Variations in free protein amino acid compositions of grass leaves

BiochemlcalSystematlcsandEcology,Vol. 10, No. 1, pp. 55-63, 1982.

~-1978/82/010055-09 $0G.00/0 © 1982PergamonPress Ltd.

Printed in Great Britain.

Variations in Free Protein Amino Acid Compositions of Grass Leaves HOCK-HIN YEOH and LESLIE WATSON Taxonomy Unit, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601, Australia

Key Word Index - Poaceae; grasses; leaves; free protein amino acids; taxonomic variation. A b e t r a c t - Total free protein amino acids in grass leaves varied from 0.02 to 0.76 g % fr. wt and were present in greater amounts in grasses (especially C3 forms) collected from the field during a hot, dry summer (for 31 species/28 genera; 0.28 _+0.21 g % fr. wt) than those grown in the greenhouse (for 48 species/36 genera; 0.10 _+0.08 g % fr. wt). Variation in free proline was generally greater in field-collected grasses (0.5-47.0% total free amino acids) than in greenhouse-grown grasses (0.4-8.4%). Taxonomic patterns are detectable in the free protein amino acid compositions of grasses grown in the greenhouse. Among the taxa, chloridoids, danthonioids, Aristida, eu-panicoids and andropogonoids exhibit closely similar free protein amino acid profiles which are distinguishable from those of pooids, while Oryza, Stipeae and Ehrharteae share certain features of their free protein amino acid patterns. Variation in free alanine is clearly associated with the different photosynthetic pathways, C3 grasses being generally lower in alanine than C4 grasses, irrespective of taxonomic groupings and growing conditions.

Irm'oduction The presence or absence of a particular nonprotein amino acid is generally regarded as being of greater value as a taxonomic character than is the free protein amino acid composition, and certainly there is more published information showing the taxonomic value of non-protein amino acids than is available for free protein amino acids [1-3]. Two factors tend to suggest that free protein amino acids lack potential in this respect; (a) the same protein amino acids occur universally, and (b) free protein acid compositions are readily influenced by the physiological status of organs and by changes in the environmental and nutritional conditions [4]. Nevertheless, there are a few reports which show some correlation between free protein amino acid compositions of plants and taxonomy [4-8]. There are numerous reports on the free protein amino acid compositions in leaves of grass species, but most of them are not taxonomically orientated. For example, Dabrowska and Przybylska [9] found no pattern in the free protein amino acid compositions of leaves from 34 grass species, comprising 32 pooids, a eu-panicoid and a chloridoid (see Table 1 for classification). Reddi and Phipps [10], who investigated the free protein amino acid compositions in the flowers of one grass tribe, the Arundinelleae, concluded that variations in the patterns were useless as taxonomic characters. However, considering the restricted ranges of

grass genera examined in these earlier studies, and the semi-quantitative nature of the analytical methods employed in one of them [9], one cannot safely assume from these results that there is no taxonomic pattern to be found in the family as a whole. Furthermore, the requisite information cannot be compiled from the numerous published studies on individual species and genera because of differences in methodology and because the accumulated sample still represents inadequate taxonomic coverage. This paper describes a preliminary attempt to explore the extent to which free protein amino acid compositions of grass leaves are taxonomically related at subfamily and tribal levels, and whether they are related to variation in photosynthetic pathways. The topic poses daunting problems over sampling of plant material, bearing in mind that marked fluctuation in free protein amino acid levels may be expected to occur in leaves of the same plant at different times and under different environmental conditions [4]. However, some effort has been made to standardize sampling and experimental procedures, and the sample presented here represents a better coverage of the grass family than has previously been achieved.

Results and discussion Variation in Free Protein Amino Acid Contents

The free protein amino acid contents of leaves from 48 grass species (36 genera) grown in the greenhouse range from 0.02 to 0.38 g % fr. wt

{Received 26 January 1981 ) 55

56

HOCK-HIN YEOH AND LESLIE WATSON

~o .

<

D

35

, ~

~ .

~$~z~88~ .

.

.

.

.

.

.

.

.

.

.

.

.

°~ -

.

.

B

.

.

~SX .

.

~ 0

O0

0000000~0000

O0

0

0000

000

O0

dOOOdOd~OddO

~

o

o0~o

~ d

dd

dddoddddddod

~0

o

dddo

o

oo

ooo

oo

ooo

oo

ooo

O

OO

OOO

N

N~

s~

0

~E

z_ Z 0

3 m

09

u. m <

Q0 Z

o

o 0.

0n

xl

0

< 0

x:

_z <

I

Z p0

N

i :>~

.c:

FREE PROTEIN AMINO ACID COMPOSITIONSOF GRASS LEAVES

57

c

8~

<

~oo

o

ooo

~

ooo

0

~

~

E

ooo o

OO0

0

000

000

0

000

000

0

000

000 000

0 0

000 000

0 0 0

~

0 0 0

0 0 0 0 0 0 0 0 0

O0

~

J

9 CJ

N

<

II

o . . . . . .~x (.9

~0

~

~ O ~ O ~ m

~ m ~

~

o

I ~ '~" ~'~ ~ ~ '~" O00'~ t'~

~

÷

0'~ ~'~ '~~"

CO

~1~ 0 ~'~

< =g

o ~~~ ~ ~0

o~

~.~.~.~.~.~.~ ~ ~

~

z~

~.~ ~.~ <

HOCK-HIN YEOH AND LESLIE WATSON

58

.E

LS~ oo0

0ooo

O0

000o

000

!T:

<

E

9

~-I N

I-o

o

@ ,_J

)

< n.oe r

~mmm

om~

~ m

Z

_o o

©

cr~ +

< ©

_z < Z

~o 0

FREE PROTEINAMINO ACID COMPOSITIONSOF GRASS LEAVES

(mean±s.d.=0.10±0.08); and of these, only four exceeded the value of 0.2 g % fr. wt (Table 1). By contrast, leaves of 31 grass species (28 genera) collected from the field during the hot and dry Canberra summer of 1979--80 have shown a much wider range, 0.04-0.76 g % fr. wt (mean ±s.d. =0.28±0.21), and more than half (17/31) had a value greater than 0.2 g % fr. wt (Table 2). The difference in mean free protein amino acid content between greenhouse-grown and fieldcollected samples is thus significant at the 5% probability level. Comparisons among grass species for which both greenhouse and field samples were analysed for total free protein amino acid contents (Tables 1 and 2, asterisked), show 7 out of 13 species grown in the greenhouse (including 5 out of 6 pooids) having very much lower free protein amino acid contents than their field-collected counterparts. Greenhouse-grown materials kept in the dark for 17 h prior to extraction were not significantly different in free amino acid contents from those of the same species grown in normal greenhouse conditions and harvested at midday (Table 3). The total free protein amino acid contents of the Co grasses grown in the greenhouse range from (~.04 to 0.38 g % fr. wt and those of the C4 species range from 0.02 to 0.20 g % fr. wt (Table 1). The mean value for the C3 species (0.11 g % fr. wt) is higher than that for the C4 species (0.07 g % fr. wt), but the difference is not statistically significant. Among the field-collected grasses, the C3 species show a bigge~ variation in the total free protein amino acid contents (0.06-0.76 g % fr. wt), compared with the range for the C4 species (0.04-0.34 g % fr. wt); but in this case, the difference between the means is significant (0.32 g % fr. wt for C3 species; 0.14 g % fr. wt for C4 species); i.e. field-collected C3 grasses tended to have higher free protein amino acid contents than field-collected C4 species (Table 2). Among the eu-panicoid grasses grown in the greenhouse, a C3 species, Oplismenus aemulus and its C3-C4 intermediate relative, Panicum milioides, showed free protein amino acid contents (0.13 and 0.11 g % fr. wt, respectively) which were higher than 5 out of 7 of their C4 relatives (0.02 to 0.04 g % fr. wt); the two exceptions being Pennisetumtyphoidesand Spinifex hirsutus (Table 1). However, the same trend (i.e. for C3 eu-panicoids to show higher levels of free protein amino acids than their C4 eu-panicoid relatives) is more convincingly seen in the eu-panicoid species collected from the field; here, the C3 species, Entolasiamarginataand/sachne globosa, are about three times higher in total free protein

59

amino acid content than the C4 species, Paspa/um disdchumand Spinifexhirsutus (Table 2). Despite the small samples and the need to be cautious in the face of environmental variations, these data have yielded three interesting observations. First, grasses collected from the field in the hot and dry conditions of a Canberra summer have tended to accumulate free protein amino acids at higher levels than their greenhouse-grown counterparts. This observation is in accord with reports that leaves of Lolium spp. and Paspa/um dilatatum grown in the greenhouse are lower in total contents of free protein amino acids than those of the same species grown outside the greenhouse [4]. Second, the highest levels of free protein amino acids and the largest differences between field-collected and greenhouse-grown samples involve members of the Pooideae (Pooideae being the main grass group represented in cool and temperate regions [11-14]). Third, C.~ grasses from both greenhouse and field have tended to show higher free protein amino acid contents than have C4 species. In view of these observations, and published reports that levels of free protein amino acids in plants increase under moisture stress [15], plus the fact that C3 plants generally have lower moisture and temperature tolerance levels than do C4 plants [16], it would be worthwile investigating (a) whether increase in total free protein amino acid contents of C3 and C4 grass leaves is an indication of a physiological response to stress conditions and (b), whether the tendency to accumulate free protein amino acids at higher levels in C3 grasses than in C4 grasses is related to the lower moisture and temperature tolerance levels of C3 grasses. Plants grown under moisture stress conditions are known to accumulate free proline (Pro) [15]. Therefore, it may be no coincidence, (a) that there is more variation in free Pro in leaves of fieldcollected grass material (0.5-47.0% total free protein amino acids) than in greenhouse-grown material (0.4-8.4%) (cf. Tables 1 and 2); and (b), that of the 3t field-collected species, only 10 had Pro levels less than 3%. By contrast, 39 out of 48 greenhouse-grown species had less than 3% Pro level, the difference between the two means (11.5% Pro for field material; 2.0% for greenhouse material) being highly significant. Referring to the free Pro contents of 13 species sampled from both field and greenhouse (see Tables 1 and 2, indicated by asterisks), 10 of the field-collected samples have given higher Pro contents than their greenhouse counterparts. Increase in Pro alone, however, is not sufficient to account for the overall higher content of free

60

HOCK-HIN YEOH AND LESLIE WATSON

8~

SS~£~888S88S

,0~

o0o0000o0o00

c ~ o 0 c~ O d c~ c 5 c 5 , ~ 0 0

0 0 0 ~ 0 0 0 0 ~ 0 0 0

00o0oooo0o00

~ 0 0 0 ~ 0 0 0 0 ~ 0 0 "r

¢~1000

0

~0

O0

0

~

0

0

O O O O O O O O O ~ O O

,.-: d ,~ o o , ~ o o , - : c~ d 0

~ 0 0 0 0 0 0 0 ~ 0

oodoO00oO000 ....

~

~

(.3

o

,-: c~ e6 o ,-: c~ c5 o ,.~ o ,.-:o _x

2

4-

I--t.~

E ~'~

,

{,o

~ ~

,

~ .~.~.~

~.~°

~

~.~

I .-~

.a ,~

.~.~

~.

FREE PROTEIN AMINO ACID COMPOSITIONSOF GRASS LEAVES

protein amino acids in field-collected grasses (see above). Reference to data on species sampled from both field and greenhouse (see Tables 1 and 2, indicated by asterisks), shows (on converting the data from % total free amino acids to g % fr. wt) that the increase in total free protein amino acid content is due to an overall increase in the amino acid levels.

Variations in Free Protein Amino Acid Compositions General considerations. The free protein amino acid compositions of leaves from 31 grass species collected from the field (Table 2) are different from those of 48 grass species grown in the greenhouse (Table 1); and in some cases, the differences (even for members of the same species) are very great indeed: see in particular the contrasting results for Agrostis avenacea, Holcus lanatus, Amphibromus neesii, Glyceria declinata, Bfiza maxima, Danthonia pallida, Eragrostis benthamii, Spinifex hirsutus, Nassella trichotoma, Stipa falcata, Ehrharta erecta and Microlaena stipoides in Tables 1 and 2. The amino acid fractions Asx, Thr+ Ser, GIx and Ala constitute the bulk of the total free protein amino acid composition in all leaves, but the field-collected material shows greater fluctuations in Asx, Thr+Ser, GIx, Pro and Ala, and shows generally higher levels of Val, lie and Phe. These facts are consistent with published data on free protein amino acids in leaves, which show that such variations in compositions may be caused by differences in conditions of growth [4]. Free protein amino acid compositions of the 12 taxonomically diverse grass species (including C3 and C4 types) kept in the dark prior to extraction, are consistently high in Asx and Val and low in GIx and Gly, compared with those for the same species grown in normal greenhouse conditions and harvested at midday (Table 3). These differences may well be attributable to diurnal changes in free protein amino acids, which in leaves of rice cultivars fluctuate independently of one another [17]. Taxonomic patterns. Although conditions of growth and physiological status of leaves affect the free protein amino acid compositions, it seems that there is some taxonomic predictability about the free protein amino acid patterns of leaves of greenhouse-grown grasses (Table 1; taxonomic summary with mean values for grass groups in Table 4). Group-by-group comparisons among pooids, chloridoids and panicoids sensu lato, show that the pooid species as a group exhibit significantly higher Asx and GIx and lower Ala

61

than the rest (the difference being significant at the 5% level). The panicoids (i.e. eu-panicoids and andropogonoids [18]), however, have significantly higher Tyr and Lys levels than have the chloridoids; and the danthonioid species sampled show two features of the chloridoid and panicoid pattern, i.e. low Asx and Glx. Oryza, Stipeae and Ehrharteae constitute a loose but interesting assemblage of taxonomic oddments, sharing certain features of bambusiod spikelet morphology, such as three Iodicules (Stipeae), stamens in excess of three and reduced glumes (Ehrhartaand Microlaena) [18]. If regarded for the present purpose as a taxonomic group, they are seen to differ from the pooids in having lower GIx levels (22.3-27.0%; pooids=30.845.4%), and from the danthonioids, chlorodoids and panicoids in their higher Asx levels (21.025.7%, others=16.7-18.3%) (see Table 4). However, there are differences within this "group". In particular, Ehrharta and Microlaena (the Ehrarteae) have higher Ala (10.3 and 15.7%, respectively) than the rest (5.0--8.3%). The free protein amino acid pattern of Afistida is high in Ala and low in Asx and GIx, and it comes closest to those of the panicoids. Within the pooids, differences in the free protein amino acid compositions are found. The two Bromus spp. (representing the Bromeae) have given the highest level in Asx, while two genera of the Aveneae and three Triticeae have manifested lower Asx levels than have 12 Agrostideae (cf. [19]). Considering samples collected from the field, no taxonomic patterns have emerged, with the notable exception that chloridoids and panicoids proved consistently high in alanine (Ala) (see discussion of variation in free Ala). With reference to the grasses kept in the dark prior to extraction (Table 3), the eu-panicoid and andropogonoid species tested are higher in Ala and lower in GIx than are Oryza and the pooid genera. The same eu-panicoid and andropogonoid species grown in normal greenhouse conditions and extracted at midday, still maintain the profile of high Ala and low GIx, but in addition, the eupanicoids show a lower Asx level. In view of the different experimental treatments, one has to be wary of quantitatively comparing these data (Table 3) with those of greenhouse-grown material (Table 1) discussed earlier. Nevertheless, characteristic features of the free protein amino acid composition of the panicoids grown in the greenhouse are also detected here (i.e. high Ala and low GIx; see Table 3). Systematic variations in the free protein amino acid compositions have proved readily detectable

62

HOCK-HIN YEOH AND LESLIE WATSON

TABLE 4. FREE PROTEIN AMINO ACID COMPOSITIONS OF GRASS LEAVES: TAXONOMIC GROUP MEANS OF DATA IN TABLE 1 Amino acid composition (g % total amino acids) Major groups/tribes (no, spp./no, genera) POOIDS(24/15) Triticeae(3/3) Bromeae(2/1) Agrostideae(12/4) Aveneae(2/2) Meliceae (1/1) Poeae (4/4)

Asx PP.0 16,7 29.4 22.7 16.4 22.4 23,1

BAMBUSOIDS, ETC. Oryzoids(1/1) Ehrharteae(2/2)

25.7 12.6 27.0 2.1 21.0 25.8 22.3 4.3

STIPEAE(3/2) DANTHONIOIDS(3/2) ARISTIDEAE(1/1) CHLORIDOIDS (3/3)

Thr + Ser 18.3 32.3 11.2 15.0 24.9 17.1 18.2

GIx 41,5 30.8 43.5 44,0 39.3 45.4 41,0

Pro 1.2 2,4 0.8 0.9 1.5 0.6 1,1

Cys 0.0 0.1 0.0 0,0 0,0 0.1 0,0

Val 1.9 1.8 1,7 1.9 2.2 1.4 1,9

Met 0.1 0.1 0.1 0,2 0.1 0,1 0,1

lie 11 0.8 0.8 1,2 1,3 1.0 1.0

Leu 0.9 1.1 0.8 0.7 1.2 1.0 0.9

Tyr 0.8 0.6 0.7 0,9 1.0 10 0,8

Phe 1,2 1.3 1.3 1.1 1.3 1.1 1.3

His 0,6 1.1 0,2 0.6 0.5 0.3 0.4

Lys 2.1 1.9 2,1 2.2 2.1 2.0 2.1

Trp 0,4 0.2 0.1 0.4 1,7 0.1 0.4

Arg 0.5 0.9 0.5 0,5 0,6 03 0,4

1.9 7.4 6.8 13.0

0.2 0.1

2.5 1.0

0.2 0.0

1,9 2.2

1.9 0.4

2.3 0.6

2.7 0.7

2.1 0.2

7,9 1.0

0.6 0.4

0,9 0.7

22.3 33.8 26.2 2.7

1.1

6.5

0.0

1.2

0.0

0.6

0.5

0.5

0.9

0.2

2.0

0.0

1.2

18.3 19.9 35.1 3.3

0.4

13.2

0.0

2.0

0.2

1.2

0.7

0.7

1.1

0.5

2.2

0.8

0.5

9.3 37.1 19.8 8.1 17.2 20.0 37.0 4.3

PANICOIDSsensulato(11/10) 16.8 18.4 30.4 1.7 Eu-panicoids(9/8) 16.7 20.0 29.0 1.9 Andropogonoids(2/2) 17.1 10.9 37.0 0.9

Gly 0.7 1,1 0.4 0.6 0.9 0.5 0.5

Ala 6,7 6.9 6.2 7.0 5.5 5.6 7.0

1.6 13.6

0,0

1.0

0.0

5.5

0.7

0.7

1.0

0.3

1.0

0.1

0.2

0.6

14.3

0.0

1.4

0.1

0,6

0.7

0.6

0.9

0.4

1.5

0.2

0.3

1.5 18.1 1.7 18.0 0.9 18.6

0.1 0.1 0.0

1.8 1.7 2.2

0.3 0.2 0.5

0.9 0.9 1.2

1.4 1.5 0.9

1,6 1.3 3.0

1.5 1.6 1.2

0.8 0.8 0.8

3.8 3.6 4.6

0.2 0.2 0.1

0.8 0.9 0.5

with fairly minimal standardization of conditions of growth. However, the patterns found tend to separate major groups, so it is understandable that Reddi and Phipps [10] should have failed to find any taxonomic pattern within the tribe Arundinelleae. It is noteworthy that in spite of the need to equivocate in the face of the relative inconstancy of free amino acid levels, the systematic patterns found here accord quite well with those uncovered by parallel studies of caryopsis protein amino acid compositions [20], total protein amino acid analyses of leaves [21] and amino acid analyses of grass RuBP carboxylase [21 ]. Difference between C3and C~grasses. The data of Tables 1-3 have been examined in detail for any differences between C3 and C4 grasses. Correlations between photosynthetic pathway and certain amino acids are inevitable, given that the C3 and C4 photosynthetic pathways are also taxonomically correlated. However, only in the case of Ala is variation more precisely predicted by photosynthetic pathway than by taxonomy. Leaves of C3 greenhouse-grown grasses are markedly lower in Ala (for 33 species, 1.7-15.7%; mean=7.1%) than are C4 greenhouse-grown grasses (for 14 species, 9.8-28.9%; mean = 19.1%) (see Table 1), the difference (which is statistically significant at the 5% level) being apparent even between C3 and C4 species of the same subfamily. In the eu-panicoids, Oplismenus aemulus (C3) has 7.3% Ala in contrast with its C4 relatives, where the range is 15.3% (Pennisetum typhoides) to 28.9% (Setar/a glauca). Likewise,, among the danthonoids, Monachatherparadoxaand D. pal~ida

(both C3) exhibit much lower Ala levels (7.3 and 5.5%, respectively) than does the C4 representative, Tffraphismollis (26.8%). The field-collected grasses showed large variations in their free protein amino acid compositions, but even here leaves of C3 grasses are generally lower in Ala (for 24 spectes, 1.1-29.0%, mean =6.7%) than those of C4 grasses (for 7 species 10.5-31.5%; mean= 14.7%) (see Table 2), and here too the difference is statistically significant. Among eu-panicoids, the C3 species, Entolasia marginata and Isachne globosa, again exhibit markedly lower Ala (4.2 and 5.5%, respectively) than do their C4 counterparts, Paspalumpaspalodes and Spinifex hirsutus (10.5 and 31.5%, respectively). Variation in Ala level in material that had been kept in the dark shows the same trend, the C3 grasses having an Ala range of 5.1-9.3%, in contrast with the C4 range of 6.2-30.7 % (Table 3); and a similar difference is noted for material grown in normal greenhouse conditions and harvested at midday: 3.0-6.2% Ala for C3 grasses and 17.130.2% Ala for C4 grasses. It is likely that the higher Ala levels of C4 grasses relate to the fact that Ala is an intermediate metabolite in the C4 photosynthetic pathway [22-24]. On the other hand, it seems worthwile finding out whether or not the higher Ala levels in C4 grasses are a consequence of the higher photosynthetic rates generally known to exist among C4 plants [16]. It has been reported in a C3 plant (a fern; [25]) that free Ala synthesis is proportional to the rate of photosynthesis.

FREE PROTEIN AMINO ACID COMPOSITIONS OF GRASS LEAVES

Experimental Plant material. Grasses were either grown from seeds in the greenhouse (plants watered daily; maximum/minimum temperature cycle, 24°/16°; no artificial lighting) or collected from the field in the course of the hot and dry Canberra summer of 1979-80. Species were identified with reference to regional Floras. Unless otherwise stated, greenhousegrown plants were harvested in the morning, between 9 a.m. and 10 a.m. Dark experiment. Plants grown in the greenhouse were kept in a dark room for at least 17 h (overnight) prior to harvesting the leaves for free protein amino acid analysis. The extraction procedure Isee below) was here followed under safe-light conditions. For comparisons, greenhouse plants of the same species grown from the same batches of seeds were harvested on the same day at mid-day. Preparation offreeproteinaminoacids. Mature and healthy culm leaf blades (avoiding flag leaves) were excised from their sheaths at the ligule. 1 g leaf blade, usually representing several leaves, was finely cut up and ground in liquid N2, then homogenized with 5 ml 3% (w/v) sulphosalicylic acid [26]. The homogenate was allowed to stand for 30 min at 0 ° before centrifugation at 27,000 g for 30 min. The supernatant was eluted through a 1.5 x 4 cm column of Bio-Rad AG50W-X-2 (100-200 mesh) cation-exchange resin, and was washed with 12 ml H20, followed by 12 ml 10% (w/v) NH 3 soln. The eluate from the NH 3 soln washing was collected and evaporated to dryness on a rotary evaporator. The sample was taken up in 1.0 ml 2 N Na citrate pH 2.2; 10 ~1 and 100 ~1 samples were used for analysis on a Beckman amino acid analyser 119CL. Total free protein amino acid content. This was calculated from the amino acid analysis and was expressed as g % fr. wt leaf.

Acknowledgements - We thank Paul Hattersley for helpful comments, Kath Britt for performing amino acid analyses and Gillian Hines for impeccable typing.

References 1. Fowden, L. (1973) Phyrochemistry (Miller, L. P., ed.) Vol 2, p. 1. Van Nostrand-Reinhold, New York.

63 2. Smith, P. M. (1976) The Chemotaxonomy of Plants. Edward Arnold, London. 3. Pilbeam, D. J. and Bell, E. A. (1979) Phytochemistry 18, 973. 4. Hegarty, M. P. and Peterson, P. J. (1973) Chemistry and Biochemistry of Herbage (Butler, G. W. and Bailey, R. W., eds) Vol. 1, p. 1. Academic Press, London. 5. Mansford, K. and Raper, R. (1954) Nature (London) 174, 314. 6. Reuter, G. (1957) Floral45,326. 7. Impellizzeri, G., Mangiafico, S., Piattelli, M. and Sciuto, S. (1977)Biochem. Syst. Ecol. 5,77. 8. De Simone, F., Senatore, F., Sica, D. and Zollo, F. (1980) Biochem. Syst. EcoL 8, 77. 9. Dabrowska, T. and Przybylska, J. (1970) Acta Soc. Bot. Pol. 39, 445. 10. Reddi, V. B. and Phipps, J. B. (1972)Brittonia24,403. 11. Hartley, W. (1950)Aust. J. Agric. Res. 1,355. 12. Hartley, W. (1958)Aust. J. Botany6,343. 13. Hartley, W. (1951)Aust. J. Botanyg, 152. 14. Hartley, W. (1973)Aust. J. Botany21,201. 15. Hanson, A. D., Nelsen, C. E., Pedersen, A. R. and Everson, E. H. (1979)Crop Sci. 19, 489. 16. Hatch, M. D. (1976) Plant Biochemistry (Bonner, J. and Varner, J. E., eds.) p. 797. Academic Press, New York. 17. Banerlee, A. and Sircar, S. M. (1978) Indian J. Agric. ScL 48, 278. 18. Watson, L. and Dallwitz, M. J. (1980) Australian Grass Genera. Anatomy, Morphology and Keys, p. 141. The Australian National University, Canberra. 19. MacFarlane, T. D. and Watson, L. (1982) Taxon (in press). 20. Yeoh, H. H. and Watson, L. (1981) Phytochemistry ~rl, 1041. 21. Yeoh, H. H. (1980) Ph.D. thesis, Australian National University, Canberra. 22. Hatch, M. D., Kagawa, T. and Craig, S. (1975) Aust. J. Plant Physiol. 2, 111. 23. Hatch, M. D. (1979)Arch. Biochem. Biophys. 194, 117. 24. Rathnam, C. K. M. and Chollet, R. (1980) Prog. Phytochem. 6, 1. 25. Payer, H. D. (1969)Planta88, 103. 26. Yeoh, H. H. and Chew, M. Y. (1976) Maiays. Agric. J. 50, 435.