Key BIOLOGICALLY ACTIVE SECONDARY METABOLITES OF BARLEY. IV. HORDENINE PRODUCTION BY DIFFERENT BARLEY LINES

Journal of Chemical Ecology, Vol. 20, No. 8, 1994

BIOLOGICALLY ACTIVE S E C O N D A R Y METABOLITES OF BARLEY. IV. H O R D E N I N E PRODUCTION BY

D I F F E R E N T BARLEY LINES

J O H N V. L O V E T T , A N N E H . C . H O U L T , * and O L A F C H R I S T E N 1 Department of Agronomy and Soil Science

University of New England Armidale, N.S. HI. 2351, Australia

(Received September 20, 1993; accepted March 21, 1994) Abstract--Forty-three lines of barley, including ancestral (wild), landraces, Middle Eastern lines, and modem cultivars, were grown under two different sets of environmental conditions. Hordenine production in barley roots was determined at the one-leaf stage by HPLC analysis and, in two lines only, over a period of 35 days. Forty-two of the 43 lines produced significant amounts of hordenine, although there was no variation among groups. Middle Eastern lines had the highest production with 327/~g/g on a dry weight basis.

Production was, however, determined more by environmental conditions dur- ing growth than by genetic factors. Hordenine production was up to seven times higher in plants grown under lower light intensities.

Key

INTRODUCTION

Alkaloids are known to have wide biocidal activity. Hordenine and gramine, both produced by the barley plant (Hordeum spp.), are typical o f this group o f secondary metabolites. A l t h o u g h a certain a m o u n t has been published about the production and activity o f gramine in barley and other species, little is known about the production and activity o f hordenine.

Apart f r o m being phytotoxic (Overland, 1966), g m m i n e has been impli- cated in the self-defense o f cultivated barley (Hordeum vulgare) against bacteria (Sepulveda and Corcuera, 1990) and aphid infestation (Zuniga et al., 1988;

*To whom correspondence should be addressed.

I Current address: Christian Albrechts University, Kiel, Germany.

1945

0098-033119410800-194550"1.0010 © 1994 Plenum Publishing Corporation

Kanehisa et al., 1990). Gramine also inhibits the growth of the fungus, Drechs- lera teres, and armyworm, Mythimna convecta, larvae (Lovett and Hoult, 1993) and is considered to be an antipalatability factor toward grazing animals (Marten et al., 1976). In contrast, all that has been reported about hordenine is that it inhibits seedling growth of white mustard, Sinapis alba (Liu and Lovett, 1989), and Dreschslera teres and Mythimna convecta (Lovett and Hoult, 1993). These findings indicate a potential for self-defense by barley against other organisms through the production of hordenine and gramine. Consequently, the level of production of these compounds and the factors controlling their production are worthy of investigation.

The production of gramine and other alkaloids has been shown to be under genetic and environmental control (Moore et al., 1967; Gentry et al., 1969;

Hanson et al., 1981, 1983; Lovett and Hoult, 1992). Hanson et al. (1981) surveyed 23 lines of barley of different origins and found gramine production ranged from 0 mg/g to 8 mg/g dry weight and concluded that high gramine production was typical of lines originating in hot areas of the world. They subsequently showed that gramine production by barley increased with temper- ature (Hanson et al., 1983). Analogous data for hordenine are not available.

Breeding for low alkaloid content, including low gramine content, has been achieved in reed canary grass (Phalaris arundinacea) (Marten et al., 1981), the object being to reduce the adverse effects of this alkaloid on the productivity of grazing animals. Hanson et al. (1981) suggested that this should also be possible in barley forage crops. However, if the self-defense role of allelochemicals in grain crops is accepted, the ability to produce large amounts of these compounds would be of advantage and could ultimately lead to reduced use of synthetic crop protection agents. Lovett (1982) suggested that, in breeding crop plants for improved yield, self-defense capability may have been reduced or lost. To reintroduce this capability, it may be necessary to look to ancestral types to find a source of appropriate genes.

I~vett and Hoult (1992) investigated the gramine content of 43 lines of barley chosen to represent the continuum in exploitation and breeding from ancestral types (H. spontaneum and H. agriocrithon) through to contemporary cultivars, and confirmed genetic control as found by Hanson et al. (1981). In this paper we report on the hordenine content of the roots of these 43 lines and discuss this in relation to genotypic and environmental control of production and in relation to the gramine content of their leaves.

METHODS AND MATERIALS

Plant Material. Six Hordeum spontaneum lines, four H. agriocrithon lines,

eight landraces, nine lines from the Middle East representing modem lines bred

close to the center of diversity, and 16 cultivars grown in Australia past and

SECONDARY METABOLITES OF BARLEY 1947

present were used. Genetically pure seed of all lines were multiplied in Armidale prior to use.

Chemicals. AR or HPLC grade chemicals were used throughout. Authentic hordenine and gramine hemisulfate were obtained from Sigma Chemicals.

Experiment 1. Seed of cv. O'Connor and H. spontaneum var. spontaneum were sown in sand, replicated four times and grown in a glasshouse in October 1991. The plants were harvested daily from day 6 to day 9, inclusive, and thereafter at 2, 3, 4, and 5 weeks of age. The roots and shoots were separated and the remains of the grain discarded. The plant material was carefully blotted dry and stored in a freezer until representative subsamples were weighed out for analysis. Alkaloid contents are expressed as micrograms per gram fresh weight, as there was not always sufficient plant material to measure dry weight.

Experiment 2. The barley lines selected were grown under two sets of environmental conditions. One environment was a growth cabinet with a 12-hr day, 430 W / m 2, 8-18 °C, and total incident light of 5160 W/m2/day. The growth conditions were outside in March 1991; mean daylength 10.3 hr, mean total incident light 6030 W/m2/day, mean maximum incident light 890 W/m2/day, and mean temperature range 10-23 °C. Fifty grains of each line, replicated three times, were sown in sand and harvested as each line reached the stage of one fully expanded leaf (approximately eight to 10 days after sowing) as described by Hoult and Lovett (1993). Plant material was stored as in experiment 1. Grain of all lines was not immediately available in sufficient quantities; therefore, the growth chamber material consisted of 30 lines whereas the outside-grown mate- rial consisted of all 43 lines. Dormancy problems were encountered in seven of the eight landraces; all were resown at a later date (July 1991) along with cv.

Prior and H. spontaneum var. spontaneum as controls.

Extraction and HPLC Analysis. The frozen root material was extracted in 0.01% acetic acid at room temperature for 24 hr, the extracts filtered through glass wool, the pH adjusted to 9.15, and then centrifuged at 3000 rpm for 5 min before being purified and concentrated using Sep-pak CI8 cartridges. Puri- fication consisted of applying 10-ml aliquots of extract to Waters C18 Sep-pak cartridges prepared before use with 5 ml A C N (acetonitrile) and 2 ml 0.01 M KH2PO 4, pH 7. The root samples were rinsed once with 2 ml 0.05 M KH2PO 4, pH 9.5, isopropanol (85 : 15) before being eluted with 1.5 ml 0.05 M KH2PO4, pH 2.3, isopropanol (70: 30). All eluates were evaporated to dryness at 40°C under a stream of nitrogen and taken up in 1 ml mobile phase for analysis by HPLC. Recovery of hordenine from these procedures was monitored by "puri- fying" two 10-ml aliquots of standard authentic compound with each batch of plant root extracts.

Extracts were analyzed by injecting 10-/~1 aliquots into a Waters HPLC

system comprising a M40 pump, flow rate 2 ml/min, U6K injector,/~Bondapak

phenyl column 10 #m (3.9 mm x 30 cm), and UV-vis spectrophotometer

wavelength 221 nm. Results were recorded as peak areas using a Waters 745 data module. Elution was isocratic, the mobile phase consisting o f 0.025 M KH2PO 4, pH 7.15, + 0.1% triethylamine/ACN (67 : 33). The presence of hor- denine and gramine having been confirmed in barley cv. Triumph (included in this survey) by GC-MS (Liu and Lovett, 1990a), identification was by retention time relative to, and by coelution of, representative samples with authentic compound. Quantification was by peak area relative to standards of authentic compound.

RESULTS AND DISCUSSION

Hordenine production within lines was much more variable than gramine production. Table 1 shows the pattern of gramine and hordenine production by two lines, H. s p o n t a n e u m spontaneura and cv. O'Connor, over the first five weeks of growth. The gradual increase in gramine production in both lines up to day 9 after sowing indicates that sampling and estimation of gramine pro- duction was reliable. However, the production o f hordenine by the two lines followed different patterns, and the sharp peak and decline of hordenine pro- duction by H. s p o n t a n e u m made estimation o f peak production difficult. Never- theless, over three replications, production potential over the first few days of growth was probably estimated reliably. Liu and Lovett (1990b) found cv.

Triumph to exude hordenine into hydroponic solution much in the H. s p o n t a - n e u m pattern, but without knowing the pattern of production by individual lines

TABLE I. PRODUCTION OF HORDENINE AND GRAMINE OVER TIME BY Two DIFFERENT BARLEY LINES a

Hordenine content of roots

Gramine content of shoots (t~g/g fresh weight)

H. vulgare H. vulgare

Day H. spontaneurn cv. O'Connor H. spontaneum cv. O'Connor

6 13.75 abc 31.77 cd 880 def 364 ab

7 28.75 bcd 31.84 cd 936 efg 539 bc

8 t2.19 ab 35.69 d 1059 efg 596 bcd

9 8.13 a 11.77 ab 1121 fg 620 bcd

14 5.81 a 4.13 a 1185 g 457 ab

21 3.07 a 4.22 a 605 bcd 507 abc

29 4.62 a 2.25 a 451 ab 321 ab

35 6.70 a 5.15 a 796 cde 206 a

"Entries carrying the same letter do not differ from each other significantly at the 5% level.

SECONDARY METABOLITES OF BARLEY 1949

it is not possible to estimate with confidence the peak production potential of lines. We consider that the data for hordenine production presented here (Table 1) are an acceptable estimate of the production of hordenine by these lines over the first few days of growth.

All lines, with the exception of

var. Borgiyyora, in which trace amounts were found, produced hordenine in measurable quantities. This indicates that hordenine production is a normal function of the barley plant and is consistent with our finding that, contrary to a previous report (Hanson et al., 1981), gramine production is also probably the norm in barley (Lovett and Hoult, 1992).

Regression analysis of hordenine production by the 30 lines grown both in the growth cabinet and outdoors gave an r ~ value of 0.2370, indicating little genetic control of hordenine production. The corresponding r 2 value for gramine production was 0.9079, indicating strong genetic control (Lovett and Hoult, 1992). The difference in degree of genetic control over the production of the two alkaloids was exemplified by the relatively large differences in hordenine production between the growth cabinet and plants grown outdoors. Gramine production was enhanced when the plants were grown outdoors, at most by 100% and generally by much less than this amount. By comparison, hordenine production was found to be as much as seven times greater in the plants grown in the growth chamber (Table 2). The enhanced production of gramine under conditions of greater light intensity and higher temperatures may be explained by an elevated metabolic rate leading to a greater excess of substrate available for the production of secondary metabolites. Application of the model of Johnson et al. (1993) for crop photosynthesis to our data indicated that the greater mean maximum light intensity, rather than the higher temperatures encountered by the plants grown outdoors, was the major factor contributing to this greater production. This is consistent with the data of Hanson et al. (1983), who found little effect of temperature on gramine production below 21 °C.

The greater hordenine production at lower light intensity cannot be explained in this way. We suggest that at the establishment phase a plant is most vulnerable to adverse environmental conditions, competition from other species, and inva- sion by pathogens. Seedling vigor is reduced if less than ideal conditions for growth prevail. This results in a concomitant shift in survival strategy from aggression, evidenced as high competitive ability through vigorous growth, to defense, in which a greater proportion of plant product is diverted to the pro- duction of secondary metabolites. It is submitted that a maximum light intensity of one third to one half full daylight would constitute stressful conditions.

Hordenine production by

group, in general, was found

to be less than in the other groups (Table 3). However, analysis of variance for

groups of unequal replicates performed on the five groups grown outdoors

(experiment 2) showed that the difference only reached significance between the

TABLE 2. IDENTITY, ORIGIN, AND GRAMINE AND HORDENINE CONTENT OF 43 BARLEY LINES Gramine content of shoots (p,g/g fr wt) Hordenine content of roots (/,Lg/g dry wt) Aust. No./ Growth Growth Q Code Cultivar Origin cabinet Outdoors cabinet Outdoors 1-1. spontaneum 400154 spontaneum unknown N/A Borgiyyora Israel N/A Eyzaria Israel N/A Tel Hay Israel N/A Mt. Meron Israel NIA Akziv Israel H. agriocrithon 402775 unknown 402776 unknown 402777 unknown 402779 unknown H. vulgare 1 LQ89 landrace Syria 2LQ89 landrace Syria 3LQ89 landrace Syria 4LQ89 landrace Syria 5LQ89 landrace Syria 6LQ89 landrace Syria 7LQ89 landrace Syria 8LQ89 landrace Syria 1147 1506(1158)" 717 135 1725 1 1312 145 1254 1028 1600 222 844 1215 921 217 1679 75 229 285 871 169 866 87 I100 1522 2275 435 719 126 (746) (238) (773) (160) (854) (353) (697) (235) (787) (250) (1069) (83) 851 (717) (64) (964) (272)

400110 N/A Ethiopia 700 885 1361 130 400117 N/A Ethiopia 915 1061 1100 151 400120 N/A Ethiopia 5 4 1042 203 400039 N/A Syria 18 7 2141 599 490224 N/A Syria 0 5 1820 322 490314 N/A Syria 449 679 763 151 490187 N/A UAR 793 1434 2038 315 490281 N/A UAR 40 1208 2080 247 490288 N/A UAR 558 1075 1919 356 401786 Prior England 640 746(565) 2078 226 401781 Proctor England 3 5 1236 341 400189 Triumph England 3 2 1274 83 401834 Resibee Australia 595 913 2307 178 401259 Lara Australia 548 741 2325 224 402239 Weeah Australia 449 717 1680 150 400188 O'Connor Australia 435 509 2625 265 400190 Clipper Australia 329 306 1604 172 402996 Yagan Australia 205 394 2200 378 400180 Forrest Australia 1 24 1534 274 400187 Schooner Australia 1 5 1200 153 400182 Galleon Ausralia 1 5 1585 206 400186 Grimmett Australia 0 3 2133 256 402997 Windich Australia 0 8 1314 458 402713 Moondyne Australia 0 4 1842 357 403001 Skiff Australia 0 3 634 133 °Data in parentheses refer to lines sown in July 1991.

o o z o o t~

TABLE 3. MEAN GRAMINE CONTENT OF SHOOTS AND HORDENINE CONTENT OF ROOTS OF FIVE GROUPS OF BARLEY LINES GROWN OUTDOORS

Hordenine Gramine content content Group (#g/g fresh weight) (~g/g dry weight)

H. spontaneum

1410 133

H. agriocrithon 848 204

H vulgare

Landraces 830 207

Middle Eastern 706 327

Australian 274 241

H. spontaneum

and the Middle Eastern group (P > 0.05). Recent reviews indicate that allelochemicals may confer fungal (Rizvi and Rizvi, 1992) and nematode (Hasan, 1992) resistance on a plant. Grodzinsky (1992) reported that barley root exudates inhibited germination and germ tube growth of

It is possible, therefore, that the greater hordenine production by bred lines of barley may be explained in terms o f defensive benefits conferred on the barley plant. W e infer, therefore, that hordenine pro- duction has been favored by selection for pathogen resistance.

In plants grown outdoors, production o f gramine and hordenine were inde- pendent o f each other with a slight trend towards a negative relationship. The exception to this was

group, where the negative relationship was significant (r 2 = 0.8848). A similar independence was found in the growth of cabinet-grown plants, with the only relationship approaching significance being a positive one in the group Australian cultivars (r 2 = 0.4398). This evidence o f a marked change in the balance of hordenine and gramine production in response to environmental changes was interpreted to be another manifestation o f the effects o f stress (i.e., low light intensity) on the metabolism of the plant and, hence, on its potential to produce allelochemicals.

CONCLUSION

Hordenine production by barley roots appears to be the norm, and there is a tendency to greater production by modern cultivars. This is in contrast to gramine production by the shoots in which alkaloid production is greatest in the ancestral lines and may be suppressed almost entirely in some modern cultivars.

It is possible that hordenine production in modern cultivars has been favored

because it confers on the barley plant a degree of resistance to fungal pathogens

SECONDARY METABOLITES OF BARLEY 1953 o r o t h e r p e s t s . F u r t h e r m o r e , in c o n t r a s t t o g r a m i n e p r o d u c t i o n , h o r d e n i n e p r o - d u c t i o n d o e s n o t s e e m to b e u n d e r s t r o n g g e n e t i c c o n t r o l b u t is m u c h m o r e r e s p o n s i v e to c h a n g e s in t h e e n v i r o n m e n t in w h i c h t h e p l a n t is g r o w n . H o r d e n i n e m a y h a v e a r o l e to p l a y in p r o t e c t i n g t h e p l a n t u n d e r s t r e s s f u l c o n d i t i o n s .

Acknowledgments--We thank the Barley Committee of the Grains Research and Development Corporation (Australia) for funding this project and the Deutsche Forschungsgemeinsehaft for sup- porting one of us (Dr. O. Christen). We also thank Dr. H. Brown, CSIRO, Canberra, and Dr. S.

Ceccarelli, ICARDA, Syria, for supplying the seeds of five H. spontaneum lines and the landraces, respectively, and Mr. M. Mackay of the Australian Winter Cereals Collection, Tamworth, NSW, for all the other lines.

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