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Carbohydrate dynamics during reproductive growth and seed yield limits in perennial ryegrass
Field Crops Research 112 (2009) 182–188
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Carbohydrate dynamics during reproductive growth and seed yield limits in perennial ryegrass J.A.K. Trethewey *, M.P. Rolston Agresearch, Lincoln Research Centre, Private Bag 4749, Christchurch, New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 December 2008 Received in revised form 27 February 2009 Accepted 1 March 2009
Forage grass seed yields are often low and variable with only 10–20% of the above ground matter harvested as seed. Seed yield is affected by the amount of carbohydrate transported to the seed. However, information regarding the storage and mobilisation of carbohydrates during reproductive development in forage grasses is limited and the contribution of stored and current assimilate to seed yield from vegetative tissues is equivocal. To identify whether the total amount of carbohydrate in the plant might be limiting seed yield, and the contribution of vegetative and reproductive tissues to seed yield, the pattern of accumulation of water-soluble carbohydrates (WSC) and their remobilisation was investigated in field-grown perennial ryegrass (Lolium perenne L.) plants. Tillers were sampled from early head emergence through to harvest. Amounts of WSC in leaf blades, leaf sheaths, internodes and reproductive heads of the tillers were measured in two ways. Firstly, WSC were estimated indirectly by changes in tissue dry weight. These changes were then used to determine any apparent translocation of WSC from vegetative tissues to the developing seed. Secondly, WSC concentration was measured directly. Low molecular weight (LMW) and high molecular-weight (HMW) WSC were extracted from vegetative and reproductive tissues and the concentrations quantified using a colorimetric anthrone assay. The seed yield of the crop was high (2950 kg h1) and the dry weights and the amount of LMW and HMW WSC in vegetative and reproductive tissues changed significantly during reproductive development. High concentrations of HMW WSC were found in the internodes post head emergence and these concentrations continued to increase during seed fill through to harvest. Total dry weight of the internodes also increased over the same period. In contrast, in the leaf blades and leaf sheaths, total dry weight decreased only slightly while the initial low concentrations of WSC steadily declined. In the heads, WSC increased significantly during seed fill and subsequently declined while the dry weight continued to increase through to harvest. The ratio of LMW and HMW WSC also changed during development depending on the tissue type and growth stage. The present study indicates that the seed head itself maybe an important factor driving seed fill. Knowledge of the mechanisms that underlie carbohydrate partitioning to the seed could in future result in significantly higher yields. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Apparent translocation Lolium perenne L. Seed yield Water-soluble carbohydrates
1. Introduction Perennial ryegrass (Lolium perenne L.) harvest index (seed as a percentage of above ground dry material) is only 10–20% (Elgersma, 1990; Rolston et al., 2005). Harvest index is directly influenced by the plant’s reproductive efficiency defined as floret site utilisation (FSU), the difference between potential seed yield and actual seed yield. Potential seed yield is a function of the total
* Corresponding author. Tel.: +64 3 321 8800x3603; fax: +64 3 321 8811. E-mail address: [email protected] (J.A.K. Trethewey). Abbreviations: FSU, floret site utilisation; GS, growth stage; HMW, high molecularweight; Int, internode; Lb, leaf blade; LMW, low molecular-weight; Ls, leaf sheath; WSC, water-soluble carbohydrates. 0378-4290/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2009.03.001
number of florets present at anthesis and corresponds to the maximum possible number of seeds per unit area (Elgersma, 1990). However, in perennial ryegrass the actual seed yield is low and variable. While approximately 60% of florets develop into a seed prior to harvest, only 10–30% of florets produce saleable seeds (Hampton and Hebblethwaite, 1985; Elgersma, 1990). Whether a floret develops into a saleable seed can be affected by the amount of carbohydrate transported to the seed. Light non-recovered seeds may not accumulate sufficient carbohydrate during seed fill. Therefore, knowledge of the different types and amounts of carbohydrates that are synthesised in different tissues and transported to the seed is fundamental for a better understanding of factors limiting seed yield. Forage grasses and cereals contain the most diverse structural and nonstructural carbohydrates of all known plants (Bacic et al., 1988;
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Carpita and Gibeaut, 1993; Trethewey and Harris, 2002; Harris, 2005) and in seeds and grains of forage grasses and cereals, starch is the major non-structural storage carbohydrate (Pollock and Cairns, 1991). However, for much of the growing season starch is often only a minor component of the total reserve carbohydrate in the plant. Vegetative tissues of cereals and temperate grasses such as perennial ryegrass and tall fescue (Festuca arundinacea Schreb.) accumulate water-soluble carbohydrates (WSC), often in high concentrations (Chatterton et al., 1989). These carbohydrates, stored in leaves and stems, are mainly in the form of low molecular-weight (LMW) sucrose and high molecular-weight (HMW) sucrose-derived polymers of fructose (fructans) (Pollock and Cairns, 1991). While much is known about the distribution of WSC and their function during vegetative growth of grasses under conditions that limit photosynthesis, such as defoliation (Cairns and Pollock, 1988; Morvan-Bertrand et al., 1999; Turner et al., 2001; Amiard et al., 2003) the mobilisation of WSC from vegetative organs to the seed during reproductive development is less well understood. As in the leaf and sheath, the predominant carbohydrates accumulated in the stems of grasses and cereals are HMW WSC and, although WSC are remobilised from the stem during seed filling, the reported contribution varies. In wheat (Triticum aestivum L.), the contribution of WSC from stems to final grain weight can vary between 10% and 80% depending upon cultivar and growing conditions (Meier and Reid, 1982; Borrell et al., 1989; Bonnett and Incoll, 1992). In winter barley (Hordeum sativum L.), HMW WSC comprise approximately 30% (dry weight) of the stem at the time of maximum stem mass (Bonnett and Incoll, 1993). As in cereals, stems of forage grasses accumulate high levels of WSC during reproductive development (Griffith, 2000). In perennial ryegrass, at final harvest WSC accounted for 25% (dry weight) of the stem (Warringa and Marinissen, 1997). Griffith (1992) showed for Italian ryegrass (Lolium multiflorum Lam.) that stem assimilates are remobilised to support both seed development and vegetative growth when plant source-sink relations are artificially modified. In many of these studies the amount of stored carbohydrate contributed by vegetative organs to the seed has been inferred from decreasing levels of leaf and stem total WSC and increasing dry weight of the seed head. The pattern of accumulation, remobilisation and significance of individual carbohydrates in specific organs and tissues during reproductive growth is not known. The amount of carbohydrate remobilised during seed filling in perennial ryegrass is variable and factors such as secondary vegetative tillering may influence the amount of carbohydrate that reaches the seed. The results of Warringa and Marinissen (1997) and Matthew (2002) with perennial ryegrass indicate that there is little net translocation of 13C between main tillers and secondary tillers. In contrast, Clemence and Hebblethwaite (1984) found that while the main export of assimilated carbon moved from the subtending leaf of the flag leaf to the flag leaf and then to the stem over time, allocation of 14C to younger tillers increased to 24% during seed fill. The competition for photosynthate between plant organs of economic importance and the remaining vegetative structures is therefore especially important. Stored and remobilized WSC can be estimated indirectly as apparent translocation by changes in stem dry weight from post anthesis to harvest (Borrell et al., 1993; Ehdaie et al., 2006a) or measured directly through WSC concentration (Kiniry, 1993; Ehdaie et al., 2006b). The objective of this research was to determine the seed filling capacity of perennial ryegrass by investigating carbohydrate dynamics through changes in dry matter and the accumulation and mobilisation of LMW and HMW WSC during reproductive development.
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2. Materials and methods 2.1. Plant material A trial was positioned in a first year perennial tetraploid ryegrass (cv. Grasslands Sterling) crop on a farm in Canterbury, New Zealand (438360 S, 1728240 E) between September 2005 and January 2006. The soil type was a Wakanui silt loam (20% clay, 50% silt). Spring nitrogen (N) was applied in two applications as urea during October giving a total of 110 kg h1 applied N. The plots (1 m 2 m) were monitored using moisture probes and irrigated as required to ensure plants were not under moisture stress. Weeds were controlled with Jaguar (250 g L1 difluferican, 25 g L1 bromoxynil) at 1.5 L h1 and Pasture-Kleen (520 g L1 2,4-D) at 1.25 L h1 applied on 31 May. Moddus plant growth regulator (trinexapac ethyl 250 g L1) at 1.2 L h1 and the fungicide Folicur (tebuconazole 430 g kg1) at 400 mL h1 were applied as one application at Zadoks growth stage (GS) 32 (second node visible) (Zadoks et al., 1974). A fungicide mixture of Folicur 300 mL h1 + carbendazim (500 g L1 at 1 L h1) + Amistar (azoxystrobin 250 g L1 at 80 mL h1) was applied on 30 September and again on 27 December. 2.2. Sampling procedure The experiment was a randomised block design with four replicates. Weekly samples (0.25 m2) were collected from the start of head emergence (GS 50) in November 2005 through to harvest in January 2006. At each sampling date tillers were cut 5 mm above ground level at the same time of day (between 9:00 am and 10:00 am) to minimise diurnal changes in WSC. A subsample was weighed and dried (80 8C, 18 h) to obtain fresh and dry weights. The developmental stage of tillers was recorded and a group of comparable tillers (25 tillers per replicate) were frozen, freeze-dried and dissected into various vegetative and reproductive fractions for chemical analysis. Growth stages chosen for analysis were as follows: early head emergence (GS 50), full head emergence (GS 57), post anthesis (GS 67) mid seed fill (GS 80) and harvest (GS 92). Tissues were numbered basipetally with the flag leaf (uppermost leaf) designated leaf blade 1 (Lb 1), the leaf sheath of the flag leaf designated leaf sheath 1 (Ls 1) and the internode below the head designated internode 1 (Int 1). The dry weight of each of these tissue fractions was recorded and combined values for each fraction (leaf blade, leaf sheath, internode and seed head) were also calculated. 2.3. Extraction and analysis of water-soluble carbohydrates Freeze-dried tissues were individually ground to pass a 0.5 mm screen and the WSC extracted as follows. Samples (20 mg) were extracted twice with 1 mL 80% (v/v) aqueous ethanol (85 8C, 30 min). This fraction is termed the ‘mobile’ fraction and is composed of LMW WSC, predominantly sucrose and monosaccharides (Carpita et al., 1989). Extracts were centrifuged (13,000 g, 10 min), combined and evaporated under vacuum to remove pigments then resuspended in water (2 mL). The residue remaining after ethanol extraction was extracted twice with 1 mL distilled water (65 8C, 30 min). This fraction is termed the ‘storage’ fraction and is composed of polymeric and HMW carbohydrates such as fructans (Prudhomme et al., 1992). Carbohydrates were determined using a colorimetric anthrone assay (Jermyn, 1956) using sucrose standards whereby 10 mL extracts were mixed with 250 mL of anthrone reagent (62.5% sulphuric acid, 37.5% ethanol, 0.00125% anthrone) and incubated (100 8C, 20 min). Absorbance was read at 620 nm.
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2.4. Calculation of assimilate storage and mobilisation The amount of current assimilates contributed to seed yield was estimated from the difference between seed yield and the amount of mobilized WSC to the seed. The amount of stored or mobilized reserves in each tissue was determined based on post-anthesis changes in tissue dry weight and in WSC content (Bonnett and Incoll, 1992; Ehdaie et al., 2008). The difference between postanthesis maximum and minimum dry weight and WSC content was used to estimate the amount of stored or mobilized reserves in each tissue. The total amount of reserves in each tissue was calculated by summing those of all separate fractions. Head dry weight measured between flowering and harvest was used to monitor post-anthesis changes in head seed weight. Final seed yield per head was calculated as the difference between head dry weight at harvest and at flowering. Seed was harvested when seed moisture content was between 38% and 40%. GenStat (version 10) was used for statistical analysis using a general ANOVA model. Individual and combined fractions were designated as treatments and replicates designated as blocks. 3. Results A seed yield of 2950 kg h1 machine dressed seed was achieved for the trial site field. The trial site had a harvest index (seed yield/ mass) of 23% and a FSU (actual number of saleable seeds/potential number of florets) of 32%. 3.1. Changes in dry weights Overall, a small decrease in the total dry weights of the combined leaf blade fractions and leaf sheath fractions was observed from early head emergence to harvest (Fig. 1). In contrast there was a large overall increase in the dry weights of internodes and heads over the same period. In the leaf blades, there was a decrease between early head emergence and full head emergence and another decrease between mid seed fill and harvest. For leaf sheaths, a decrease was observed between early head emergence and full head emergence. In internodes and heads there was an increase between early head emergence and full head emergence and again between post anthesis and mid seed fill. In individual leaf blades and leaf sheaths there was generally an overall decrease in dry weights from early head emergence through to harvest (Fig. 2a and b,). The only departure from this pattern was the flag leaf (Lb 1) and the leaf sheath of the flag leaf (Ls 1) in which no overall decrease in dry weight was observed. Leaf
Fig. 1. Total dry weights of combined leaf blades, leaf sheaths, internodes and heads from early head emergence through to harvest. Bars = LSD (5%).
Fig. 2. Changes in dry weights of individual vegetative tissues from early head emergence through to harvest. (a) Leaf blades, (b) leaf sheaths, (c) internodes. Bars = LSD (5%).
blade 2 (Lb 2), Lb 3 and Lb 4 all decreased from early head emergence to full head emergence and again from mid seed fill to harvest. Similarly, leaf sheath 2 (Ls 2), Ls 3 and Ls 4 all decreased from early head emergence to full head emergence. At all growth stages, the dry weights of individual leaf blades generally decreased in the following order: Lb 1>Lb 2>Lb 3>Lb 4. Similarly, for leaf sheaths the proportion of the dry weights of individual tissues remained constant throughout development, the dry weights decreasing sequentially from Ls 1 to Ls 4. The leaf sheath of the flag leaf (Ls 1) was markedly and significantly heavier than the remaining leaf sheaths and accounted for between 45% and 52% of the total dry weight of leaf sheaths. In contrast to the leaf blades and leaf sheaths, the dry weights of individual internodes generally increased through to harvest (Fig. 2c). Internode 1 (Int 1, internode directly below head) significantly increased from early head emergence through to mid seed fill. Internode 2 (Int 2) increased from early head emergence to full head emergence, decreased through to flowering and increased again through to harvest. Internode 3 (Int 3) increased from early head emergence to full head emergence. The exception to this trend was Int 4 (basal internode) which increased during seed fill and subsequently declined through to harvest. During
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Table 1 Changes in dry weight of reproductive heads from early head emergence through to harvest. Growth stage of tiller
Dry weight (mg tiller1)
Early head emergence Full head emergence Post anthesis Mid seed fill Harvest
99a 171b 169b 310c 329c
Means followed by the same letter are not significantly different at 5% LSD.
seed fill 31% of the total dry weight of internodes was accumulated. The dry weights of the heads increased during reproductive development from early head emergence through to end of flowering and again during seed fill (Table 1). The relationship between internode dry weight and head dry weight was determined during reproductive development. There was a significant positive correlation (R2 = 0.86) between the two characters. 3.2. Changes in WSC The total WSC concentration in the combined tissue fractions changed significantly from early head emergence through to harvest (Fig. 3). In leaf blades and leaf sheaths total WSC steadily declined throughout reproductive development. In heads the concentration of WSC decreased from early head emergence to full head emergence, increased during seed fill and decreased again from mid seed fill to harvest. In contrast to the leaf blades, leaf sheaths, and heads, total WSC in internodes increased from early head emergence through to mid seed fill and remained high through to harvest. The concentrations and ratio between LMW ‘mobile’ (80% ethanol-extracted) and HMW ‘storage’ (water extracted) WSC in vegetative and reproductive tissues also changed considerably during reproductive development. The ratio of LMW to HMW WSC varied during development depending on the tissue type and growth stage. In leaf blades the concentration of both LMW and HMW WSC decreased significantly from early head emergence to full head emergence and remained low through to harvest (Fig. 4a). During head emergence, LMW WSC were the predominant form of carbohydrate. As for the leaf blades, total WSC in leaf sheaths decreased from early head emergence to full head emergence (Fig. 4b) and this trend continued through to harvest. At all growth stages HMW WSC were the predominant carbohydrate.
In reproductive heads at the start of head emergence, the concentrations of both ‘mobile’ LMW and ‘storage’ HMW WSC were similar (Fig. 5). The concentration of LMW WSC significantly increased between post-anthesis and mid-seed fill followed by a subsequent and significant decline through to harvest. In contrast HMW WSC significantly decreased during head emergence and remained low through to harvest. In internodes, as for the reproductive heads, the concentrations of both LMW and HMW WSC were similar at the start of head emergence (Fig. 6). However, the internodes showed a significant increase in the total concentration of HMW WSC from head emergence through to seed fill to harvest (Fig. 6). At harvest, these high concentrations of HMW WSC remained in the internodes.
Fig. 3. Total WSC of combined leaf blades, leaf sheaths, internodes and heads from early head emergence through to harvest. Bars = LSD (5%).
Fig. 5. Concentration of ‘mobile’ and ‘storage’ WSC in heads of perennial ryegrass during reproductive development. Bars = LSD (5%).
Fig. 4. Concentration of LMW ‘mobile’ and HMW ‘storage’ WSC in leaf blades (a) and leaf sheaths (b) of perennial ryegrass during reproductive development. Bars = LSD (5%).
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Fig. 6. Concentration of ‘mobile’ and ‘storage’ WSC in internodes of perennial ryegrass during reproductive development. Bars = LSD (5%).
From full head emergence to post anthesis LMW WSC decreased and remained low through to harvest. As high concentrations of HMW WSC were found in the combined internodes post anthesis the concentrations of WSC in individual internodes (Int 1–4) were investigated in more detail. Concentrations of HMW WSC in individual internodes increased substantially during reproductive development (Fig. 7a) with the majority of HMW WSC accumulating in the two most basal internodes (internodes three and four below the head). At the end of flowering, mid seed fill and harvest, HMW (storage) WSC in internodes 3 and 4 accounted for 76, 72 and 67%, respectively, of total combined internode HMW WSC. During head emergence and flowering, Int 3 and Int 4 were the only internodes to have a
significant increase in HMW WSC. In contrast, from post anthesis to mid seed fill, Int 2, Int 3, and Int 4 had a significant increase in HMW WSC (Fig. 7a). Of all internodes at all stages of development, Int 4 was the only internode to have a decrease in the concentration of HMW WSC, whereby the concentration decreased from seed fill to harvest. Although the concentrations of LMW WSC were small there were significant differences during development (Fig. 7b). Internode 1 showed a significant increase from post anthesis to mid seed fill followed by a significant decline to harvest. Internode 2 significantly increased from early head emergence to full head emergence followed by a decrease to post anthesis. A similar, although non-significant, trend was observed for Int 3 with an increase during mid head emergence followed by a decline through to harvest. In Int 4 there was a significant decrease in LMW WSC from early head emergence to post anthesis that remained low through to harvest. 3.3. Apparent translocation and WSC during seed fill The total amount of stored or mobilised reserves in tissues was calculated from the net loss or gain of WSC and dry weights of tissues from anthesis through to harvest (Fig. 8). The flag leaf blade (Lb 1) was the only leaf blade to gain dry weight in the post anthesis to harvest period, and, overall leaf blades had a total net loss of 11 mg tiller1. Leaf sheaths 1 and 2 had a combined dry weight gain of approximately 3 mg tiller1 whereas leaf sheaths 3 and 4 lost approximately 2 mg tiller1. All leaf blades and leaf sheaths had a net loss of WSC with a greater total net loss observed in leaf sheaths (5.7 mg tiller1). Reproductive heads also had a small net loss (4.5 mg tiller1) of WSC during seed fill. The internodes were the only tissue to have a net total increase (49 mg tiller1) in WSC during seed fill (Fig. 8). All internodes increased in WSC with internodes 2 and 3 having the largest increase (17 and 19 mg tiller1, respectively). Of the total final WSC in internodes 38% was accumulated during seed fill. The dry weight of heads and internodes considerably increased post anthesis to harvest with internodes gaining a net total 107 mg tiller1 and heads gaining 160 mg tiller1. All internodes increased in dry weight with internodes 1, 2, and 3 increasing substantially more than internode 4 (Fig. 8). There was a significant positive correlation (R2 = 0.90) between internode dry weight and internode WSC content during reproductive development. 4. Discussion
Fig. 7. Concentration of ‘storage’ (a) and ‘mobile’ (b) WSC in individual internodes of perennial ryegrass during reproductive development. Internodes are numbered basipetally: 1 = first internode below head (upper most internode); 4 = fourth internode below head (lower most internode). Bars = LSD (5%).
In perennial ryegrass the difference between potential seed yield and actual seed yield is large and variable with only 10–30% of available florets producing saleable seeds (Hampton and Hebblethwaite, 1985; Elgersma, 1990; Rolston et al., 2007). The results of this study suggest that the total amount of WSC in vegetative tissues following anthesis is not limiting seed yield. During seed fill large amounts of HMW ‘storage’ WSC accumulated in the basal internodes of perennial ryegrass, and even in the upper internodes, WSC continued to accumulate, though to a lesser degree. Similarly, Warringa and Marinissen (1997) found that in perennial ryegrass at final harvest WSC, measured as total reducing sugars, accounted for 25% (dry weight) of the stem. While the total amount of assimilate in the tiller may not be limiting seed yield, the distribution and remobilisation of available WSC to the seed is an important factor limiting seed yield. In the present study, there was a strong positive relationship between internode WSC and dry weight throughout reproductive development. During seed fill there was a net gain in dry weight and WSC in internodes with a concomitant increase in head dry weight. This suggests that perennial ryegrass has the capacity to fill
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Fig. 8. Net post anthesis loss or gain in WSC and dry weight in (a) combined and (b) individual vegetative and reproductive tissues. Gains in dry weight or WSC are indicated in bold and by shading.
available seed and accumulate WSC simultaneously with little or no remobilisation of carbohydrate from internodes to the developing seeds. In perennial ryegrass, reducing light intensity by 76% following anthesis reduced stem WSC but had virtually no effect on the relative contribution of seeds to total tiller weight (Warringa and Marinissen, 1996). In contrast, in cereals it is generally accepted that stems contribute a significant amount of assimilates to the developing grain (Borrell et al., 1989). In winter barley ‘storage’ WSC comprise approximately 30% (dry weight) of the stem at the time of maximum stem mass (Bonnett and Incoll, 1993) and in wheat, severe shading during seed fill was shown to reduce stem dry weight and grain yield (Kiniry, 1993). A strong positive relationship between stem dry weight and WSC has also been shown in wheat (Ehdaie et al., 2008). However, in contrast to the present study, both WSC and dry weight of wheat stems decreased during grain fill. Assimilation of WSC and their remobilisation within different organs of perennial ryegrass in a variety of stressful conditions needs to be examined in more detail. In the present study the proportion of LMW and HMW WSC changed considerably in vegetative and reproductive tissues during reproductive development. In all tissues, both LMW and HMW WSC generally decreased, however, large amounts of WSC accumulated in the basal internodes of perennial ryegrass. This agrees with results of Griffith (1992, 2000) who showed that postanthesis WSC increased in lower internodes of Italian ryegrass and perennial ryegrass. Previous reports suggest that younger daughter tillers may compete with seeds for carbohydrate during reproductive development. In perennial ryegrass Clemence and Hebblethwaite (1984)
found that allocation of 14C to younger tillers increased to 24% during seed development. In the present study the dry weight and WSC concentration of the most basal internode (Int 4) decreased from mid seed fill to harvest. However, secondary tillering was minimal (data not presented) and the increase in total dry weight and high concentrations of HMW storage WSC in the internodes during reproductive development suggest competition for assimilate by secondary tillers was weak. The amount of carbon assimilated into various structural and non-structural carbohydrates in different tissues during different stages of reproductive development and their possible remobilisation into the seed requires further investigation. 5. Conclusion The results of this study indicate that the seed head itself maybe an important factor driving seed fill. The decrease in dry weight and low levels of WSC in leaf blades and leaf sheaths combined with the increases in dry weight and WSC of internodes suggest the seed head has sufficient capacity to fill available seeds without contribution from vegetative tissues. This will need to be tested under a variety of conditions that reduce the photosynthetic capacity of potential source organs and tissues from anthesis through to harvest. Breeding for increased seed yield through improved source-sink relationships in the leaf, internode and inflorescence can be effective only when the factors that determine the priority for allocation of assimilates to each sink during reproductive development are understood, along with the contribution of carbohydrate from each organ. This information can
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then be used for evaluating and enhancing seed yield in forage grasses in the future Acknowledgement This research was funded by the Foundation for Arable Research, Lincoln, New Zealand. References Amiard, V., Morvan-Bertrand, A., Billard, J.-P., Huault, C., Prud’homme, M.-P., 2003. Fate of fructose supplied to leaf sheaths after defoliation of Lolium perenne L assessment by 13C-fructose labelling. J. Exp. Bot. 54, 1231–1243. Bacic, A., Harris, P.J., Stone, B.A., 1988. Structure and function of plant cell walls. In: Preiss, J. (Ed.), The Biochemistry of Plants Carbohydrates, 14. Academic Press, San Diego, pp. 297–371. Bonnett, G.B., Incoll, L.D., 1992. The potential pre-anthesis and post-anthesis contributions of stem internodes to grain yield in crops of winter barley. Ann. Bot. (London) 69, 219–225. Bonnett, G.B., Incoll, L.D., 1993. Effects on the stem of winter barley of manipulating the source and sink during grain-filling II. Changes in the composition of watersoluble carbohydrates of internodes. J. Exp. Bot. 44, 83–91. Borrell, A., Incoll, L.D., Dalling, M.J., 1993. The influence of the Rht1 and Rht2 alleles on the deposition and use of stem reserve in wheat. Ann. Bot. (London) 71, 317– 326. Borrell, A.K., Incoll, L.D., Simpson, R.L., Dalling, M.J., 1989. Partitioning of dry matter and the deposition and use of stem reserves in semi-dwarf wheat crop. Ann. Bot. (London) 63, 527–539. Cairns, A.J., Pollock, C.J., 1988. Fructan biosynthesis in excised leaves of Lolium temulentum L I. Chromatographic characterisation of oligofructans and their labelling patterns following 14CO2 feeding. New Phytol. 109, 399–405. Carpita, N.C., Gibeaut, D.M., 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3, 1–30. Carpita, N.C., Kanabus, J., Housley, T.L., 1989. Linkage structure of fructans and fructan oligomers from Tritcum aestivum and Festuca arundinacea leaves. J. Plant Physiol. 134, 162–168. Chatterton, N.J., Harrison, P.A., Bennett, J.A., Asay, K.H., 1989. Carbohydrate partitioning in 185 accessions of Gramineae grown under warm and cool temperatures. J. Plant Physiol. 134, 169–179. Clemence, T.G.A., Hebblethwaite, P.D., 1984. An appraisal of ear, leaf and stem 14CO2 assimilation 14C-assimilate distribution and growth in reproductive seed crop of amenity Lolium perenne. Ann. Appl. Biol. 105, 319–327. Ehdaie, B., Alloush, G.A., Madore, M.A., Waines, J.G., 2006a. Genotypic variation for stem reserves and mobilization in wheat I. Postanthesis changes in internode dry matter. Crop Sci. 46, 735–746. Ehdaie, B., Alloush, G.A., Modare, M.A., Waines, J.G., 2006b. Genotypic variation for stem reserves and mobilization in wheat: II Post anthesis changes in internode water soluble carbohydrates to grain yield in wheat. Crop Sci. 46, 2093–2103.
Ehdaie, B., Alloush, G.A., Waines, J.G., 2008. Genotypic variation in linear rate of grain growth and contribution of stem reserves to grain yield in wheat. Field Crops Res. 106, 34–43. Elgersma, A., 1990. Seed yield related to crop development and to yield components in nine cultivars of perennial ryegrass (Lolium perenne L.). Euphytica 49, 141–154. Griffith, S.M., 1992. Changes in post-anthesis assimilates in stem and spike components of Italian ryegrass (Lolium multiflorum Lam.). I. Water soluble carbohydrates. Ann. Bot. (London) 69, 243–248. Griffith, S.M., 2000. Changes in dry matter, carbohydrate and seed yield resulting from lodging in three temperate grass species. Ann. Bot. (London) 85, 675–680. Hampton, J.G., Hebblethwaite, 1985. The effect of growth retardant application on floret site utilization and assimilate distribution in ears of perennial ryegrass cv. S 24. Ann. Appl. Biol. 107, 127–136. Harris, P.J., 2005. Diversity in plant cell walls. In: Henry, R.J. (Ed.), Plant Diversity and Evolution: Genotypic and Phenotypic Variation in Higher Plants. CAB International, Wallingford, UK, pp. 201–227. Jermyn, M.A., 1956. A new method for determining ketohexoses in the presence of aldohexoses. Nature 177, 38–39. Kiniry, J.R., 1993. Nonstructural carbohydrates of wheat stems as influenced by sink-source manipulations. J. Plant Physiol. 134, 243–250. Matthew, C., 2002. Translocation from flowering to daughter tillers in perennial ryegrass (Lolium perenne L.). Aust. J. Agric. Res. 53, 21–28. Meier, H., Reid, J.S.G., 1982. Reserve polysaccharides other than starch in higher plants. In: Loewus, F.A., Tanner, W. (Eds.), Encyclopedia of Plant Physiology, 13. Springer-Verlag, pp. 418–471. Morvan-Bertrand, A., Boucaud, J., Prud’homme, M., 1999. Influence of initial levels of carbohydrates, fructans, nitrogen, and soluble proteins on regrowth of Lolium perenne L. cv Bravo following defoliation. J Exp. Bot. 50, 1817–1826. Pollock, C.J., Cairns, A.J., 1991. Fructan metabolism in grasses and cereals. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 77–101. Prud’homme, M.P., Gonzalez, B., Billard, J.P., Boucaud, J., 1992. Carbohydrate content, function and sucrose enzyme-activities in roots, stubble and leaves of ryegrass (Lolium perenne L.) as affected by source sink modification after cutting. J. Plant Physiol. 140, 282–291. Rolston, M., Archie, P., Rumball, W.J.W., 2005. Branched inflorescence perennial ryegrass (Lolium perenne L.) - seed yield evaluated in field trials and response to nitrogen and trinexapac-ethyl plant growth regulator. NZ. J. Agric. Res. 48, 87–92. Rolston, M.P., Trethewey, J.A.K., McCloy, B., Chynoweth, R., 2007.In: Achieving forage ryegrass seed yields of 3000 kg/ha and limitations to higher yields, Proceedings of the 6th International Herbage Seed Conference, Norway. Trethewey, J.A.K., Harris, P.J., 2002. Location of (1!3)- and (1!3) (!4)-b-D-glucans in vegetative cell walls of barley (Hordeum vulgare) using immunolabelling. New Phytol. 154, 347–358. Turner, L.B., Humphreys, M.O., Cairns, A.J., Pollock, C.J., 2001. Comparison of growth and carbohydrate accumulation in seedlings of two varieties of Lolium perenne. J. Plant Physiol. 158, 891–897. Warringa, J.W., Marinissen, M.J., 1996. The effect of light intensity after anthesis on dry matter distribution and seed yield of Lolium perenne L. Grass Forage Sci. 51, 103–110. Warringa, J.W., Marinissen, M.J., 1997. Sink-source and sink-sink relations during reproductive development in Lolium perenne L. Neth. J. Agric. Sci. 45, 505–520. Zadoks, J.C., Chang, T.T., Zonzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415–421.
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Carbohydrate dynamics during reproductive growth and seed yield limits in perennial ryegrass J.A.K. Trethewey *, M.P. Rolston Agresearch, Lincoln Research Centre, Private Bag 4749, Christchurch, New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 December 2008 Received in revised form 27 February 2009 Accepted 1 March 2009
Forage grass seed yields are often low and variable with only 10–20% of the above ground matter harvested as seed. Seed yield is affected by the amount of carbohydrate transported to the seed. However, information regarding the storage and mobilisation of carbohydrates during reproductive development in forage grasses is limited and the contribution of stored and current assimilate to seed yield from vegetative tissues is equivocal. To identify whether the total amount of carbohydrate in the plant might be limiting seed yield, and the contribution of vegetative and reproductive tissues to seed yield, the pattern of accumulation of water-soluble carbohydrates (WSC) and their remobilisation was investigated in field-grown perennial ryegrass (Lolium perenne L.) plants. Tillers were sampled from early head emergence through to harvest. Amounts of WSC in leaf blades, leaf sheaths, internodes and reproductive heads of the tillers were measured in two ways. Firstly, WSC were estimated indirectly by changes in tissue dry weight. These changes were then used to determine any apparent translocation of WSC from vegetative tissues to the developing seed. Secondly, WSC concentration was measured directly. Low molecular weight (LMW) and high molecular-weight (HMW) WSC were extracted from vegetative and reproductive tissues and the concentrations quantified using a colorimetric anthrone assay. The seed yield of the crop was high (2950 kg h1) and the dry weights and the amount of LMW and HMW WSC in vegetative and reproductive tissues changed significantly during reproductive development. High concentrations of HMW WSC were found in the internodes post head emergence and these concentrations continued to increase during seed fill through to harvest. Total dry weight of the internodes also increased over the same period. In contrast, in the leaf blades and leaf sheaths, total dry weight decreased only slightly while the initial low concentrations of WSC steadily declined. In the heads, WSC increased significantly during seed fill and subsequently declined while the dry weight continued to increase through to harvest. The ratio of LMW and HMW WSC also changed during development depending on the tissue type and growth stage. The present study indicates that the seed head itself maybe an important factor driving seed fill. Knowledge of the mechanisms that underlie carbohydrate partitioning to the seed could in future result in significantly higher yields. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Apparent translocation Lolium perenne L. Seed yield Water-soluble carbohydrates
1. Introduction Perennial ryegrass (Lolium perenne L.) harvest index (seed as a percentage of above ground dry material) is only 10–20% (Elgersma, 1990; Rolston et al., 2005). Harvest index is directly influenced by the plant’s reproductive efficiency defined as floret site utilisation (FSU), the difference between potential seed yield and actual seed yield. Potential seed yield is a function of the total
* Corresponding author. Tel.: +64 3 321 8800x3603; fax: +64 3 321 8811. E-mail address: [email protected] (J.A.K. Trethewey). Abbreviations: FSU, floret site utilisation; GS, growth stage; HMW, high molecularweight; Int, internode; Lb, leaf blade; LMW, low molecular-weight; Ls, leaf sheath; WSC, water-soluble carbohydrates. 0378-4290/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2009.03.001
number of florets present at anthesis and corresponds to the maximum possible number of seeds per unit area (Elgersma, 1990). However, in perennial ryegrass the actual seed yield is low and variable. While approximately 60% of florets develop into a seed prior to harvest, only 10–30% of florets produce saleable seeds (Hampton and Hebblethwaite, 1985; Elgersma, 1990). Whether a floret develops into a saleable seed can be affected by the amount of carbohydrate transported to the seed. Light non-recovered seeds may not accumulate sufficient carbohydrate during seed fill. Therefore, knowledge of the different types and amounts of carbohydrates that are synthesised in different tissues and transported to the seed is fundamental for a better understanding of factors limiting seed yield. Forage grasses and cereals contain the most diverse structural and nonstructural carbohydrates of all known plants (Bacic et al., 1988;
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Carpita and Gibeaut, 1993; Trethewey and Harris, 2002; Harris, 2005) and in seeds and grains of forage grasses and cereals, starch is the major non-structural storage carbohydrate (Pollock and Cairns, 1991). However, for much of the growing season starch is often only a minor component of the total reserve carbohydrate in the plant. Vegetative tissues of cereals and temperate grasses such as perennial ryegrass and tall fescue (Festuca arundinacea Schreb.) accumulate water-soluble carbohydrates (WSC), often in high concentrations (Chatterton et al., 1989). These carbohydrates, stored in leaves and stems, are mainly in the form of low molecular-weight (LMW) sucrose and high molecular-weight (HMW) sucrose-derived polymers of fructose (fructans) (Pollock and Cairns, 1991). While much is known about the distribution of WSC and their function during vegetative growth of grasses under conditions that limit photosynthesis, such as defoliation (Cairns and Pollock, 1988; Morvan-Bertrand et al., 1999; Turner et al., 2001; Amiard et al., 2003) the mobilisation of WSC from vegetative organs to the seed during reproductive development is less well understood. As in the leaf and sheath, the predominant carbohydrates accumulated in the stems of grasses and cereals are HMW WSC and, although WSC are remobilised from the stem during seed filling, the reported contribution varies. In wheat (Triticum aestivum L.), the contribution of WSC from stems to final grain weight can vary between 10% and 80% depending upon cultivar and growing conditions (Meier and Reid, 1982; Borrell et al., 1989; Bonnett and Incoll, 1992). In winter barley (Hordeum sativum L.), HMW WSC comprise approximately 30% (dry weight) of the stem at the time of maximum stem mass (Bonnett and Incoll, 1993). As in cereals, stems of forage grasses accumulate high levels of WSC during reproductive development (Griffith, 2000). In perennial ryegrass, at final harvest WSC accounted for 25% (dry weight) of the stem (Warringa and Marinissen, 1997). Griffith (1992) showed for Italian ryegrass (Lolium multiflorum Lam.) that stem assimilates are remobilised to support both seed development and vegetative growth when plant source-sink relations are artificially modified. In many of these studies the amount of stored carbohydrate contributed by vegetative organs to the seed has been inferred from decreasing levels of leaf and stem total WSC and increasing dry weight of the seed head. The pattern of accumulation, remobilisation and significance of individual carbohydrates in specific organs and tissues during reproductive growth is not known. The amount of carbohydrate remobilised during seed filling in perennial ryegrass is variable and factors such as secondary vegetative tillering may influence the amount of carbohydrate that reaches the seed. The results of Warringa and Marinissen (1997) and Matthew (2002) with perennial ryegrass indicate that there is little net translocation of 13C between main tillers and secondary tillers. In contrast, Clemence and Hebblethwaite (1984) found that while the main export of assimilated carbon moved from the subtending leaf of the flag leaf to the flag leaf and then to the stem over time, allocation of 14C to younger tillers increased to 24% during seed fill. The competition for photosynthate between plant organs of economic importance and the remaining vegetative structures is therefore especially important. Stored and remobilized WSC can be estimated indirectly as apparent translocation by changes in stem dry weight from post anthesis to harvest (Borrell et al., 1993; Ehdaie et al., 2006a) or measured directly through WSC concentration (Kiniry, 1993; Ehdaie et al., 2006b). The objective of this research was to determine the seed filling capacity of perennial ryegrass by investigating carbohydrate dynamics through changes in dry matter and the accumulation and mobilisation of LMW and HMW WSC during reproductive development.
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2. Materials and methods 2.1. Plant material A trial was positioned in a first year perennial tetraploid ryegrass (cv. Grasslands Sterling) crop on a farm in Canterbury, New Zealand (438360 S, 1728240 E) between September 2005 and January 2006. The soil type was a Wakanui silt loam (20% clay, 50% silt). Spring nitrogen (N) was applied in two applications as urea during October giving a total of 110 kg h1 applied N. The plots (1 m 2 m) were monitored using moisture probes and irrigated as required to ensure plants were not under moisture stress. Weeds were controlled with Jaguar (250 g L1 difluferican, 25 g L1 bromoxynil) at 1.5 L h1 and Pasture-Kleen (520 g L1 2,4-D) at 1.25 L h1 applied on 31 May. Moddus plant growth regulator (trinexapac ethyl 250 g L1) at 1.2 L h1 and the fungicide Folicur (tebuconazole 430 g kg1) at 400 mL h1 were applied as one application at Zadoks growth stage (GS) 32 (second node visible) (Zadoks et al., 1974). A fungicide mixture of Folicur 300 mL h1 + carbendazim (500 g L1 at 1 L h1) + Amistar (azoxystrobin 250 g L1 at 80 mL h1) was applied on 30 September and again on 27 December. 2.2. Sampling procedure The experiment was a randomised block design with four replicates. Weekly samples (0.25 m2) were collected from the start of head emergence (GS 50) in November 2005 through to harvest in January 2006. At each sampling date tillers were cut 5 mm above ground level at the same time of day (between 9:00 am and 10:00 am) to minimise diurnal changes in WSC. A subsample was weighed and dried (80 8C, 18 h) to obtain fresh and dry weights. The developmental stage of tillers was recorded and a group of comparable tillers (25 tillers per replicate) were frozen, freeze-dried and dissected into various vegetative and reproductive fractions for chemical analysis. Growth stages chosen for analysis were as follows: early head emergence (GS 50), full head emergence (GS 57), post anthesis (GS 67) mid seed fill (GS 80) and harvest (GS 92). Tissues were numbered basipetally with the flag leaf (uppermost leaf) designated leaf blade 1 (Lb 1), the leaf sheath of the flag leaf designated leaf sheath 1 (Ls 1) and the internode below the head designated internode 1 (Int 1). The dry weight of each of these tissue fractions was recorded and combined values for each fraction (leaf blade, leaf sheath, internode and seed head) were also calculated. 2.3. Extraction and analysis of water-soluble carbohydrates Freeze-dried tissues were individually ground to pass a 0.5 mm screen and the WSC extracted as follows. Samples (20 mg) were extracted twice with 1 mL 80% (v/v) aqueous ethanol (85 8C, 30 min). This fraction is termed the ‘mobile’ fraction and is composed of LMW WSC, predominantly sucrose and monosaccharides (Carpita et al., 1989). Extracts were centrifuged (13,000 g, 10 min), combined and evaporated under vacuum to remove pigments then resuspended in water (2 mL). The residue remaining after ethanol extraction was extracted twice with 1 mL distilled water (65 8C, 30 min). This fraction is termed the ‘storage’ fraction and is composed of polymeric and HMW carbohydrates such as fructans (Prudhomme et al., 1992). Carbohydrates were determined using a colorimetric anthrone assay (Jermyn, 1956) using sucrose standards whereby 10 mL extracts were mixed with 250 mL of anthrone reagent (62.5% sulphuric acid, 37.5% ethanol, 0.00125% anthrone) and incubated (100 8C, 20 min). Absorbance was read at 620 nm.
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2.4. Calculation of assimilate storage and mobilisation The amount of current assimilates contributed to seed yield was estimated from the difference between seed yield and the amount of mobilized WSC to the seed. The amount of stored or mobilized reserves in each tissue was determined based on post-anthesis changes in tissue dry weight and in WSC content (Bonnett and Incoll, 1992; Ehdaie et al., 2008). The difference between postanthesis maximum and minimum dry weight and WSC content was used to estimate the amount of stored or mobilized reserves in each tissue. The total amount of reserves in each tissue was calculated by summing those of all separate fractions. Head dry weight measured between flowering and harvest was used to monitor post-anthesis changes in head seed weight. Final seed yield per head was calculated as the difference between head dry weight at harvest and at flowering. Seed was harvested when seed moisture content was between 38% and 40%. GenStat (version 10) was used for statistical analysis using a general ANOVA model. Individual and combined fractions were designated as treatments and replicates designated as blocks. 3. Results A seed yield of 2950 kg h1 machine dressed seed was achieved for the trial site field. The trial site had a harvest index (seed yield/ mass) of 23% and a FSU (actual number of saleable seeds/potential number of florets) of 32%. 3.1. Changes in dry weights Overall, a small decrease in the total dry weights of the combined leaf blade fractions and leaf sheath fractions was observed from early head emergence to harvest (Fig. 1). In contrast there was a large overall increase in the dry weights of internodes and heads over the same period. In the leaf blades, there was a decrease between early head emergence and full head emergence and another decrease between mid seed fill and harvest. For leaf sheaths, a decrease was observed between early head emergence and full head emergence. In internodes and heads there was an increase between early head emergence and full head emergence and again between post anthesis and mid seed fill. In individual leaf blades and leaf sheaths there was generally an overall decrease in dry weights from early head emergence through to harvest (Fig. 2a and b,). The only departure from this pattern was the flag leaf (Lb 1) and the leaf sheath of the flag leaf (Ls 1) in which no overall decrease in dry weight was observed. Leaf
Fig. 1. Total dry weights of combined leaf blades, leaf sheaths, internodes and heads from early head emergence through to harvest. Bars = LSD (5%).
Fig. 2. Changes in dry weights of individual vegetative tissues from early head emergence through to harvest. (a) Leaf blades, (b) leaf sheaths, (c) internodes. Bars = LSD (5%).
blade 2 (Lb 2), Lb 3 and Lb 4 all decreased from early head emergence to full head emergence and again from mid seed fill to harvest. Similarly, leaf sheath 2 (Ls 2), Ls 3 and Ls 4 all decreased from early head emergence to full head emergence. At all growth stages, the dry weights of individual leaf blades generally decreased in the following order: Lb 1>Lb 2>Lb 3>Lb 4. Similarly, for leaf sheaths the proportion of the dry weights of individual tissues remained constant throughout development, the dry weights decreasing sequentially from Ls 1 to Ls 4. The leaf sheath of the flag leaf (Ls 1) was markedly and significantly heavier than the remaining leaf sheaths and accounted for between 45% and 52% of the total dry weight of leaf sheaths. In contrast to the leaf blades and leaf sheaths, the dry weights of individual internodes generally increased through to harvest (Fig. 2c). Internode 1 (Int 1, internode directly below head) significantly increased from early head emergence through to mid seed fill. Internode 2 (Int 2) increased from early head emergence to full head emergence, decreased through to flowering and increased again through to harvest. Internode 3 (Int 3) increased from early head emergence to full head emergence. The exception to this trend was Int 4 (basal internode) which increased during seed fill and subsequently declined through to harvest. During
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Table 1 Changes in dry weight of reproductive heads from early head emergence through to harvest. Growth stage of tiller
Dry weight (mg tiller1)
Early head emergence Full head emergence Post anthesis Mid seed fill Harvest
99a 171b 169b 310c 329c
Means followed by the same letter are not significantly different at 5% LSD.
seed fill 31% of the total dry weight of internodes was accumulated. The dry weights of the heads increased during reproductive development from early head emergence through to end of flowering and again during seed fill (Table 1). The relationship between internode dry weight and head dry weight was determined during reproductive development. There was a significant positive correlation (R2 = 0.86) between the two characters. 3.2. Changes in WSC The total WSC concentration in the combined tissue fractions changed significantly from early head emergence through to harvest (Fig. 3). In leaf blades and leaf sheaths total WSC steadily declined throughout reproductive development. In heads the concentration of WSC decreased from early head emergence to full head emergence, increased during seed fill and decreased again from mid seed fill to harvest. In contrast to the leaf blades, leaf sheaths, and heads, total WSC in internodes increased from early head emergence through to mid seed fill and remained high through to harvest. The concentrations and ratio between LMW ‘mobile’ (80% ethanol-extracted) and HMW ‘storage’ (water extracted) WSC in vegetative and reproductive tissues also changed considerably during reproductive development. The ratio of LMW to HMW WSC varied during development depending on the tissue type and growth stage. In leaf blades the concentration of both LMW and HMW WSC decreased significantly from early head emergence to full head emergence and remained low through to harvest (Fig. 4a). During head emergence, LMW WSC were the predominant form of carbohydrate. As for the leaf blades, total WSC in leaf sheaths decreased from early head emergence to full head emergence (Fig. 4b) and this trend continued through to harvest. At all growth stages HMW WSC were the predominant carbohydrate.
In reproductive heads at the start of head emergence, the concentrations of both ‘mobile’ LMW and ‘storage’ HMW WSC were similar (Fig. 5). The concentration of LMW WSC significantly increased between post-anthesis and mid-seed fill followed by a subsequent and significant decline through to harvest. In contrast HMW WSC significantly decreased during head emergence and remained low through to harvest. In internodes, as for the reproductive heads, the concentrations of both LMW and HMW WSC were similar at the start of head emergence (Fig. 6). However, the internodes showed a significant increase in the total concentration of HMW WSC from head emergence through to seed fill to harvest (Fig. 6). At harvest, these high concentrations of HMW WSC remained in the internodes.
Fig. 3. Total WSC of combined leaf blades, leaf sheaths, internodes and heads from early head emergence through to harvest. Bars = LSD (5%).
Fig. 5. Concentration of ‘mobile’ and ‘storage’ WSC in heads of perennial ryegrass during reproductive development. Bars = LSD (5%).
Fig. 4. Concentration of LMW ‘mobile’ and HMW ‘storage’ WSC in leaf blades (a) and leaf sheaths (b) of perennial ryegrass during reproductive development. Bars = LSD (5%).
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Fig. 6. Concentration of ‘mobile’ and ‘storage’ WSC in internodes of perennial ryegrass during reproductive development. Bars = LSD (5%).
From full head emergence to post anthesis LMW WSC decreased and remained low through to harvest. As high concentrations of HMW WSC were found in the combined internodes post anthesis the concentrations of WSC in individual internodes (Int 1–4) were investigated in more detail. Concentrations of HMW WSC in individual internodes increased substantially during reproductive development (Fig. 7a) with the majority of HMW WSC accumulating in the two most basal internodes (internodes three and four below the head). At the end of flowering, mid seed fill and harvest, HMW (storage) WSC in internodes 3 and 4 accounted for 76, 72 and 67%, respectively, of total combined internode HMW WSC. During head emergence and flowering, Int 3 and Int 4 were the only internodes to have a
significant increase in HMW WSC. In contrast, from post anthesis to mid seed fill, Int 2, Int 3, and Int 4 had a significant increase in HMW WSC (Fig. 7a). Of all internodes at all stages of development, Int 4 was the only internode to have a decrease in the concentration of HMW WSC, whereby the concentration decreased from seed fill to harvest. Although the concentrations of LMW WSC were small there were significant differences during development (Fig. 7b). Internode 1 showed a significant increase from post anthesis to mid seed fill followed by a significant decline to harvest. Internode 2 significantly increased from early head emergence to full head emergence followed by a decrease to post anthesis. A similar, although non-significant, trend was observed for Int 3 with an increase during mid head emergence followed by a decline through to harvest. In Int 4 there was a significant decrease in LMW WSC from early head emergence to post anthesis that remained low through to harvest. 3.3. Apparent translocation and WSC during seed fill The total amount of stored or mobilised reserves in tissues was calculated from the net loss or gain of WSC and dry weights of tissues from anthesis through to harvest (Fig. 8). The flag leaf blade (Lb 1) was the only leaf blade to gain dry weight in the post anthesis to harvest period, and, overall leaf blades had a total net loss of 11 mg tiller1. Leaf sheaths 1 and 2 had a combined dry weight gain of approximately 3 mg tiller1 whereas leaf sheaths 3 and 4 lost approximately 2 mg tiller1. All leaf blades and leaf sheaths had a net loss of WSC with a greater total net loss observed in leaf sheaths (5.7 mg tiller1). Reproductive heads also had a small net loss (4.5 mg tiller1) of WSC during seed fill. The internodes were the only tissue to have a net total increase (49 mg tiller1) in WSC during seed fill (Fig. 8). All internodes increased in WSC with internodes 2 and 3 having the largest increase (17 and 19 mg tiller1, respectively). Of the total final WSC in internodes 38% was accumulated during seed fill. The dry weight of heads and internodes considerably increased post anthesis to harvest with internodes gaining a net total 107 mg tiller1 and heads gaining 160 mg tiller1. All internodes increased in dry weight with internodes 1, 2, and 3 increasing substantially more than internode 4 (Fig. 8). There was a significant positive correlation (R2 = 0.90) between internode dry weight and internode WSC content during reproductive development. 4. Discussion
Fig. 7. Concentration of ‘storage’ (a) and ‘mobile’ (b) WSC in individual internodes of perennial ryegrass during reproductive development. Internodes are numbered basipetally: 1 = first internode below head (upper most internode); 4 = fourth internode below head (lower most internode). Bars = LSD (5%).
In perennial ryegrass the difference between potential seed yield and actual seed yield is large and variable with only 10–30% of available florets producing saleable seeds (Hampton and Hebblethwaite, 1985; Elgersma, 1990; Rolston et al., 2007). The results of this study suggest that the total amount of WSC in vegetative tissues following anthesis is not limiting seed yield. During seed fill large amounts of HMW ‘storage’ WSC accumulated in the basal internodes of perennial ryegrass, and even in the upper internodes, WSC continued to accumulate, though to a lesser degree. Similarly, Warringa and Marinissen (1997) found that in perennial ryegrass at final harvest WSC, measured as total reducing sugars, accounted for 25% (dry weight) of the stem. While the total amount of assimilate in the tiller may not be limiting seed yield, the distribution and remobilisation of available WSC to the seed is an important factor limiting seed yield. In the present study, there was a strong positive relationship between internode WSC and dry weight throughout reproductive development. During seed fill there was a net gain in dry weight and WSC in internodes with a concomitant increase in head dry weight. This suggests that perennial ryegrass has the capacity to fill
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Fig. 8. Net post anthesis loss or gain in WSC and dry weight in (a) combined and (b) individual vegetative and reproductive tissues. Gains in dry weight or WSC are indicated in bold and by shading.
available seed and accumulate WSC simultaneously with little or no remobilisation of carbohydrate from internodes to the developing seeds. In perennial ryegrass, reducing light intensity by 76% following anthesis reduced stem WSC but had virtually no effect on the relative contribution of seeds to total tiller weight (Warringa and Marinissen, 1996). In contrast, in cereals it is generally accepted that stems contribute a significant amount of assimilates to the developing grain (Borrell et al., 1989). In winter barley ‘storage’ WSC comprise approximately 30% (dry weight) of the stem at the time of maximum stem mass (Bonnett and Incoll, 1993) and in wheat, severe shading during seed fill was shown to reduce stem dry weight and grain yield (Kiniry, 1993). A strong positive relationship between stem dry weight and WSC has also been shown in wheat (Ehdaie et al., 2008). However, in contrast to the present study, both WSC and dry weight of wheat stems decreased during grain fill. Assimilation of WSC and their remobilisation within different organs of perennial ryegrass in a variety of stressful conditions needs to be examined in more detail. In the present study the proportion of LMW and HMW WSC changed considerably in vegetative and reproductive tissues during reproductive development. In all tissues, both LMW and HMW WSC generally decreased, however, large amounts of WSC accumulated in the basal internodes of perennial ryegrass. This agrees with results of Griffith (1992, 2000) who showed that postanthesis WSC increased in lower internodes of Italian ryegrass and perennial ryegrass. Previous reports suggest that younger daughter tillers may compete with seeds for carbohydrate during reproductive development. In perennial ryegrass Clemence and Hebblethwaite (1984)
found that allocation of 14C to younger tillers increased to 24% during seed development. In the present study the dry weight and WSC concentration of the most basal internode (Int 4) decreased from mid seed fill to harvest. However, secondary tillering was minimal (data not presented) and the increase in total dry weight and high concentrations of HMW storage WSC in the internodes during reproductive development suggest competition for assimilate by secondary tillers was weak. The amount of carbon assimilated into various structural and non-structural carbohydrates in different tissues during different stages of reproductive development and their possible remobilisation into the seed requires further investigation. 5. Conclusion The results of this study indicate that the seed head itself maybe an important factor driving seed fill. The decrease in dry weight and low levels of WSC in leaf blades and leaf sheaths combined with the increases in dry weight and WSC of internodes suggest the seed head has sufficient capacity to fill available seeds without contribution from vegetative tissues. This will need to be tested under a variety of conditions that reduce the photosynthetic capacity of potential source organs and tissues from anthesis through to harvest. Breeding for increased seed yield through improved source-sink relationships in the leaf, internode and inflorescence can be effective only when the factors that determine the priority for allocation of assimilates to each sink during reproductive development are understood, along with the contribution of carbohydrate from each organ. This information can
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then be used for evaluating and enhancing seed yield in forage grasses in the future Acknowledgement This research was funded by the Foundation for Arable Research, Lincoln, New Zealand. References Amiard, V., Morvan-Bertrand, A., Billard, J.-P., Huault, C., Prud’homme, M.-P., 2003. Fate of fructose supplied to leaf sheaths after defoliation of Lolium perenne L assessment by 13C-fructose labelling. J. Exp. Bot. 54, 1231–1243. Bacic, A., Harris, P.J., Stone, B.A., 1988. Structure and function of plant cell walls. In: Preiss, J. (Ed.), The Biochemistry of Plants Carbohydrates, 14. Academic Press, San Diego, pp. 297–371. Bonnett, G.B., Incoll, L.D., 1992. The potential pre-anthesis and post-anthesis contributions of stem internodes to grain yield in crops of winter barley. Ann. Bot. (London) 69, 219–225. Bonnett, G.B., Incoll, L.D., 1993. Effects on the stem of winter barley of manipulating the source and sink during grain-filling II. Changes in the composition of watersoluble carbohydrates of internodes. J. Exp. Bot. 44, 83–91. Borrell, A., Incoll, L.D., Dalling, M.J., 1993. The influence of the Rht1 and Rht2 alleles on the deposition and use of stem reserve in wheat. Ann. Bot. (London) 71, 317– 326. Borrell, A.K., Incoll, L.D., Simpson, R.L., Dalling, M.J., 1989. Partitioning of dry matter and the deposition and use of stem reserves in semi-dwarf wheat crop. Ann. Bot. (London) 63, 527–539. Cairns, A.J., Pollock, C.J., 1988. Fructan biosynthesis in excised leaves of Lolium temulentum L I. Chromatographic characterisation of oligofructans and their labelling patterns following 14CO2 feeding. New Phytol. 109, 399–405. Carpita, N.C., Gibeaut, D.M., 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3, 1–30. Carpita, N.C., Kanabus, J., Housley, T.L., 1989. Linkage structure of fructans and fructan oligomers from Tritcum aestivum and Festuca arundinacea leaves. J. Plant Physiol. 134, 162–168. Chatterton, N.J., Harrison, P.A., Bennett, J.A., Asay, K.H., 1989. Carbohydrate partitioning in 185 accessions of Gramineae grown under warm and cool temperatures. J. Plant Physiol. 134, 169–179. Clemence, T.G.A., Hebblethwaite, P.D., 1984. An appraisal of ear, leaf and stem 14CO2 assimilation 14C-assimilate distribution and growth in reproductive seed crop of amenity Lolium perenne. Ann. Appl. Biol. 105, 319–327. Ehdaie, B., Alloush, G.A., Madore, M.A., Waines, J.G., 2006a. Genotypic variation for stem reserves and mobilization in wheat I. Postanthesis changes in internode dry matter. Crop Sci. 46, 735–746. Ehdaie, B., Alloush, G.A., Modare, M.A., Waines, J.G., 2006b. Genotypic variation for stem reserves and mobilization in wheat: II Post anthesis changes in internode water soluble carbohydrates to grain yield in wheat. Crop Sci. 46, 2093–2103.
Ehdaie, B., Alloush, G.A., Waines, J.G., 2008. Genotypic variation in linear rate of grain growth and contribution of stem reserves to grain yield in wheat. Field Crops Res. 106, 34–43. Elgersma, A., 1990. Seed yield related to crop development and to yield components in nine cultivars of perennial ryegrass (Lolium perenne L.). Euphytica 49, 141–154. Griffith, S.M., 1992. Changes in post-anthesis assimilates in stem and spike components of Italian ryegrass (Lolium multiflorum Lam.). I. Water soluble carbohydrates. Ann. Bot. (London) 69, 243–248. Griffith, S.M., 2000. Changes in dry matter, carbohydrate and seed yield resulting from lodging in three temperate grass species. Ann. Bot. (London) 85, 675–680. Hampton, J.G., Hebblethwaite, 1985. The effect of growth retardant application on floret site utilization and assimilate distribution in ears of perennial ryegrass cv. S 24. Ann. Appl. Biol. 107, 127–136. Harris, P.J., 2005. Diversity in plant cell walls. In: Henry, R.J. (Ed.), Plant Diversity and Evolution: Genotypic and Phenotypic Variation in Higher Plants. CAB International, Wallingford, UK, pp. 201–227. Jermyn, M.A., 1956. A new method for determining ketohexoses in the presence of aldohexoses. Nature 177, 38–39. Kiniry, J.R., 1993. Nonstructural carbohydrates of wheat stems as influenced by sink-source manipulations. J. Plant Physiol. 134, 243–250. Matthew, C., 2002. Translocation from flowering to daughter tillers in perennial ryegrass (Lolium perenne L.). Aust. J. Agric. Res. 53, 21–28. Meier, H., Reid, J.S.G., 1982. Reserve polysaccharides other than starch in higher plants. In: Loewus, F.A., Tanner, W. (Eds.), Encyclopedia of Plant Physiology, 13. Springer-Verlag, pp. 418–471. Morvan-Bertrand, A., Boucaud, J., Prud’homme, M., 1999. Influence of initial levels of carbohydrates, fructans, nitrogen, and soluble proteins on regrowth of Lolium perenne L. cv Bravo following defoliation. J Exp. Bot. 50, 1817–1826. Pollock, C.J., Cairns, A.J., 1991. Fructan metabolism in grasses and cereals. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 77–101. Prud’homme, M.P., Gonzalez, B., Billard, J.P., Boucaud, J., 1992. Carbohydrate content, function and sucrose enzyme-activities in roots, stubble and leaves of ryegrass (Lolium perenne L.) as affected by source sink modification after cutting. J. Plant Physiol. 140, 282–291. Rolston, M., Archie, P., Rumball, W.J.W., 2005. Branched inflorescence perennial ryegrass (Lolium perenne L.) - seed yield evaluated in field trials and response to nitrogen and trinexapac-ethyl plant growth regulator. NZ. J. Agric. Res. 48, 87–92. Rolston, M.P., Trethewey, J.A.K., McCloy, B., Chynoweth, R., 2007.In: Achieving forage ryegrass seed yields of 3000 kg/ha and limitations to higher yields, Proceedings of the 6th International Herbage Seed Conference, Norway. Trethewey, J.A.K., Harris, P.J., 2002. Location of (1!3)- and (1!3) (!4)-b-D-glucans in vegetative cell walls of barley (Hordeum vulgare) using immunolabelling. New Phytol. 154, 347–358. Turner, L.B., Humphreys, M.O., Cairns, A.J., Pollock, C.J., 2001. Comparison of growth and carbohydrate accumulation in seedlings of two varieties of Lolium perenne. J. Plant Physiol. 158, 891–897. Warringa, J.W., Marinissen, M.J., 1996. The effect of light intensity after anthesis on dry matter distribution and seed yield of Lolium perenne L. Grass Forage Sci. 51, 103–110. Warringa, J.W., Marinissen, M.J., 1997. Sink-source and sink-sink relations during reproductive development in Lolium perenne L. Neth. J. Agric. Sci. 45, 505–520. Zadoks, J.C., Chang, T.T., Zonzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415–421.