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STARCH ACCUMULATION IN TEMPERATE FORAGE GRASSES STARCH ACCUMULATION IN TEMPERATE Andy Cairns

S ugars are the primary source of metabolic energy Available carbohydrate limits nitrogen use

FORAGE GRASSES in food and underpin agricultural productivity. Our efficiency work on the improvement of sugar content in The reason for this poor nitrogen conversion is temperate grass forage has, in the past, concentrated primarily carbohydrate (energy) limitation. The on the soluble sugars, sucrose and fructan, the microbes in the rumen require readily-available predominant natural reserve carbohydrates in these carbohydrates for growth and, in turn, nitrogen species. Although starch, the more common leaf uptake. We know that increasing the fermentable reserve in the plant kingdom, accumulates to high sugar content of forages greatly improves animal concentrations in grass seeds, (as is clear from crops performance. For example, research at IGER has such as wheat and barley), it does not reach high shown that a 6% increase in sugar content resulted in concentrations in stems and leaves. So the temperate a 16% increase in liveweight gain in lambs. So cereals and grasses clearly have the genetic potential increasing sugar content in forage has the potential for starch accumulation but its expression is subject for significant increases in animal performance. We to differential developmental regulation. Why starch are interested in all aspects of plant sugar is a minor component of the reserve in grass forage metabolism which may assist in improving the is not known. Understanding the manufacture and provision of nutritional energy for animals. regulation of starch accumulation in forage tissue may provide insights into means of improving its Improving fermentable carbohydrate in accumulation. This article summarises why sugars forage grasses and starch are important in ruminant production, Our best forage grass varieties yield an average of what is currently known about starch accumulation about 200 g / kg dry matter in the form of the in temperate-grass leaves and the possible fermentable sugars, sucrose and fructan. Leaf tissue significance of starch in the evolution of reserve has the known physiological capacity to accumulate metabolism in the fructan-grasses. at least twice this amount, so there is the potential for improvement. There is a significant natural variation Forage feeding of ruminants in the capacity for soluble sugar accumulation within Post-BSE there is an increasing impetus to feed populations of our cultivated varieties. Historically, ruminant animals with fresh forage, hay and silage in our research has concentrated on understanding the preference to manufactured concentrate. There is, physiological basis for the differences in sugar however, an inherent difficulty because the content and exploiting the variation by genetic efficiency of forage nitrogen conversion into meat mapping (see the contribution of Humphreys/Turner, and milk is currently only 20-25%. This is this issue). We are now diversifying our interests to inefficient in terms of productivity and also means explore the potential of structural sugars in the cell that the remainder is excreted, with attendant, wall and starch, as alternative means for potentially adverse, environmental consequences.

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improvement of energy provision. Here we The pattern of starch accumulation in the temperate concentrate on starch accumulation. forage grasses is unusual.

Starch accumulation in leaves of the The pattern of accumulation is quite distinct from STARCH ACCUMULATION IN TEMPERATE temperate forage grasses that in most plants, where starch is usually the Asurvey revealed a surprisingly small literature on predominant form of stored carbon. In Lolium, starch accumulation and its regulation in leaves of starch is a minor component, secondary to sucrose. A plants in general. Even less information is available In addition, once sucrose reaches high levels the FORAGE GRASSES concerning starch in grass tissues. This prompted an production of fructans is induced. In the experiment experimental study of the pattern of starch shown in Figure 1.1, fructan accumulation continued accumulation in the model ryegrass, Lolium when starch accumulation slowed, indicating that the temulentum. When leaves were continuously supply of photosynthetic products was not the illuminated, the pattern of starch, sucrose and fructan limiting factor. From the perspective of forage accumulation was as shown in Figure 1.1. agronomy the most interesting question is "what limits starch accumulation?" If we could understand the limitation, we may be able to beneficially increase starch content in leaves. 130 Sucrose On the basis of the data in Figure 1.1, we might speculate that continued sucrose accumulation is required for continued starch accumulation.

65 Alternatively, switching on fructan synthesis may Fructan inhibit starch accumulation, or fructan synthesis may compete with and divert the flow of photosynthetic

rmentable carbohydrate (g/kg DM) carbohydrate rmentable Starch

Fe products away from starch. Results from 0 experiments to test these ideas are shown in Figures 01020301.2 and 1.3. Time (h)

Figure 1.1. Accumulation of reserves in leaves of Lolium in What happens to starch accumulation when continuous light. fructan synthesis is blocked? Figure 1.2 shows the result of preventing fructan Four interesting features emerged: synthesis by using the antibiotic, cycloheximide. The (i) Starch accumulation was immediate from the experiment provides a number of useful facts about beginning of the photoperiod indicating that the the regulation of starch accumulation. Firstly, enzymes were present at all times. (This is unlike sucrose accumulated to three times the "normal" fructan synthesis which began only after about 8 level but did not result in enhanced starch synthesis. hours.) Secondly, the abolition of fructan synthesis did not (ii) Starch was synthesised "from new" without the enhance starch accumulation, so competition for the need of a starch primer. products of photosynthesis by fructan does not (iii) The rate of accumulation was five-ten fold less provide an explanation for the limitation of starch than that for sucrose and fructan. accumulation. (iv) Starch accumulation ceased when it reached 35g / kg dry matter at 16 h.

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A B 250 Sucrose

STARCH ACCUMULATION IN TEMPERATE Sucrose 200

150 tan

n FORAGE GRASSES

a

t Fruc c 100

u

r Sucrose

F

Carbohydrate levels (g/kg DM) levels Carbohydrate 50 Starch Starch tan Fruc - + 0 WF G WF G Cycloheximide SStFr SStFr Control Cycloheximide TREATMENT

Figure 1.2 Effect of cycloheximide on the accumulation of reserves in leaves of Lolium in continuous light. A) Shown by chromatography. B) Changes in the amounts of sugars. The important changes occurred in sucrose (= S), fructan (= F) and starch (= St), G, Fr and W = glucose, fructose and total water-soluble sugar, respectively.

What happens to starch accumulation when sucrose synthesis is blocked? 125 Mannose has a similar chemical structure to glucose but, when fed into leaves of most starch- 100 accumulating plants, it interferes with metabolic communication reducing sucrose synthesis and 75 increasing starch accumulation. When fed to grass leaves at low concentration, mannose has a marked 50 inhibitory effect on sucrose synthesis but inhibits,

NSC (G/KG DM/7H) rather than enhances, starch accumulation (Figure 25 1.3). In common with the data in Figures 1.1 and 1.2, the effect of mannose shows that the regulation of 0 starch synthesis in Lolium leaves is quite distinct 0h W M M+P from that in most plants. Treatment

Where does the starch occur? Figure 1.3. Effect of mannose on the accumulation of sucrose ( ) Figure 1.4 shows a microscopic section through a and starch ( ) in leaves of Lolium in continuous light. Effect of grass leaf after accumulation of carbohydrate for 24 10mM mannose (M) and 10mM each of mannose and phosphate supplied simultaneously (M+P), compared to a water only control hours. The starch accumulates predominantly in the (W). Mannose inhibits sucrose and starch accumulation. chloroplasts of mesophyll cells. This is the same location as sucrose and fructan synthesis and implies

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What is the molecular structure of Lolium leaf starch? Leaf starch has been analysed by various

chromatographies and its composition probed by its STARCH ACCUMULATION IN TEMPERATE sensitivity to enzymatic digestion (Figure 1.5). The native starch has a molecular mass of 970 kDa but 99% of the mass is sensitive hydrolysis by isoamylase (de- branching enzyme) and is converted to polymers FORAGE GRASSES smaller than 6.4 kDa. The starch is unusual in that it is predominantly a highly-branched amylopectin-like polymer containing very little long chain amylose.

Conclusions Leaves of the temperate grasses have a distinctive pattern of reserve metabolism based on soluble sugars. They also accumulate starch at low rates and to a low maximal concentration. The overall pattern of Figure 1.4. Microscopic section through a Lolium leaf which had accumulation with respect to sucrose differs from that accumulated carbohydrate for 24 hours. Starch granules (S, arrows) are visible within the chloroplasts of mesophyll cells. in many starch-accumulating plants. Treatments that modify sucrose accumulation, resulting in increased starch in most higher plants, do not release the

6 70 a limitation and enhance starch accumulation in Lolium.

Log The mechanism of this limitation is unknown and its Native starch 5 10

oeua asCH Molecular mass elucidation is of considerable interest as a potential 4 35 route to improvement in forage energy content. This is Isoamylase especially important in ensiling, because starch persists fragments in the silo and is present in the finished product. Refractive index (% FSD) Refractive 0 Increased starch in grass tissues holds out the 01020 30 possibility of high-energy silage. The pattern of starch b 10 100 0.4 accumulation in the fructan-grasses contributes to our 3

COONa eluant (M) fundamental understanding of carbohydrate partitioning in plants because it provides an example of 50 0.2 20 a regulatory organisation distinctive from the rest of the 30 plant kingdom.

Detector response (nC) response Detector Fructan accumulation occurs in about 15% of plant 0 0.0 010203040 species, but the reason why these plants have this Time (min) distinctive metabolism is not known. The above results

Figure 1.5. Analysis of starch structure. a) Size exclusion suggest an explanation. If the ancestral fructan grasses chromatography shows the native starch is a large polymer, 99% were limited in their ability to accumulate reserves as of which is sensitive to isoamylase digestion and converted to starch, there may have been a selective pressure small fragments. b) Isoamylase fragments containing between 5- towards an alternative storage form, resulting in the 50 sugar residues shown by High Performance Anion Exchange Chromatography. evolution of fructan accumulation.

that the regulatory processes governing carbohydrate Contact: [email protected] partitioning in leaves all occur in the same cell.

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