$20 billion in the United States in 2013 (Euromonitor, 2013). The grains and vegetative parts of the plant may also be valuable as biofuel feedstocks (Rooney et al., 2007). An understanding of the polysaccharide composition of the grain will assist in future assessments of its nutritional and renewable feedstock potential. Sorghum spp. belong to the Poaceae and, like other cereals, the sorghum grain is predominantly carbohydrate. Starch comprises 55e75% of the dry grain weight (reviewed by Taylor and Emmambux, 2010), and cellulose constitutes another 3e4% (Bach Knudsen et al., 1988). However, where wheat, barley and oats contain upwards of 10% w/w non-cellulosic polysaccharides such as
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(1,3;1,4)-b-glucans and arabinoxylans (reviewed by Collins et al., 2010), sorghum grain contains only 3.4e7.3% w/w (Bach Knudsen et al., 1988; Verbruggen et al., 1993). The remaining grain mass is composed of protein (8e12%), fat, structural minerals and tannins (Bach Knudsen et al., 1988; U.S. Department of Agriculture (2013)). (1,3;1,4)-b-glucan is a chain of glucose moieties, joined predominantly with b(1 / 4) linkages. Scattered throughout the chain in a non-random yet non-repeating manner, are b(1 / 3) linkages that introduce kinks into the otherwise linear molecule. These kinks ensure the molecules cannot align with each other to form large, insoluble complexes like cellulose; instead, the molecules remain soluble, forming a gel-like matrix. Most commonly, the b(1 / 3) linkages occur after every three or four glucose residues. The ratio of these trisaccharides to tetrasaccharides (degree of polymerisation (DP) 3 and 4, respectively) within the molecule gives an indication of its solubility, which is considered to increase as the DP3:DP4 ratio approaches 1:1 (Burton and Fincher, 2012). The amount of (1,3;1,4)-b-glucan in wholegrain flours varies widely between cereal species, ranging from 4 to 10% (w/w) in barley and oats; around 1% (w/w) in wheat and maize; down to approximately 0.1% (w/w) in sorghum and rice (Collins et al., 2010; Niba and Hoffman, 2003). There has been little focus on characterising (1,3;1,4)-b-glucan from whole sorghum grain. Previous researchers have isolated only small molecules from waterinsoluble endosperm and grain fractions (Ramesh and Tharanathan, 1998; Verbruggen et al., 1993; Woolard et al., 1976); or have looked at only a single, unidentified sorghum variety (Niba and Hoffman, 2003). There have been no previous reports of DP3:DP4 ratio of (1,3;1,4)-b-glucan in sorghum grain: in other species, the DP3:DP4 ratio in oats is 1.8e2.4:1; in barley and rye is 2.7e3.3:1; and in wheat is 3.0e4.5:1 (reviewed by Collins et al., 2010). Arabinoxylan consists of a backbone of b(1 / 4) linked xylose residues, substituted on the O2 and/or O3 atoms with arabinose moieties. In some tissues, these xylan backbones may be further decorated with glucuronic acid residues, creating glucuronoarabinoxylan (GAX). The size and structure of the heteroxylan molecule will determine its solubility, but generally arabinoxylan is less soluble than (1,3;1,4)-b-glucan. Arabinoxylan comprises 4e9% w/w of the whole grain in wheat, but this proportion is much lower in rice and maize, at around 2e5% w/w (Collins et al., 2010). The structure of sorghum GAX from water-insoluble endosperm fractions has been well characterised (Verbruggen et al., 1998a, 1995, 1993, 1998b). It is highly substituted compared with wheat and barley and has complex backbone substituents of arabinose and glucuronic acids. Unusually in a cereal, some of these substituents appear to be short arabinan chains (Verbruggen et al., 1995, 1998b). Estimates of arabinoxylan amount in total grain range from 2.0 to 2.8% (w/w) (inferred from Bach Knudsen et al., 1988; Verbruggen et al., 1993). Other agronomically important cereals, such as wheat, barley and rice, have well-characterised cell walls, especially in the grain that is so important for human use. Increased fibre in human diets has significant health benefits, reducing the incidence and severity of diabetes, colorectal cancers, cardiovascular and inflammatory diseases. Likewise, higher levels of soluble polysaccharides, such as (1,3;1,4)-b-glucan, in a biofuel feedstock improves saccharification to provide more fermentable sugars for conversion into bioethanol; whereas lower amounts of viscous polysaccharides are preferred for brewing end uses. Characterisation of cell wall polysaccharides in sorghum whole grain is thus of vital importance in understanding their effect on human and animal nutrition and biofuel generation. Here, we present a systematic analysis of nutritionally and energetically important non-cellulosic polysaccharides in a diverse
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range of S. bicolor grains, and examine how environmental conditions can affect (1,3;1,4)-b-glucan and arabinoxylan accumulation and structure. 2. Materials and methods 2.1. Sorghum growth and harvesting A diverse set of sorghum genotypes (see Table A.1) were grown at the Hermitage Research Facility, Queensland, Australia. The plants were grown in a vertisol soil with ample water, row spacing of 1 m and within-row spacing of 0.2e0.5 m. The grain selfpollinated and ripened under paper bags to ensure purity. A smaller set of ten genotypes were grown in five environments (five different locations in two different seasons; see Tables A.2 and A.3 for details of weather, site location and approximate harvest date). All were sown with row spacing of 1 m and within-row spacing of 0.2e0.5 m. Grain was hand-harvested when the latestmaturing genotypes were mature. Grain was manually de-glumed before grinding in a dental amalgam mixer to a fine flour. 2.2. (1,3;1,4)-b-glucan and starch assays (1,3;1,4)-b-glucan was measured on 25 mg sorghum flour using a scaled down version of the standard method developed for cereal grains (Burton et al., 2011). A portion of the lichenase digest was retained before b-glucosidase treatment for DP3:DP4 analysis. Starch was measured on 20 mg sorghum flour using a small scale version of the Megazyme Total Starch Assay (amyloglucosidase/a-amylase method; Burton et al., 2011). 2.3. Monosaccharide analysis Polysaccharides were quantified as described in Burton et al. (2011). This method is considered to fully hydrolyse noncellulosic polysaccharides, although estimates of neutral sugar amount are slightly underestimated due to degradation during hydrolysis. Chromatography was performed using a Phenomenex Kinetex C18, 2.6 mm, 100 3 mm at 30 C on an Agilent 1200 LC. Using eluents A (10% acetonitrile, 40 mM ammonium acetate pH 6.8) and B (70% acetonitrile), a gradient of 8e16.5% B over 9.3 min at a flow rate of 0.8 mL/min was used to separate the PMP-monosaccharides. Sample peak areas were compared to calibration curves of monosaccharide standard solutions. 2.4. (1,3;1,4)-b-glucan oligosaccharide analysis Enzyme digests were purified on a solid phase extraction (SPE) cartridge packed with graphitized carbon (Varian Bond Elut Carbon 50 mg/1 mL columns). The oligosaccharides bound to the column were washed with 1 mL water, eluted with 55% acetonitrile, dried under vacuum, and dissolved in water. (1,3;1,4)-b-glucan-oligosaccharides DP3 and DP4 were separated using high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS-5000 chromatograph. Aliquots of 25 mL were injected onto a Dionex CarboPAC PA-200 column (3 250 mm) with guard (3 50 mm) kept at 30 C and operated at a flow rate of 0.5 mL/min. The eluents were (A) 0.1 M sodium hydroxide and (B) 0.1 M sodium hydroxide with 1 M sodium acetate. The gradient was 1e13.3% B over 14 min followed by a 1 min wash with 100% B and re-equilibration at 1% B. The detector was kept at 20 C and data collection was at 2 Hz, using the Gold Standard PAD waveform for carbohydrates. The
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integrated peak areas were compared with DP3 and DP4 standard curves. 2.5. Microscopy and antibody labelling Five varieties, all grown at the Narrabri trial site, were selected for microscopy: IS8525; BTx623; ICSV400; MR Buster; and SC49. Grain was sliced in half, then fixed, dehydrated in an ethanol series, infiltrated with LR White Resin and polymerized (Burton et al., 2011). Survey sections were stained with toluidine blue. Labelling was performed on thin sections and visualised under the CM100 transmission electron microscope (TEM) as previously described in Burton et al. (2011) with the following antibodies: (1,3;1,4)-bglucan using BG1; arabinoxylan using LM10 and LM11; cellulose using CBM3a; xyloglucan using LM15; and pectin using LM20. All antibodies were purchased from PlantProbes (Leeds, UK) except for BG1, which was purchased from Biosupplies (Australia). 2.6. Statistical design and analysis Twenty-seven S. bicolor genotypes were grown at five trial sites in northern New South Wales (NSW) and southern Queensland (Qld). In the 2009e2010 growing season, sites at Jimbour, Gatton, Narrabri and Liverpool Plains were used; in the 2010e2011 season, a single site at Tulloona was chosen. The sorghum genotypes were planted as a two replicate trial in a randomised complete block design. Grain from a diverse subset of ten genotypes was retained from each of the two replicate plots, forming the biological replicates in this study. A total of 95 grain samples were collected: two biological replicates of ten genotypes sourced from five locations, allowing for five missing samples. Approximately thirty grains from each sample were ground into flour, which was used for analysis. These ground flour samples were split into laboratory replicates and randomly assigned to batches for processing. A statistical analysis was conducted for genotype and environment effects on (1,3;1,4)-b-glucan, DP3:DP4 ratio, arabinoxylan and starch measurements. These data were analysed in a linear mixed model framework where environment (E) was fitted as a fixed effect and genotype (G) and G E effects were fitted as random terms. A term for variation between biological replicates was included as a random effect together with batch effects in the laboratory, and variation between laboratory replicates formed the residual variance term. Estimates of variance parameters were obtained using residual maximum likelihood (REML; Patterson and Thompson, 1971) and best linear unbiased predictions (BLUPS; Robinson, 1991) were obtained for G E effects. The program ASReml-R (Butler et al., 2007) was used to fit the linear model and form predictions. 2.7. Weather analysis Weather data, including daily rainfall (mm), temperature maxima and minima ( C) and solar exposure (MJ m2) were sourced from the Australian Government Bureau of Meteorology (http:// www.bom.gov.au/climate/data/?ref¼ftr). Weather station locations were chosen as being the closest to the planting sites based on GPS coordinates (Table A.3). 3. Results 3.1. Quantification of the content and structure of non-cellulosic polysaccharides We chose a highly diverse range of 27 S. bicolor varieties, with origins from all over the world: six from Asia (China, Japan, India
and Russia); nine from Africa and the Middle East; seven from the United States; three from Australia, including two elite breeding lines; and two of unknown origin (Table A.1). In addition, we tested two related wild accessions: S. bicolor ssp. verticilliflorum from Australia and S. bicolor ssp. drummondii from North-East Africa. Grain morphology, including colour, size and shape, varied widely (see Fig. 1 for example grains). A broad range in (1,3;1,4)-b-glucan levels was detected, from 0.06 to 0.43% (w/w) (Fig. 1), with the mean across the 27 cultivated varieties of 0.20% (w/w). The sweet sorghum Rio had a slightly lower than average level of 0.12% (w/w). The two wild accessions had (1,3;1,4)-b-glucan levels toward the lower end of the range exhibited by the cultivated S. bicolor varieties. The structure of (1,3;1,4)-b-glucan molecules was assessed by measuring the ratio of cellotriose (DP3) to cellotetraose (DP4) generated using lichenase in the (1,3;1,4)-b-glucan assay. DP3:DP4 ratios also exhibited a broad range of values, from 1.8 to 2.9 across the 29 varieties (Fig. 1, Table A.4). There was no relationship between (1,3;1,4)-b-glucan amount and DP3:DP4 ratio (data not shown). Levels of neutral and acidic monosaccharides were consistent with previous reports (Bach Knudsen et al., 1988; Verbruggen et al., 1993): arabinose was the most prevalent, closely followed by xylose. Galactose and glucuronic acid were present at lower levels, while other sugars were only detected at levels that could not be accurately quantitated (Tables 1 and A.4). Arabinose levels averaged 1.3% (w/w), but ranged from 1.0 to 2.3% (w/w). Xylose levels averaged 1.1% (w/w) and ranged from 0.7 to 1.9% (w/w). Adding arabinose and xylose quantities to provide an estimate of arabinoxylan amount, values ranged from 1.5 to 3.6% w/w, which expands the range inferred from previous work (Bach Knudsen et al., 1988; Verbruggen et al., 1993). The resulting arabinose:xylose ratios of 1.0e1.3 agree with Verbruggen et al. (1995), although the presence of occasional arabinan side chains on the xylan backbones renders this ratio a less useful indicator of substitution (Verbruggen et al., 1998b). There was no detectable correlation between arabinoxylan amount or structure (data not shown). Starch levels ranged from 64 to 76% (w/w) (Table 1), consistent with previous reports (Bach Knudsen et al., 1988; Verbruggen et al., 1993) and other cereal grains (Burton and Fincher, 2012). Free glucose, measured in a parallel experiment without hydrolytic enzymes, ranged from 0.9 to 1.3% (w/w), which was approximately half the levels found in three varieties by Bach Knudsen et al. (1988). 3.2. Microscopic analysis of non-cellulosic polysaccharides in sorghum grain Antibody labelling of grain sections confirmed low levels of (1,3;1,4)-b-glucan and arabinoxylan in cell walls of different tissues of the grain (Fig. 2). Labelling of (1,3;1,4)-b-glucan appeared evenly throughout cell walls of the testa, aleurone and endosperm (Fig. 2AeC). Arabinoxylans were screened with both LM10 and LM11 antibodies, which were previously reported unable to detect sorghum GAX (McCartney et al., 2005). Here, LM10, which detects xylan with no or low levels of arabinose substitution (McCartney et al., 2005), did not label any polysaccharides in the sorghum grain (data not shown). However LM11, which detects xylans with higher levels of arabinose substituents such as those found in wheat grains (McCartney et al., 2005), labelled cell walls in the testa, aleurone and endosperm (Fig. 2DeF) with occasional concentration of label in the middle lamella (Fig. 2F). Low levels of labelling were observed in testa, aleurone and endosperm walls with an anti-cellulose antibody (endosperm shown in 2G). Low levels of xyloglucan were also detected in the
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Fig. 1. (1,3;1,4)-b-glucan amounts in selected diverse sorghum lines. The image above the graph shows grain morphology. The number below the graph gives the DP3:DP4 ratio of the (1,3;1,4)-b-glucan in that line. For (1,3;1,4)-b-glucan content, presented as % w/w of the grain, all samples are the average of two assays except for LR2490-3, which was assayed three times.
aleurone and testa, but not the endosperm (aleurone shown in Fig. 2H). Labelling with LM20, an antibody that detects esterified homogalacturonan, revealed the presence of pectin in the cells walls and the middle lamella region of walls belonging to testa and endosperm, but not aleurone, cells (Fig. 2IeK). For reference, a survey section of sorghum grain stained with toluidine blue is shown in Fig. 2L. 3.3. G E effect on non-cellulosic polysaccharides Ten S. bicolor genotypes were compared across five trial sites in northern New South Wales (NSW) and southern Queensland (Qld).
Fig. A.1 shows the location for each site and Tables A.2 and A.3 give meteorological data for the growing season. Jimbour in particular was subject to high levels of rainfall in March, at the onset of grain fill, after an otherwise dry season. Tulloona had the highest solar exposure over March and April, with the highest average minima and maxima over the growing season. (1,3;1,4)-b-glucan amounts ranged from 0.07 to 0.41% (w/w), within the range previously observed within the 29 diverse lines (Table A.5). There was an obvious environmental effect: (1,3;1,4)-bglucan amounts were generally higher at the three NSW sites compared with the Qld sites, and the 2011 trial produced the highest (1,3;1,4)-b-glucan levels (Fig. 3A). Overall, 74% of the
Table 1 Non-cellulosic polysaccharides in grain of 29 diverse sorghum genotypes. Masses are given as % w/w ± standard deviation (SD); ratios are given as x:1. Component
Cultivated S. bicolor Average ± SD
(1,3;1,4)-b-glucan DP3:DP4 ratio (:1) Arabinose Xylose Galactose Glucuronic acid A:X ratio Starch Free glucose
0.20 2.51 1.35 1.12 0.36 0.33 1.21 70.4 1.01
± ± ± ± ± ± ± ± ±
0.10 0.31 0.33 0.30 0.05 0.08 0.07 3.8 0.09
ssp. verticilliflorum
ssp. drummondii
Minimum
Maximum
Average ± SD
Average ± SD
0.06 1.84 0.95 0.75 0.29 0.25 0.95 64.5 0.89
0.43 2.90 2.27 1.87 0.47 0.52 1.31 76.3 1.28
0.06 1.96 1.44 1.11 0.45 0.40 1.29 66.4 1.0
± ± ± ± ± ± ± ±
0.00 0.02 0.02 0.01 0.00 0.01 0.01 7.1
0.07 1.77 1.00 0.88 0.39 0.48 1.14 72.7 0.9
± ± ± ± ± ± ± ±
0.01 0.02 0.02 0.01 0.01 0.05 0.00 2.7
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Fig. 2. Microscopic analysis of cell wall polysaccharides in sorghum grain. All sections are taken from mature S. bicolor grain. Scale bars as marked. BTx623 testa (A), aleurone (B) and endosperm (C) labelled with BG1, revealing small amounts of (1,3;1,4)-b-glucan in the cell wall. BTx623 testa (D), aleurone (E) and endosperm (F) labelled with LM11, indicating the presence of arabinoxylan in the cell walls and middle lamella. (G) BTx623 endosperm labelled with CBM3a, showing small amounts of cellulose in the cell wall. (H) SC49 aleurone labelled with LM15, showing small amounts of xyloglucan in the cell wall. (I) BTx623 testa, (J) aleurone and (K) endosperm labelled with LM20, revealing small amounts of pectin in all of these tissues. (L) Cross-section of sorghum grain stained with toluidine blue, showing morphological features.
variation was attributable to genotype effects and 14% to the environment (Table 2). Amounts of arabinose and xylose, individually and cumulatively, were also within the ranges observed within the 29 diverse lines (Fig. 3B and Table A.6). Only 3.7% of the overall variation was attributable to the environment (Table 2). The various genotypes also reacted differently to environmental factors. Over 5% of the variation in (1,3;1,4)-b-glucan content was due to a G E interaction. At Jimbour, seven of the ten genotypes exhibited their lowest (1,3;1,4)-b-glucan amounts but QL12
reversed the trend and produced its equal highest amount for the sites in 2010 (Fig. 3A). Conversely, nine of the ten genotypes produced the most (1,3;1,4)-b-glucan at Tulloona in 2011, with the exception of K-34 (Fig. 3A). For arabinoxylan amounts, 3.7% of the total variation was due to a G E effect. Nine genotypes produced their lowest amounts at Gatton except for KS115, which was unaffected by the trial site location. The DP3:DP4 ratio observed at the five sites ranged from 1.9 to 3.0, which was just higher than ratios observed within the original 29 lines. This variation was almost entirely attributable to
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Fig. 3. G E effect on non-cellulosic polysaccharides in sorghum grain. (A) Average (1,3;1,4)-b-glucan amounts for each genotype at the 5 trial sites. For standard error (SE) values, see Table A.5. (B) Average arabinoxylan amounts for each genotype at the five trial sites. For standard error (SE) values, see Table A.6.
genotype, with almost no influence from environmental, G E or other factors (Table 2). The A:X ratio was also slightly higher, ranging from 1.1 to 1.3 for the G E lines but again, this variation was almost entirely determined by genotype (Table 2).
Starch levels for two genotypes were analysed at four planting sites in the 2009e2010 growing season. There were no significant differences in starch levels between the test genotypes or between the trial locations, so further testing was not performed (Fig. A.2). 4. Discussion
Table 2 Sources of variation for amount and structure of (1,3;1,4)-b-glucan in sorghum grains (% of total variation). Sample refers to differences between biological repeats of the same grain; Batch refers to differences between experimental batches; and Residual is unexplained experimental variation.
Genotype (G) Environment (E) GE Sample Batch and residual
(1,3; 1,4)-b-glucan
Arabinoxylan
Amount
DP3:DP4 ratio
Amount
A:X ratio
73.8 13.8 5.4 4.3 2.6
83.4 0.8 1.7 7.2 6.9
66.7 3.7 3.7 12.2 13.7
64.5 17.8 1.4 4.2 12.0
(1,3;1,4)-b-glucan content in grain from the sorghum varieties examined here ranges from 0.06 to 0.43% (w/w) (Fig. 1), which is consistent with the single finding of Niba and Hoffman (2003) who reported a level of 0.12% (w/w). Sorghum therefore appears to be more similar to wheat, rice and maize than to barley, oats and rye, whose (1,3;1,4)-b-glucan content can reach up to 20% (w/w) (Collins et al., 2010). The DP3:DP4 ratio also showed a wide range in values from 1.8 to 3.1:1, but these numbers are consistent with values from barley, oats and rye. The (1,3;1,4)-b-glucan in sorghum grain was also detected by the BG1 antibody that detects (1,3;1,4)-b-glucan in other grains, suggesting sorghum (1,3;1,4)-b-glucan is similar in
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structure to other cereal grains and therefore reasonably soluble in water (Fig. 2AeC). Previous reports of extracting extremely small (1,3;1,4)-b-glucan molecules (DP20e26) from sorghum grain that focus solely on grain fractions after extensive water extractions are therefore unlikely to represent the total (1,3;1,4)-b-glucan profile (Ramesh and Tharanathan, 1998; Woolard et al., 1976). The structure of heteroxylans in sorghum grain has been subject to much scrutiny (Bach Knudsen et al., 1988; Verbruggen et al., 1998a, 1995, 1993, 1998b). Again, Verbruggen and associates focussed on water-unextractable grain fractions, and it is likely they lost some soluble, less substituted forms of heteroxylan in their analyses. We observed labelling of arabinoxylan in the wall of endosperm, aleurone and testa cells with the LM11 antibody, which had not previously been successful in detecting highly-substituted sorghum GAX (41% xylose, 46% arabinose and 10% glucuronic acid; Fig. 2DeF; McCartney et al., 2005). Total amounts of arabinose and xylose in the grain were found to range from 1.5 to 3.6% (w/w); if glucuronic acid is added to these totals, the range becomes 2.0e4.6% (w/w) (Table A.4). By comparison, past research implies a narrower range of 2.0e2.8% (w/w) (Bach Knudsen et al., 1988; Verbruggen et al., 1993). Verbruggen et al. (1993) also found polishing the sorghum grain d removing the outside 20% of the grain by mass d decreased the non-starch polysaccharides in the grain by 70%. In contrast, our antibody labelling suggested (1,3;1,4)-b-glucan and arabinoxylan are distributed throughout the endosperm, aleurone and testa (Fig. 2AeF). However, we have found sorghum glumes, which are often attached to the grain, contain over 20% w/w xylose (unpublished data). Previous work on determining GAX structure assumed all xylose, arabinose and uronic acid moieties were associated with xylans. Here, TEM analysis detected xyloglucans and pectins, which contain uronic acids, in the cell walls of sorghum grain tissues (Fig. 2HeK). While xyloglucans and pectins are not usually reported in the cell walls of monocotyledonous plants, pectins have recently been found in rice and wheat endosperm (Chateigner-Boutin et al., 2014). Sorghum grain may therefore be more complex than previously supposed. While genotype had the largest influence on (1,3;1,4)-b-glucan and arabinoxylan accumulation in the grain, there was still a marked environmental and G E effect (Table 2). A similar hierarchy of effects was observed when genotypic, environmental and G E effects on sorghum grain popping ability were assessed (Rooney and Rooney, 2013). (1,3;1,4)-b-Glucan levels in sorghum were lower at Gatton and Jimbour, which both experienced more rainfall during grain filling than the other three sites (Fig. 3A and Table A.2). This result is consistent with reports that rain during barley grain filling is generally correlated with lower (1,3;1,4)-bglucan levels, which is desirable for malting and subsequent brewing (Zhang et al., 2001). Grain harvested at Gatton also contained the lowest arabinoxylan amounts (Fig. 3B). While in wheat, higher levels of arabinoxylan in the grain has been linked with heat and drought stress after flowering (Rakszegi et al., 2014 and references therein), there was no obvious weather-related effect at Gatton. Starch levels were stable across all environmental sites tested (Fig. A.2), indicating that sorghum would be a reliable crop in most climates to provide starch for human food, animal feed, brewing, or biofuel generation. Rooney (2004) reported little genetic variation exists in sorghum for polysaccharide content and that most variation comes from environmental factors. By contrast, our study showed there was a large genotypic component that governs (1,3;1,4)-b-glucan and arabinoxylan accumulation in sorghum grain, suggesting there may be more genotypic variation in wild races and landraces from diverse geographic origins than previously thought. With the
recent sequencing of the sorghum genome (Mace et al., 2013; Paterson et al., 2009), sorghum may provide a novel system to search for genes responsible for regulating the synthesis of cell wall polysaccharides. Acknowledgements Thanks to Geoffrey Fincher and Helen Collins for advice; Kuok Yap for assistance with starch assays and HPLC analysis; and Sally Norton (Department of Environment and Primary Industries) for information on sorghum accessions. This work was supported by grants from the Australian Research Council (CE110001007). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jcs.2015.02.007. References Agu, R.C., Palmer, G.H., 1998. A reassessment of sorghum for lager-beer brewing. Bioresour. Technol. 66, 253e261. Bach Knudsen, K., Munck, L., Eggum, B., 1988. Effect of cooking, pH and polyphenol level on carbohydrate composition and nutritional quality of a sorghum (Sorghum bicolor (L.) Moench) food, ugali. Br. J. Nutr. 59, 31e47. Burton, R., Fincher, G., 2012. Current challenges in cell wall biology in the cereals and grasses. Front. Plant Sci. 3. Burton, R.A., Collins, H.M., Kibble, N.A.J., Smith, J.A., Shirley, N.J., Jobling, S.A., Henderson, M., Singh, R.R., Pettolino, F., Wilson, S.M., Bird, A.R., Topping, D.L., Bacic, A., Fincher, G.B., 2011. Over-expression of specific HvCslF cellulose synthase-like genes in transgenic barley increases the levels of cell wall (1,3;1,4)-b-D-glucans and alters their fine structure. Plant Biotechnol. J. 9, 117e135. Butler, D., Cullis, B.R., Gilmour, A., Gogel, B., 2007. ASReml-R Reference Manual. Queensland Department of Primary Industries and Fisheries. Chateigner-Boutin, A.-L., Bouchet, B., Alvarado, C., Bakan, B., Guillon, F., 2014. The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution. PLoS ONE 9, e89620. Collins, H.M., Burton, R.A., Topping, D.L., Liao, M.-L., Bacic, A., Fincher, G.B., 2010. Variability in fine structures of noncellulosic cell wall polysaccharides from cereal grains: potential importance in human health and nutrition. Cereal Chem. 87, 272e282. Euromonitor, 2013. Pet Food Sales. Pet Food Institute. http://www.petfoodinstitu te.org/?page¼PetFoodSales. Gelski, J., 2014. Ancient Grains for Modern Trends. Food Business News. Mace, E.S., Tai, S., Gilding, E.K., Li, Y., Prentis, P.J., Bian, L., Campbell, B.C., Hu, W., re, C., Zhang, H., Hunt, C.H., Innes, D.J., Han, X., Cruickshank, A., Dai, C., Fre Wang, X., Shatte, T., Wang, M., Su, Z., Li, J., Lin, X., Godwin, I.D., Jordan, D.R., Wang, J., 2013. Whole-genome sequencing reveals untapped genetic potential in Africa's indigenous cereal crop sorghum. Nat. Commun. 4. McCartney, L., Marcus, S.E., Knox, J.P., 2005. Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J. Histochem. Cytochem. 53, 543e546. Niba, L.L., Hoffman, J., 2003. Resistant starch and b-glucan levels in grain sorghum (Sorghum bicolor M.) are influenced by soaking and autoclaving. Food Chem. 81, 113e118. Paterson, A.H., Bowers, J.E., Bruggmann, R., Dubchak, I., Grimwood, J., Gundlach, H., Haberer, G., Hellsten, U., Mitros, T., Poliakov, A., Schmutz, J., Spannagl, M., Tang, H., Wang, X., Wicker, T., Bharti, A.K., Chapman, J., Feltus, F.A., Gowik, U., Grigoriev, I.V., Lyons, E., Maher, C.A., Martis, M., Narechania, A., Otillar, R.P., Penning, B.W., Salamov, A.A., Wang, Y., Zhang, L., Carpita, N.C., Freeling, M., Gingle, A.R., Hash, C.T., Keller, B., Klein, P., Kresovich, S., McCann, M.C., Ming, R., Peterson, D.G., Mehboob ur, R., Ware, D., Westhoff, P., Mayer, K.F.X., Messing, J., Rokhsar, D.S., 2009. The Sorghum bicolor genome and the diversification of grasses. Nature 457, 551e556. Patterson, H.D., Thompson, R., 1971. Recovery of inter-block information when block sizes are unequal. Biometrika 58, 545e554. ng, L., Bedo }, Z., Veisz, O., Shewry, P.R., 2014. Rakszegi, M., Lovegrove, A., Balla, K., La Effect of heat and drought stress on the structure and composition of arabinoxylan and b-glucan in wheat grain. Carbohydr. Polym. 102, 557e565. Ramesh, H.P., Tharanathan, R.N., 1998. Structural characteristics of a mixed linkage b-D-glucan from sorghum (Sorghum bicolor). Carbohydr. Res. 308, 239e243. Robinson, G.K., 1991. That BLUP is a good thing: the estimation of random effects. Stat. Sci. 6, 15e32. Rooney, T.E., Rooney, W.L., 2013. Genotype and environment effects on the popping characteristics of grain sorghum. J. Crop Improv. 27, 460e468. Rooney, W.L., 2004. Sorghum improvementdintegrating traditional and new technology to produce improved genotypes. In: Sparks, D.L. (Ed.), Advances in Agronomy. Academic Press, UK, pp. 37e109.
N.S. Betts et al. / Journal of Cereal Science 63 (2015) 64e71 Rooney, W.L., Blumenthal, J., Bean, B., Mullet, J.E., 2007. Designing sorghum as a dedicated bioenergy feedstock. Biofuels, Bioprod. Biorefining 1, 147e157. Taylor, J.R.N., Emmambux, M.N., 2010. Developments in our understanding of sorghum polysaccharides and their health benefits. Cereal Chem. J. 87, 263e271. U.S. Department of Agriculture, 2013. A.R.S., USDA National Nutrient Database for Standard Reference, Release 26. Nutrient Data Laboratory Home Page. Verbruggen, M.A., Beldman, G., Voragen, A.G.J., 1995. The selective extraction of glucuronoarabinoxylans from sorghum endosperm cell walls using barium and potassium hydroxide solutions. J. Cereal Sci. 21, 271e282. Verbruggen, M.A., Beldman, G., Voragen, A.G.J., 1998a. Enzymic degradation of sorghum glucuronoarabinoxylans leading to tentative structures. Carbohydr. Res. 306, 275e282.
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Verbruggen, M.A., Beldman, G., Voragen, A.G.J., Hollemans, M., 1993. Water-unextractable cell wall material from sorghum: isolation and characterization. J. Cereal Sci. 17, 71e82. Verbruggen, M.A., Spronk, B.A., Schols, H.A., Beldman, G., Voragen, A.G.J., Thomas, J.R., Kamerling, J.P., Vliegenthart, J.F.G., 1998b. Structures of enzymically derived oligosaccharides from sorghum glucuronoarabinoxylan. Carbohydr. Res. 306, 265e274. Woolard, G.R., Rathbone, E.B., Novellie, L., 1976. A hemicellulosic b-D-glucan from the endosperm of sorghum grain. Carbohydr. Res. 51, 249e252. Zhang, G., Chen, J., Wang, J., Ding, S., 2001. Cultivar and environmental effects on (1/3,1/4)-b-D-glucan and protein content in malting barley. J. Cereal Sci. 34, 295e301.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Non-cellulosic cell wall polysaccharides are subject to genotype environment effects in sorghum (Sorghum bicolor) grain Natalie S. Betts a, Glen P. Fox b, Alison M. Kelly c, Alan W. Cruickshank d, Jelle Lahnstein a, Marilyn Henderson a, David R. Jordan b, Rachel A. Burton a, * a
ARC Centre of Excellence in Plant Cell Walls, Waite Campus, University of Adelaide, Glen Osmond 5064, Australia Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Centre for Nutrition and Food Science, Queensland 4350, Australia Department of Agriculture, Fisheries and Forestry Queensland, Leslie Research Facility, Queensland 4350, Australia d Department of Agriculture, Fisheries and Forestry Queensland, Hermitage Research Facility, Queensland 4072, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2014 Received in revised form 20 February 2015 Accepted 23 February 2015 Available online 1 April 2015
Sorghum is a staple food for half a billion people and, through growth on marginal land with minimal inputs, is an important source of feed, forage and increasingly, biofuel feedstock. Here we present information about non-cellulosic cell wall polysaccharides in a diverse set of cultivated and wild Sorghum bicolor grains. Sorghum grain contains predominantly starch (64e76%) but is relatively deficient in other polysaccharides present in wheat, oats and barley. Despite overall low quantities, sorghum germplasm exhibited a remarkable range in polysaccharide amount and structure. Total (1,3;1,4)-b-glucan ranged from 0.06 to 0.43% (w/w) whilst internal cellotriose:cellotetraose ratios ranged from 1.8 to 2.9:1. Arabinoxylan amounts fell between 1.5 and 3.6% (w/w) and the arabinose:xylose ratio, denoting arabinoxylan structure, ranged from 0.95 to 1.35. The distribution of these and other cell wall polysaccharides varied across grain tissues as assessed by electron microscopy. When ten genotypes were tested across five environmental sites, genotype (G) was the dominant source of variation for both (1,3;1,4)-b-glucan and arabinoxylan content (69e74%), with environment (E) responsible for 5e14%. There was a small G E effect for both polysaccharides. This study defines the amount and spatial distribution of polysaccharides and reveals a significant genetic influence on cell wall composition in sorghum grain. Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.
Keywords: Sorghum Grain GE Cell wall
1. Introduction An increasing global population and our changing climate have led to a concomitant decrease in arable land area. Sorghum bicolor (L.) Moench has long been used as a food and feed staple in Africa and India due to its ratoon habit and ability to grow in marginal areas with minimal inputs (Rooney, 2004), but it is also an
Abbreviations: A, arabinose; DP3, degree of polymerisation of 3 (cellotriose); DP4, degree of polymerisation of 4 (cellotetraose); E, environment; G, genotype; GAX, glucuronoarabinoxylan; G E, genotype environment; HPLC, high performance liquid chromatography; PAD, pulsed amperometric detection; X, xylose. * Corresponding author. Level 4 WIC Building, Waite Campus, The University of Adelaide, Urrbrae, SA 5064, Australia. Tel.: þ61 8 8313 1057; fax: þ61 8 8303 7116. E-mail addresses: [email protected] (N.S. Betts), [email protected] (G.P. Fox), [email protected] (A.M. Kelly), [email protected]. au (A.W. Cruickshank), [email protected] (J. Lahnstein), [email protected] (M. Henderson), [email protected] (D.R. Jordan), [email protected] (R.A. Burton). http://dx.doi.org/10.1016/j.jcs.2015.02.007 0733-5210/Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.
attractive crop for other purposes. It is commonly used to make European lager-style beers in areas where more traditional malting cereals are unavailable (Agu and Palmer, 1998). Sorghum's popularity among coeliac patients and health-conscious consumers is rising due to its gluten-free nature and 'ancient grains' appeal (Gelski, 2014); and it is a cheap replacement for rice in the pet food industry, worth over US$20 billion in the United States in 2013 (Euromonitor, 2013). The grains and vegetative parts of the plant may also be valuable as biofuel feedstocks (Rooney et al., 2007). An understanding of the polysaccharide composition of the grain will assist in future assessments of its nutritional and renewable feedstock potential. Sorghum spp. belong to the Poaceae and, like other cereals, the sorghum grain is predominantly carbohydrate. Starch comprises 55e75% of the dry grain weight (reviewed by Taylor and Emmambux, 2010), and cellulose constitutes another 3e4% (Bach Knudsen et al., 1988). However, where wheat, barley and oats contain upwards of 10% w/w non-cellulosic polysaccharides such as
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(1,3;1,4)-b-glucans and arabinoxylans (reviewed by Collins et al., 2010), sorghum grain contains only 3.4e7.3% w/w (Bach Knudsen et al., 1988; Verbruggen et al., 1993). The remaining grain mass is composed of protein (8e12%), fat, structural minerals and tannins (Bach Knudsen et al., 1988; U.S. Department of Agriculture (2013)). (1,3;1,4)-b-glucan is a chain of glucose moieties, joined predominantly with b(1 / 4) linkages. Scattered throughout the chain in a non-random yet non-repeating manner, are b(1 / 3) linkages that introduce kinks into the otherwise linear molecule. These kinks ensure the molecules cannot align with each other to form large, insoluble complexes like cellulose; instead, the molecules remain soluble, forming a gel-like matrix. Most commonly, the b(1 / 3) linkages occur after every three or four glucose residues. The ratio of these trisaccharides to tetrasaccharides (degree of polymerisation (DP) 3 and 4, respectively) within the molecule gives an indication of its solubility, which is considered to increase as the DP3:DP4 ratio approaches 1:1 (Burton and Fincher, 2012). The amount of (1,3;1,4)-b-glucan in wholegrain flours varies widely between cereal species, ranging from 4 to 10% (w/w) in barley and oats; around 1% (w/w) in wheat and maize; down to approximately 0.1% (w/w) in sorghum and rice (Collins et al., 2010; Niba and Hoffman, 2003). There has been little focus on characterising (1,3;1,4)-b-glucan from whole sorghum grain. Previous researchers have isolated only small molecules from waterinsoluble endosperm and grain fractions (Ramesh and Tharanathan, 1998; Verbruggen et al., 1993; Woolard et al., 1976); or have looked at only a single, unidentified sorghum variety (Niba and Hoffman, 2003). There have been no previous reports of DP3:DP4 ratio of (1,3;1,4)-b-glucan in sorghum grain: in other species, the DP3:DP4 ratio in oats is 1.8e2.4:1; in barley and rye is 2.7e3.3:1; and in wheat is 3.0e4.5:1 (reviewed by Collins et al., 2010). Arabinoxylan consists of a backbone of b(1 / 4) linked xylose residues, substituted on the O2 and/or O3 atoms with arabinose moieties. In some tissues, these xylan backbones may be further decorated with glucuronic acid residues, creating glucuronoarabinoxylan (GAX). The size and structure of the heteroxylan molecule will determine its solubility, but generally arabinoxylan is less soluble than (1,3;1,4)-b-glucan. Arabinoxylan comprises 4e9% w/w of the whole grain in wheat, but this proportion is much lower in rice and maize, at around 2e5% w/w (Collins et al., 2010). The structure of sorghum GAX from water-insoluble endosperm fractions has been well characterised (Verbruggen et al., 1998a, 1995, 1993, 1998b). It is highly substituted compared with wheat and barley and has complex backbone substituents of arabinose and glucuronic acids. Unusually in a cereal, some of these substituents appear to be short arabinan chains (Verbruggen et al., 1995, 1998b). Estimates of arabinoxylan amount in total grain range from 2.0 to 2.8% (w/w) (inferred from Bach Knudsen et al., 1988; Verbruggen et al., 1993). Other agronomically important cereals, such as wheat, barley and rice, have well-characterised cell walls, especially in the grain that is so important for human use. Increased fibre in human diets has significant health benefits, reducing the incidence and severity of diabetes, colorectal cancers, cardiovascular and inflammatory diseases. Likewise, higher levels of soluble polysaccharides, such as (1,3;1,4)-b-glucan, in a biofuel feedstock improves saccharification to provide more fermentable sugars for conversion into bioethanol; whereas lower amounts of viscous polysaccharides are preferred for brewing end uses. Characterisation of cell wall polysaccharides in sorghum whole grain is thus of vital importance in understanding their effect on human and animal nutrition and biofuel generation. Here, we present a systematic analysis of nutritionally and energetically important non-cellulosic polysaccharides in a diverse
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range of S. bicolor grains, and examine how environmental conditions can affect (1,3;1,4)-b-glucan and arabinoxylan accumulation and structure. 2. Materials and methods 2.1. Sorghum growth and harvesting A diverse set of sorghum genotypes (see Table A.1) were grown at the Hermitage Research Facility, Queensland, Australia. The plants were grown in a vertisol soil with ample water, row spacing of 1 m and within-row spacing of 0.2e0.5 m. The grain selfpollinated and ripened under paper bags to ensure purity. A smaller set of ten genotypes were grown in five environments (five different locations in two different seasons; see Tables A.2 and A.3 for details of weather, site location and approximate harvest date). All were sown with row spacing of 1 m and within-row spacing of 0.2e0.5 m. Grain was hand-harvested when the latestmaturing genotypes were mature. Grain was manually de-glumed before grinding in a dental amalgam mixer to a fine flour. 2.2. (1,3;1,4)-b-glucan and starch assays (1,3;1,4)-b-glucan was measured on 25 mg sorghum flour using a scaled down version of the standard method developed for cereal grains (Burton et al., 2011). A portion of the lichenase digest was retained before b-glucosidase treatment for DP3:DP4 analysis. Starch was measured on 20 mg sorghum flour using a small scale version of the Megazyme Total Starch Assay (amyloglucosidase/a-amylase method; Burton et al., 2011). 2.3. Monosaccharide analysis Polysaccharides were quantified as described in Burton et al. (2011). This method is considered to fully hydrolyse noncellulosic polysaccharides, although estimates of neutral sugar amount are slightly underestimated due to degradation during hydrolysis. Chromatography was performed using a Phenomenex Kinetex C18, 2.6 mm, 100 3 mm at 30 C on an Agilent 1200 LC. Using eluents A (10% acetonitrile, 40 mM ammonium acetate pH 6.8) and B (70% acetonitrile), a gradient of 8e16.5% B over 9.3 min at a flow rate of 0.8 mL/min was used to separate the PMP-monosaccharides. Sample peak areas were compared to calibration curves of monosaccharide standard solutions. 2.4. (1,3;1,4)-b-glucan oligosaccharide analysis Enzyme digests were purified on a solid phase extraction (SPE) cartridge packed with graphitized carbon (Varian Bond Elut Carbon 50 mg/1 mL columns). The oligosaccharides bound to the column were washed with 1 mL water, eluted with 55% acetonitrile, dried under vacuum, and dissolved in water. (1,3;1,4)-b-glucan-oligosaccharides DP3 and DP4 were separated using high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS-5000 chromatograph. Aliquots of 25 mL were injected onto a Dionex CarboPAC PA-200 column (3 250 mm) with guard (3 50 mm) kept at 30 C and operated at a flow rate of 0.5 mL/min. The eluents were (A) 0.1 M sodium hydroxide and (B) 0.1 M sodium hydroxide with 1 M sodium acetate. The gradient was 1e13.3% B over 14 min followed by a 1 min wash with 100% B and re-equilibration at 1% B. The detector was kept at 20 C and data collection was at 2 Hz, using the Gold Standard PAD waveform for carbohydrates. The
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integrated peak areas were compared with DP3 and DP4 standard curves. 2.5. Microscopy and antibody labelling Five varieties, all grown at the Narrabri trial site, were selected for microscopy: IS8525; BTx623; ICSV400; MR Buster; and SC49. Grain was sliced in half, then fixed, dehydrated in an ethanol series, infiltrated with LR White Resin and polymerized (Burton et al., 2011). Survey sections were stained with toluidine blue. Labelling was performed on thin sections and visualised under the CM100 transmission electron microscope (TEM) as previously described in Burton et al. (2011) with the following antibodies: (1,3;1,4)-bglucan using BG1; arabinoxylan using LM10 and LM11; cellulose using CBM3a; xyloglucan using LM15; and pectin using LM20. All antibodies were purchased from PlantProbes (Leeds, UK) except for BG1, which was purchased from Biosupplies (Australia). 2.6. Statistical design and analysis Twenty-seven S. bicolor genotypes were grown at five trial sites in northern New South Wales (NSW) and southern Queensland (Qld). In the 2009e2010 growing season, sites at Jimbour, Gatton, Narrabri and Liverpool Plains were used; in the 2010e2011 season, a single site at Tulloona was chosen. The sorghum genotypes were planted as a two replicate trial in a randomised complete block design. Grain from a diverse subset of ten genotypes was retained from each of the two replicate plots, forming the biological replicates in this study. A total of 95 grain samples were collected: two biological replicates of ten genotypes sourced from five locations, allowing for five missing samples. Approximately thirty grains from each sample were ground into flour, which was used for analysis. These ground flour samples were split into laboratory replicates and randomly assigned to batches for processing. A statistical analysis was conducted for genotype and environment effects on (1,3;1,4)-b-glucan, DP3:DP4 ratio, arabinoxylan and starch measurements. These data were analysed in a linear mixed model framework where environment (E) was fitted as a fixed effect and genotype (G) and G E effects were fitted as random terms. A term for variation between biological replicates was included as a random effect together with batch effects in the laboratory, and variation between laboratory replicates formed the residual variance term. Estimates of variance parameters were obtained using residual maximum likelihood (REML; Patterson and Thompson, 1971) and best linear unbiased predictions (BLUPS; Robinson, 1991) were obtained for G E effects. The program ASReml-R (Butler et al., 2007) was used to fit the linear model and form predictions. 2.7. Weather analysis Weather data, including daily rainfall (mm), temperature maxima and minima ( C) and solar exposure (MJ m2) were sourced from the Australian Government Bureau of Meteorology (http:// www.bom.gov.au/climate/data/?ref¼ftr). Weather station locations were chosen as being the closest to the planting sites based on GPS coordinates (Table A.3). 3. Results 3.1. Quantification of the content and structure of non-cellulosic polysaccharides We chose a highly diverse range of 27 S. bicolor varieties, with origins from all over the world: six from Asia (China, Japan, India
and Russia); nine from Africa and the Middle East; seven from the United States; three from Australia, including two elite breeding lines; and two of unknown origin (Table A.1). In addition, we tested two related wild accessions: S. bicolor ssp. verticilliflorum from Australia and S. bicolor ssp. drummondii from North-East Africa. Grain morphology, including colour, size and shape, varied widely (see Fig. 1 for example grains). A broad range in (1,3;1,4)-b-glucan levels was detected, from 0.06 to 0.43% (w/w) (Fig. 1), with the mean across the 27 cultivated varieties of 0.20% (w/w). The sweet sorghum Rio had a slightly lower than average level of 0.12% (w/w). The two wild accessions had (1,3;1,4)-b-glucan levels toward the lower end of the range exhibited by the cultivated S. bicolor varieties. The structure of (1,3;1,4)-b-glucan molecules was assessed by measuring the ratio of cellotriose (DP3) to cellotetraose (DP4) generated using lichenase in the (1,3;1,4)-b-glucan assay. DP3:DP4 ratios also exhibited a broad range of values, from 1.8 to 2.9 across the 29 varieties (Fig. 1, Table A.4). There was no relationship between (1,3;1,4)-b-glucan amount and DP3:DP4 ratio (data not shown). Levels of neutral and acidic monosaccharides were consistent with previous reports (Bach Knudsen et al., 1988; Verbruggen et al., 1993): arabinose was the most prevalent, closely followed by xylose. Galactose and glucuronic acid were present at lower levels, while other sugars were only detected at levels that could not be accurately quantitated (Tables 1 and A.4). Arabinose levels averaged 1.3% (w/w), but ranged from 1.0 to 2.3% (w/w). Xylose levels averaged 1.1% (w/w) and ranged from 0.7 to 1.9% (w/w). Adding arabinose and xylose quantities to provide an estimate of arabinoxylan amount, values ranged from 1.5 to 3.6% w/w, which expands the range inferred from previous work (Bach Knudsen et al., 1988; Verbruggen et al., 1993). The resulting arabinose:xylose ratios of 1.0e1.3 agree with Verbruggen et al. (1995), although the presence of occasional arabinan side chains on the xylan backbones renders this ratio a less useful indicator of substitution (Verbruggen et al., 1998b). There was no detectable correlation between arabinoxylan amount or structure (data not shown). Starch levels ranged from 64 to 76% (w/w) (Table 1), consistent with previous reports (Bach Knudsen et al., 1988; Verbruggen et al., 1993) and other cereal grains (Burton and Fincher, 2012). Free glucose, measured in a parallel experiment without hydrolytic enzymes, ranged from 0.9 to 1.3% (w/w), which was approximately half the levels found in three varieties by Bach Knudsen et al. (1988). 3.2. Microscopic analysis of non-cellulosic polysaccharides in sorghum grain Antibody labelling of grain sections confirmed low levels of (1,3;1,4)-b-glucan and arabinoxylan in cell walls of different tissues of the grain (Fig. 2). Labelling of (1,3;1,4)-b-glucan appeared evenly throughout cell walls of the testa, aleurone and endosperm (Fig. 2AeC). Arabinoxylans were screened with both LM10 and LM11 antibodies, which were previously reported unable to detect sorghum GAX (McCartney et al., 2005). Here, LM10, which detects xylan with no or low levels of arabinose substitution (McCartney et al., 2005), did not label any polysaccharides in the sorghum grain (data not shown). However LM11, which detects xylans with higher levels of arabinose substituents such as those found in wheat grains (McCartney et al., 2005), labelled cell walls in the testa, aleurone and endosperm (Fig. 2DeF) with occasional concentration of label in the middle lamella (Fig. 2F). Low levels of labelling were observed in testa, aleurone and endosperm walls with an anti-cellulose antibody (endosperm shown in 2G). Low levels of xyloglucan were also detected in the
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Fig. 1. (1,3;1,4)-b-glucan amounts in selected diverse sorghum lines. The image above the graph shows grain morphology. The number below the graph gives the DP3:DP4 ratio of the (1,3;1,4)-b-glucan in that line. For (1,3;1,4)-b-glucan content, presented as % w/w of the grain, all samples are the average of two assays except for LR2490-3, which was assayed three times.
aleurone and testa, but not the endosperm (aleurone shown in Fig. 2H). Labelling with LM20, an antibody that detects esterified homogalacturonan, revealed the presence of pectin in the cells walls and the middle lamella region of walls belonging to testa and endosperm, but not aleurone, cells (Fig. 2IeK). For reference, a survey section of sorghum grain stained with toluidine blue is shown in Fig. 2L. 3.3. G E effect on non-cellulosic polysaccharides Ten S. bicolor genotypes were compared across five trial sites in northern New South Wales (NSW) and southern Queensland (Qld).
Fig. A.1 shows the location for each site and Tables A.2 and A.3 give meteorological data for the growing season. Jimbour in particular was subject to high levels of rainfall in March, at the onset of grain fill, after an otherwise dry season. Tulloona had the highest solar exposure over March and April, with the highest average minima and maxima over the growing season. (1,3;1,4)-b-glucan amounts ranged from 0.07 to 0.41% (w/w), within the range previously observed within the 29 diverse lines (Table A.5). There was an obvious environmental effect: (1,3;1,4)-bglucan amounts were generally higher at the three NSW sites compared with the Qld sites, and the 2011 trial produced the highest (1,3;1,4)-b-glucan levels (Fig. 3A). Overall, 74% of the
Table 1 Non-cellulosic polysaccharides in grain of 29 diverse sorghum genotypes. Masses are given as % w/w ± standard deviation (SD); ratios are given as x:1. Component
Cultivated S. bicolor Average ± SD
(1,3;1,4)-b-glucan DP3:DP4 ratio (:1) Arabinose Xylose Galactose Glucuronic acid A:X ratio Starch Free glucose
0.20 2.51 1.35 1.12 0.36 0.33 1.21 70.4 1.01
± ± ± ± ± ± ± ± ±
0.10 0.31 0.33 0.30 0.05 0.08 0.07 3.8 0.09
ssp. verticilliflorum
ssp. drummondii
Minimum
Maximum
Average ± SD
Average ± SD
0.06 1.84 0.95 0.75 0.29 0.25 0.95 64.5 0.89
0.43 2.90 2.27 1.87 0.47 0.52 1.31 76.3 1.28
0.06 1.96 1.44 1.11 0.45 0.40 1.29 66.4 1.0
± ± ± ± ± ± ± ±
0.00 0.02 0.02 0.01 0.00 0.01 0.01 7.1
0.07 1.77 1.00 0.88 0.39 0.48 1.14 72.7 0.9
± ± ± ± ± ± ± ±
0.01 0.02 0.02 0.01 0.01 0.05 0.00 2.7
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Fig. 2. Microscopic analysis of cell wall polysaccharides in sorghum grain. All sections are taken from mature S. bicolor grain. Scale bars as marked. BTx623 testa (A), aleurone (B) and endosperm (C) labelled with BG1, revealing small amounts of (1,3;1,4)-b-glucan in the cell wall. BTx623 testa (D), aleurone (E) and endosperm (F) labelled with LM11, indicating the presence of arabinoxylan in the cell walls and middle lamella. (G) BTx623 endosperm labelled with CBM3a, showing small amounts of cellulose in the cell wall. (H) SC49 aleurone labelled with LM15, showing small amounts of xyloglucan in the cell wall. (I) BTx623 testa, (J) aleurone and (K) endosperm labelled with LM20, revealing small amounts of pectin in all of these tissues. (L) Cross-section of sorghum grain stained with toluidine blue, showing morphological features.
variation was attributable to genotype effects and 14% to the environment (Table 2). Amounts of arabinose and xylose, individually and cumulatively, were also within the ranges observed within the 29 diverse lines (Fig. 3B and Table A.6). Only 3.7% of the overall variation was attributable to the environment (Table 2). The various genotypes also reacted differently to environmental factors. Over 5% of the variation in (1,3;1,4)-b-glucan content was due to a G E interaction. At Jimbour, seven of the ten genotypes exhibited their lowest (1,3;1,4)-b-glucan amounts but QL12
reversed the trend and produced its equal highest amount for the sites in 2010 (Fig. 3A). Conversely, nine of the ten genotypes produced the most (1,3;1,4)-b-glucan at Tulloona in 2011, with the exception of K-34 (Fig. 3A). For arabinoxylan amounts, 3.7% of the total variation was due to a G E effect. Nine genotypes produced their lowest amounts at Gatton except for KS115, which was unaffected by the trial site location. The DP3:DP4 ratio observed at the five sites ranged from 1.9 to 3.0, which was just higher than ratios observed within the original 29 lines. This variation was almost entirely attributable to
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Fig. 3. G E effect on non-cellulosic polysaccharides in sorghum grain. (A) Average (1,3;1,4)-b-glucan amounts for each genotype at the 5 trial sites. For standard error (SE) values, see Table A.5. (B) Average arabinoxylan amounts for each genotype at the five trial sites. For standard error (SE) values, see Table A.6.
genotype, with almost no influence from environmental, G E or other factors (Table 2). The A:X ratio was also slightly higher, ranging from 1.1 to 1.3 for the G E lines but again, this variation was almost entirely determined by genotype (Table 2).
Starch levels for two genotypes were analysed at four planting sites in the 2009e2010 growing season. There were no significant differences in starch levels between the test genotypes or between the trial locations, so further testing was not performed (Fig. A.2). 4. Discussion
Table 2 Sources of variation for amount and structure of (1,3;1,4)-b-glucan in sorghum grains (% of total variation). Sample refers to differences between biological repeats of the same grain; Batch refers to differences between experimental batches; and Residual is unexplained experimental variation.
Genotype (G) Environment (E) GE Sample Batch and residual
(1,3; 1,4)-b-glucan
Arabinoxylan
Amount
DP3:DP4 ratio
Amount
A:X ratio
73.8 13.8 5.4 4.3 2.6
83.4 0.8 1.7 7.2 6.9
66.7 3.7 3.7 12.2 13.7
64.5 17.8 1.4 4.2 12.0
(1,3;1,4)-b-glucan content in grain from the sorghum varieties examined here ranges from 0.06 to 0.43% (w/w) (Fig. 1), which is consistent with the single finding of Niba and Hoffman (2003) who reported a level of 0.12% (w/w). Sorghum therefore appears to be more similar to wheat, rice and maize than to barley, oats and rye, whose (1,3;1,4)-b-glucan content can reach up to 20% (w/w) (Collins et al., 2010). The DP3:DP4 ratio also showed a wide range in values from 1.8 to 3.1:1, but these numbers are consistent with values from barley, oats and rye. The (1,3;1,4)-b-glucan in sorghum grain was also detected by the BG1 antibody that detects (1,3;1,4)-b-glucan in other grains, suggesting sorghum (1,3;1,4)-b-glucan is similar in
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structure to other cereal grains and therefore reasonably soluble in water (Fig. 2AeC). Previous reports of extracting extremely small (1,3;1,4)-b-glucan molecules (DP20e26) from sorghum grain that focus solely on grain fractions after extensive water extractions are therefore unlikely to represent the total (1,3;1,4)-b-glucan profile (Ramesh and Tharanathan, 1998; Woolard et al., 1976). The structure of heteroxylans in sorghum grain has been subject to much scrutiny (Bach Knudsen et al., 1988; Verbruggen et al., 1998a, 1995, 1993, 1998b). Again, Verbruggen and associates focussed on water-unextractable grain fractions, and it is likely they lost some soluble, less substituted forms of heteroxylan in their analyses. We observed labelling of arabinoxylan in the wall of endosperm, aleurone and testa cells with the LM11 antibody, which had not previously been successful in detecting highly-substituted sorghum GAX (41% xylose, 46% arabinose and 10% glucuronic acid; Fig. 2DeF; McCartney et al., 2005). Total amounts of arabinose and xylose in the grain were found to range from 1.5 to 3.6% (w/w); if glucuronic acid is added to these totals, the range becomes 2.0e4.6% (w/w) (Table A.4). By comparison, past research implies a narrower range of 2.0e2.8% (w/w) (Bach Knudsen et al., 1988; Verbruggen et al., 1993). Verbruggen et al. (1993) also found polishing the sorghum grain d removing the outside 20% of the grain by mass d decreased the non-starch polysaccharides in the grain by 70%. In contrast, our antibody labelling suggested (1,3;1,4)-b-glucan and arabinoxylan are distributed throughout the endosperm, aleurone and testa (Fig. 2AeF). However, we have found sorghum glumes, which are often attached to the grain, contain over 20% w/w xylose (unpublished data). Previous work on determining GAX structure assumed all xylose, arabinose and uronic acid moieties were associated with xylans. Here, TEM analysis detected xyloglucans and pectins, which contain uronic acids, in the cell walls of sorghum grain tissues (Fig. 2HeK). While xyloglucans and pectins are not usually reported in the cell walls of monocotyledonous plants, pectins have recently been found in rice and wheat endosperm (Chateigner-Boutin et al., 2014). Sorghum grain may therefore be more complex than previously supposed. While genotype had the largest influence on (1,3;1,4)-b-glucan and arabinoxylan accumulation in the grain, there was still a marked environmental and G E effect (Table 2). A similar hierarchy of effects was observed when genotypic, environmental and G E effects on sorghum grain popping ability were assessed (Rooney and Rooney, 2013). (1,3;1,4)-b-Glucan levels in sorghum were lower at Gatton and Jimbour, which both experienced more rainfall during grain filling than the other three sites (Fig. 3A and Table A.2). This result is consistent with reports that rain during barley grain filling is generally correlated with lower (1,3;1,4)-bglucan levels, which is desirable for malting and subsequent brewing (Zhang et al., 2001). Grain harvested at Gatton also contained the lowest arabinoxylan amounts (Fig. 3B). While in wheat, higher levels of arabinoxylan in the grain has been linked with heat and drought stress after flowering (Rakszegi et al., 2014 and references therein), there was no obvious weather-related effect at Gatton. Starch levels were stable across all environmental sites tested (Fig. A.2), indicating that sorghum would be a reliable crop in most climates to provide starch for human food, animal feed, brewing, or biofuel generation. Rooney (2004) reported little genetic variation exists in sorghum for polysaccharide content and that most variation comes from environmental factors. By contrast, our study showed there was a large genotypic component that governs (1,3;1,4)-b-glucan and arabinoxylan accumulation in sorghum grain, suggesting there may be more genotypic variation in wild races and landraces from diverse geographic origins than previously thought. With the
recent sequencing of the sorghum genome (Mace et al., 2013; Paterson et al., 2009), sorghum may provide a novel system to search for genes responsible for regulating the synthesis of cell wall polysaccharides. Acknowledgements Thanks to Geoffrey Fincher and Helen Collins for advice; Kuok Yap for assistance with starch assays and HPLC analysis; and Sally Norton (Department of Environment and Primary Industries) for information on sorghum accessions. This work was supported by grants from the Australian Research Council (CE110001007). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jcs.2015.02.007. References Agu, R.C., Palmer, G.H., 1998. A reassessment of sorghum for lager-beer brewing. Bioresour. Technol. 66, 253e261. Bach Knudsen, K., Munck, L., Eggum, B., 1988. Effect of cooking, pH and polyphenol level on carbohydrate composition and nutritional quality of a sorghum (Sorghum bicolor (L.) Moench) food, ugali. Br. J. Nutr. 59, 31e47. Burton, R., Fincher, G., 2012. Current challenges in cell wall biology in the cereals and grasses. Front. Plant Sci. 3. Burton, R.A., Collins, H.M., Kibble, N.A.J., Smith, J.A., Shirley, N.J., Jobling, S.A., Henderson, M., Singh, R.R., Pettolino, F., Wilson, S.M., Bird, A.R., Topping, D.L., Bacic, A., Fincher, G.B., 2011. Over-expression of specific HvCslF cellulose synthase-like genes in transgenic barley increases the levels of cell wall (1,3;1,4)-b-D-glucans and alters their fine structure. Plant Biotechnol. J. 9, 117e135. Butler, D., Cullis, B.R., Gilmour, A., Gogel, B., 2007. ASReml-R Reference Manual. Queensland Department of Primary Industries and Fisheries. Chateigner-Boutin, A.-L., Bouchet, B., Alvarado, C., Bakan, B., Guillon, F., 2014. The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution. PLoS ONE 9, e89620. Collins, H.M., Burton, R.A., Topping, D.L., Liao, M.-L., Bacic, A., Fincher, G.B., 2010. Variability in fine structures of noncellulosic cell wall polysaccharides from cereal grains: potential importance in human health and nutrition. Cereal Chem. 87, 272e282. Euromonitor, 2013. Pet Food Sales. Pet Food Institute. http://www.petfoodinstitu te.org/?page¼PetFoodSales. Gelski, J., 2014. Ancient Grains for Modern Trends. Food Business News. Mace, E.S., Tai, S., Gilding, E.K., Li, Y., Prentis, P.J., Bian, L., Campbell, B.C., Hu, W., re, C., Zhang, H., Hunt, C.H., Innes, D.J., Han, X., Cruickshank, A., Dai, C., Fre Wang, X., Shatte, T., Wang, M., Su, Z., Li, J., Lin, X., Godwin, I.D., Jordan, D.R., Wang, J., 2013. Whole-genome sequencing reveals untapped genetic potential in Africa's indigenous cereal crop sorghum. Nat. Commun. 4. McCartney, L., Marcus, S.E., Knox, J.P., 2005. Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J. Histochem. Cytochem. 53, 543e546. Niba, L.L., Hoffman, J., 2003. Resistant starch and b-glucan levels in grain sorghum (Sorghum bicolor M.) are influenced by soaking and autoclaving. Food Chem. 81, 113e118. Paterson, A.H., Bowers, J.E., Bruggmann, R., Dubchak, I., Grimwood, J., Gundlach, H., Haberer, G., Hellsten, U., Mitros, T., Poliakov, A., Schmutz, J., Spannagl, M., Tang, H., Wang, X., Wicker, T., Bharti, A.K., Chapman, J., Feltus, F.A., Gowik, U., Grigoriev, I.V., Lyons, E., Maher, C.A., Martis, M., Narechania, A., Otillar, R.P., Penning, B.W., Salamov, A.A., Wang, Y., Zhang, L., Carpita, N.C., Freeling, M., Gingle, A.R., Hash, C.T., Keller, B., Klein, P., Kresovich, S., McCann, M.C., Ming, R., Peterson, D.G., Mehboob ur, R., Ware, D., Westhoff, P., Mayer, K.F.X., Messing, J., Rokhsar, D.S., 2009. The Sorghum bicolor genome and the diversification of grasses. Nature 457, 551e556. Patterson, H.D., Thompson, R., 1971. Recovery of inter-block information when block sizes are unequal. Biometrika 58, 545e554. ng, L., Bedo }, Z., Veisz, O., Shewry, P.R., 2014. Rakszegi, M., Lovegrove, A., Balla, K., La Effect of heat and drought stress on the structure and composition of arabinoxylan and b-glucan in wheat grain. Carbohydr. Polym. 102, 557e565. Ramesh, H.P., Tharanathan, R.N., 1998. Structural characteristics of a mixed linkage b-D-glucan from sorghum (Sorghum bicolor). Carbohydr. Res. 308, 239e243. Robinson, G.K., 1991. That BLUP is a good thing: the estimation of random effects. Stat. Sci. 6, 15e32. Rooney, T.E., Rooney, W.L., 2013. Genotype and environment effects on the popping characteristics of grain sorghum. J. Crop Improv. 27, 460e468. Rooney, W.L., 2004. Sorghum improvementdintegrating traditional and new technology to produce improved genotypes. In: Sparks, D.L. (Ed.), Advances in Agronomy. Academic Press, UK, pp. 37e109.
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