Planta (1992)186:166-171 P l a n t a

9 Springer-Verlag 1992

Ontogenetic variation in levels of gibberellin A 1 in Pisum Implications for the control of stern elongation

John J. Ross*, James B. Reid, and Heidi S. Dungey Department of Plant Science, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia

Received 6 February; accepted 22 August 1991

Abstract. The levels of the biologically active gibberellin (GA), GA1, and of its precursor, GA20, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chroma- tography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of develop- ment the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA 3. Tall plants generally contained 10-18 times more GA 1 and possessed internodes 2-3 times lon- ger than dwarf plants. Further, dwarf plants contained 3-5 times more GA20 than tall plants. No conclusive evidence for the presence of GA3 or GA 5 could be ob- tained, even with the aid of [2H2]GA 3 and [2H2]GA 5 internal standards. If GA3 and GA s were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA1, respectively. Comparison of the effects of gene le on GA1 levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA 1 content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA3 were present.

Key words: Elongation (stem) - Gibberellin A 1 - - Inter- node length - Pisum (gibberellins) - Stem elongation

Introduction

For the last decade there has been considerable debate over whether plant development is predominantly con- trolled by changes in growth-substance concentration or

* To whom correspondence should be addressed Abbreviations: GA n = gibberellin A n

by changing tissue sensitivity to constant levels of these substances (e.g. Trewavas 1982; Trewavas and Cleland 1983; Guern 1987). In a number of species, the use of dwarf mutants has shown that the content of the biologi- cally active gibberellin, GA1, is an important factor in the control of stem elongation (see Reid 1990; Phinney and Spray 1990). The results obtained have established that GA1 is important for normal growth. However, they do not by themselves imply that GA1 levels actually vary in response to environmental or ontogenetic changes. Indeed, in the garden pea, complete dark, or far-red photoperiod extensions, do not appear to in- crease GA 1 levels even though both result in marked increases in internode length (Sponsel 1986; Reid et al. 1990). Rather, it is suggested that these environmental factors act by enhancing tissue sensitivity to GA 1 (Reid 1988; Reid et al. 1990). However, Potts (1986), using the rice-seedling bioassay, obtained evidence for ontogenetic variation in GAl-like activity in a tall line. Ecklund and Moore (1974) also reported that the ability of pea seed- lings to synthesise the GA precursor, ent-kaurene, in- creased during the early stages of seedling growth.

In the present paper we demonstrate parallel effects of ontogeny on both GA1 levels and internode length in Pisum. This has been achieved by monitoring GA levels at various ontogenetic stages in both wild-type (tall) plants and in Mendel's dwarf (le) mutant using deu- terated internal standards and gas chromatography-se- lected ion monitoring. Gene le is known to reduce inter- node length by partially blocking the conversion of GA20 to GA 1 (Ingram et al. 1984), and its inclusion al- lows the significance of observed changes in GA1 (and GA20 ) levels to be assessed. The effects of ontogeny on the ability of the tissue to respond to applied GA 3 are also examined. In addition, an attempt is made to deter- mine if GA 3 and GAs occur in the tall line used since these GAs have been shown to be present at low levels in vegetative tissue of other species e.g. maize (Fujioka et al. 1988a, b). Since GA 3 is active in promoting stem elongation its possible presence is of direct relevance to theories regarding the control of shoot extension.

J.J. Ross et al.: Ontogenetie variation in GA levels in Pisum 167

Materials and methods

Plant material. All experiments involved the isogenic lines 205 + (Le, tall) and 205- (le, dwarf) of Pisum sativum L., developed from the crossing program at Hobart, Australia (Reid 1988).

Growing conditions. Plants were grown either in a heated glasshouse or in a growth cabinet. For the glasshouse-grown plants the cultural conditions were as previously described (Reid et al. 1990), The natu- ral photoperiod was extended to 18 h with a mixture of fluorescent (40-W, cool-white tubes; Thorn, Sydney, Australia) and incandes- cent (100-W pearl globes; Mazda, Sydney, Australia) lamps (ap- prox. 30txmol.m-2.s -x at pot top). In the growth cabinet, light was provided by eight 40-W and two 20-W Thorn white fluorescent tubes, and four 60-W incandescent bulbs (250 ~tmol.m -2 . s - 1 at pot top). The daily light period was 18 h, and the day and night temperatures were 20 ~ C and 15 ~ C, respectively. Counting of nodes started from the cotyledons as zero.

Application of GA3. Gibberellin A3 (30 pg in 10 ~tl of ethanol) was applied either to the testa of dry seeds before planting or to the second-last expanded leaf of line 205 + (Le) plants at 10, 24, 34 and 58 d from planting.

Extraction and analysis of GA. Plants grown for examining onto- genetic variation in GA content were sown sequentially in the glass- house to enable five of the six harvests to be carried out within 1 d of each other. The remaining harvest (of plants with 21 ex- panded leaves) was performed 8 d before the other five harvests. In all cases ten plants were not harvested and (for five of the har- vests) internodes subsequently expanded by these plants grew under identical conditions (Table 1). In this experiment the harvested (api- cal) portion consisted of all tissue above the uppermost expanded leaf. For 42- and 57-d-old plants, the larger flower buds were ex- cised from the apical portion. Further details are given in Table 1, Excised portions were weighed and immediately immersed in cold ( - 2 0 ~ C)methanol. Internal standards ([17, 17-2Hz]GA1 and [17, 17-2H2]GA2o) were then added, and the tissue was homogenised and returned to the freezer. To quantify GA3 and GA s (if present), 63.3 g of apical tissue from 99 tall plants with six or seven expanded leaves, was harvested. In addition to [ 2 H 2 ] G A 1 and [2H2]GA2o , [17, 17-2H2]GA3 (50ng) and [17, 17-2H2]GA5 (50ng) were also added. The ratios of ZH o to ZH 2 in the internal standards were 0.4:99.6 (2H:GAI), 0.4:99,6 ([2H2]GA2o), 0.5:99,5 ([2H2]G'A3) and 0.2:99.8 ([2Hz]GAs). Extracts were purified using Sep-Pak C18 cartridges, solvent partitioning and high-performance liquid chromatography (HPLC) as before (Reid et al. 1990). Broad HPLC

fractions encompassing the known retention times of GA1 and GA2o were collected and dried for analysis by gas chromatography- selected ion monitoring (GC-SIM). The HPLC of methyl (Me) esters was found to be unnecessary, except to separate Me-GAs and Me-GA2o (this involved a linear gradient of 40 to 80% metha- nol in distilled water over 35 min, at a flow rate of 1.5 ml. rain- ~). Analysis by GC-SIM was performed as previously described (Reid et al. 1991). Peak-area ratios for ion pairs 506/508 (GAI) and 418/ 420 (GA2o) were determined and corrected for the abundance of natural isotopes. For GA3 and GAs the ion pairs 504/506 and 416/418 were monitored, respectively. Corrected peak-area ratios were used to calculate endogenous GA levels. In all cases other diagnostic ions were also monitored to confirm identification.

Results

Ontogenetic variation in internode length and GA levels. In m a t u r e pea p lan ts of the wi ld - type tal l (Le) line 205 +, i n t e rnode length genera l ly inc reased with increas ing in- t e rnode number , unt i l a m a x i m u m was r eached immed i - a tely be low the node bea r ing the first f lower (no rma l ly node 15 or 16, Fig. 1). I n t e r n o d e length then dec l ined rap id ly unti l cessa t ion of ap ica l g rowth occurred , a t a b o u t node 22 unde r the cond i t ions used. This p a t t e r n is s imi lar to those p rev ious ly obse rved (e.g. Re id a n d Muffe t 1984). However , a small decrease in i n t e rnode length at a b o u t node 10 was also cons is ten t ly observed. The va r i a t i on in i n t e rnode length with o n t o g e n y was bas ica l ly s imi lar in dwar f (le) line 2 0 5 - p l an t s (Fig. 1). A b o v e i n t e rnode 1-2 the in t e rnodes of tal l p l an t s were 2-3 t imes longer than those of d w a r f plants . However , this effect, while large, was ac tua l ly exceeded by changes in i n t e rnode length dur ing o n t o g e n y wi th in tal l p l an t s where i n t e rnode length va r ied by up to 15-fold (Fig. 1).

The ra te of e l onga t i on of recent ly emerged tal l (Le) seedlings was also low. The ra te of e longa t ion then grad- ual ly increased (by a fac tor of app rox . 2) unt i l a re la t ive ly cons t an t ra te was reached 150 h after sowing (Fig. 2, see also E c k lund a n d M o o r e 1974).

The levels of b o t h GA1 and GA2o in the apica l p o r - t ion of the p l an t were low on emergence (i.e. day 4) bu t

Table 1. Harvest details for tall (Le) and dwarf (le) pea plants harvested at six on- Age of togenetic stages for analysis of GA levels plants

(d)

4

9

20

30

42

57

Phenotype Number of Number FW of Amount of internal expanded of plants harvested standard added leaves harvested tissue (g) (ng)

GA1 GAzo

Tall Dwarf Tall Dwarf Tall Dwarf Tall Dwarf Tall Dwarf Tall Dwarf

1.0• 53 1.50 15 22 1.0• 54 1.33 15 27 4.0 • 0.1 40 5.06 30 75 3.8_____0.1 40 5.65 15 110 8.9 • 0.2 36 11.36 70 170 7.8 __0.1 36 15.68 19 300 14.3• 36 21.49 130 320 12.6_____0.1 38 24.62 30 500 20.6• 37 8.85 27 130 18.3 _____0.2 38 14.22 15 280 21.0_____0.0 34 3.43 15 50 19.3__+0.2 38 3.97 15 80

168 J.J. Ross et al.: Ontogenetic variation in GA levels in Pisum

14

.-.12 E 0

E

0 1"2' '4- '5 '7 -8 ' 10-"1i '13-"1~, "16-'1"I 19'-'20 ' Internode number

Fig. 1. lnternode length plotted against internode number for tall (o) and dwarf (A) pea plants grown in a daily 18-h photoperiod. Some typical SEs are shown

150

130 /

110 "--" 9 0

._~ - r

7 0

5O

3 0 O ~ ~ 1 7 6 J I i i I I I I I I i I

1 0 0 120 1 4 0 160 180 2 0 0 2 2 0

T i m e (h) Fig. 2. Height of tall (Le) pea plants (from node 0 to the tip of the stipules enclosing the apical bud) plotted against time from sowing. Plants were emerging from the soil surface at approx. 100 h. SEs were generally too small to show on the figure and averaged 1.8 mm

subsequently increased to a maximum and then declined (Table 2). The capacity for elongation of the (still expand- ing) internodes in the harvested apical portion was esti- mated by measuring the corresponding internodes (when fully expanded) of ten accompanying plants left intact at harvest. When the final length of the oldest internode present in the harvested port ion (estimated in the above manner) was plotted against log~o of the GA~ level for tall and dwarf plants together, a quantitative relationship was apparent (Fig. 3a, r=0.83, P<0.001). Importantly, the correlation between internode length and GA1 level

was also significant within the tall genotype (P<0.05). The relatively low level of GA1 in emerging seedlings was also observed in a separate experiment (data not shown).

However, no overall correlation was found between internode length and the level of GA2o (Fig. 3 b, r = 0.24, P > 0.30). Nevertheless, significant correlations occurred when the data for tall and dwarf plants were analysed separately (P < 0.05). These relationships resemble those relating growth responses of Le and le plants to varying uptake of applied (labelled) GA2o (Ingrain et al. 1986), supporting the previous conclusion that GA2o levels per se do not regulate internode length in peas (Ingram et al. 1986). The data reflect the fact that dwarf plants are much shorter but accumulate more GA2o than tall plants (Fig. 1, Table 2). In contrast, tall and dwarf genotypes with similar levels of GA 1 (e.g. 4-d-old tall and 9-d-old dwarf plants) subsequently produced internodes compa- rable in length. For example, the combined length of the three internodes formed above the leaf at which 4-d- old tall plants were harvested (8.56cm, GA~ content 1.1 ng/g FW) was similar to that of the comparable three internodes of 9-d-old dwarf plants (9.54 cm, GA 1 content 1.2ng/g FW). However, with ontogeny there also ap- peared to be major changes in the potential for internode elongation. For example, in tall plants the internodes above node 14 (GA~ content 12.0 ng/g) were considera- bly longer than those above node 4 (GA~ content 17.6 ng/g) (Table 2).

This is the first report detailing the quantification of GA1 and GA2o levels in isogenic Le and le plants using the internal-standard technique, although the effect of allele le 5839 o n GA levels has been determined by this method (Reid et al. 1990). The relative level of GA~ in le plants observed here (5-10% of the level in Le plants) agrees well with a previous estimate (Ross et al. 1989) and should finally remove any remaining doubt on the question of whether or not le plants contain less GA~ than Le plants (see Graebe 1987; Trewavas 1987). The effect of the Le/le gene difference on GA~ levels was diminished on emergence and on impending senescence. This coincided with a reduction in the difference in inter- node length between the two genotypes (Table 2).

Possible presence o f GA 3 and GA 5 in tall plants. Analysis of an extract from the apical portions of tall plants (con- taining deuterated GA 3 and GA 5 as internal standards) did not yield conclusive evidence for the presence of en- dogenous GA 3 or GAs. However, in both cases the mass chromatogram of the molecular ion for the (putative) endogenous GA contained a peak which co-eluted with the internal standard (allowing for the small shift in re- tention time attributable to the presence of two deuteri- um atoms). The peak-area ratios (deuterated to endoge- nous) for GA a and GA 5 were 9.9 and 3.6, respectively. This indicates that the maximum possible levels of GA 3 and GAs were 0.8 and 0.22 ng/g FW, respectively. The ratios of GA1 to GA 3 and of GA2o to GA5 were there- fore at least 200 to 1 and 100 to 1 in the harvested tissue, respectively (GA1 level of 16.82ng/g FW and GA20 level of 21.30 ng/g FW). A similar maximal level

J.J. Ross et al.: Ontogenetic variation in GA levels in Pisum 169

Table 2. Levels of GA1 and GA2o in apical portions of tall (Le) and dwarf (le) peas at various ontogenetic stages. Internodes l, 2 and 3 were the three internodes above the node of excision,

measured on ten unharvested plants (for each ontogenetic stage) after expansion was complete. Data are shown as mean • SE. The isotopic ratios of the GAs are also shown

Age of plants (d)

Phenotype Gibberellin

GA1 GA2o

Isotope Level Isotope Level ratio ratio 2H 2:2H 0 ng/g FW ng/plant 2H 2:2H 0 ng/g FW ng/plant

Internode length (cm)"

1 2 3

4 Tall 90.5:9.5 1.1 0.03 Dwarf >96.0:4.0 <0.5 <0.01

9 Tall 25.2:74.8 17.6 2.23 Dwarf 68.5:31.5 1.2 0.17

20 Tall 31.5:68.5 13.4 4.23 Dwarf 46.8:53.2 1.4 0.61

30 Tall 33.5:66.5 12.0 7.2 Dwarf 64.2 : 35.8 0.7 0.45

42 Tall 34.8:65.2 5.7 1.36 Dwarf 70.0 : 30.0 0.5 0.19

57 Tall >92.7:<7.3 <0.3 <0.03 Dwarf >97.5:<2.5 <0.1 0.01

79.0:21.0 61.8:38.2 49.2 : 50.8 26.7:73.3 40.0: 60.0 13.5:86.5 35.1:64.9 16.9:83.1 31.8:68.2 21.6:78.4 80.0: 20.0 60.2 : 39.8

3.9 0.11 0.5• 3.2• 12.6 0.31 0.6• 1.5• 15.3 1.94 7.4• 9.2• 53.5 7.6 2.5• 3.1• 22.4 7.1 10.4• 10.2•

122.9 53.5 3.9• 4.1• 27.5 16.0 11.7• 14.2• 99.9 64.7 4.5• 4.8• 31.6 7.6 5.8• 2.9• 71.7 26.8 3.3• 2.3•

3.7 0.37 2.2• n.p. 13.3 1.39 1.9• n.p.

4.9• 1.6• 9.2• 3.9•

10.0• 4.5•

13.3• 5.4•

n.p. n.p. n.p. n.p.

a n.p. Not present

12

~10

~6 x : 6

[ a .

o 9

o 9

b

9 9 o

0 1 0 1 2 LOgloGA 1 level Loglo GA20 level

Fig. 3a, b. Final length of the internode immediately above the node of excision plotted against loglo of the GA1 a and GA2o b levels (ng/g FW) present in apical portions of tall (e) and dwarf (0) pea plants at various ontogenetic stages. Regression lines are shown for all data in a and for tall and dwarf plants separately in b

16

14

~ 10

o 6

"E - 4

2

[3"-,~_ D

J

1-2 4 -5 7 -8 10-11 13 -14 16-17 19 -20 22 -23 25 -26 Internode number

Fig. 4. Internode length plotted against internode number for six groups of tall (Le) pea plants left untreated (o) or treated with a single application of GA a (30 ~g) applied to the dry seed (T), leaf 3 (A), leaf 8 (o), leaf 13 (m), or leaf 20 (o). The lengths of three internodes which expanded after each treatment ( . . . . . . ) and the node of GA 3 application 0,) are indicated

for GA 3 was found in a separate experiment (data not shown).

Response of tall plants to applied GA. Application of GA 3 to the dry seed or to leaf 3, 8, or 20 caused signifi- cant increases in internode length (Fig. 4). When applied to leaf 13, GA a did not significantly affect internode length (Fig. 4) but increased the overall elongation rate since the increases in total height of control and treated plants during the 9 d after application were 49.6 -t- 0.4 cm (n = 14) and 55.0 _+ 0.6 cm (n = 12) respectively (P < 0.001).

Application to leaf 3 or to leaf 8 also significantly in- creased the overall elongation rate (data not shown). The largest effects (in relative terms) on internode length were observed after GAa applications to the dry seed or to leaf 20, where subsequently expanded internodes are rel- atively short when untreated (Fig. 4). Moore (1967) and Ecklund and Moore (1968) also found that in the cultivar Alaska the first- and last-formed internodes were rela- tively more responsive to applied GA3. In the present case internode 1-2, for example, exhibited at least a three-fold increase in length in response to the (presum-

170 J.J. Ross et al.: Ontogenetic variation in GA levels in Pisum

ably) saturating dose of GA3. However, in treated plants internode 1-2 was still approx. 3 times shorter than inter- node 2-3, indicating that the former internode has less potential for elongation. The results therefore clearly demonstrate that while most internodes are capable of further elongation in response to G A 3 there are very substantial differences in their potential for elongation even when G A levels are saturating.

types, previous information on ontogenetic and spatial variation in G A levels in this species is limited (Ingram et al. 1985; Potts 1986). The present paper addresses the first deficiency and indicates a role for GA~ in regulating some aspects of the ontogenetic variation in internode elongation in wild-type plants. Information on the distri- bution of GAs around the plant is also required and may allow the role of GA~ in other developmental pro- cesses to be assessed.

Discussion

In tall (Le) plants, GA1 levels in the young apical tissues vary by up to 50-fold over the life of the plant (Table 2). This variation is potentially sufficient to cause large changes in internode length, since the mutant allele le exerts a substantial dwarfing effect by reducing the GA1 level by 10- to 18-fold (Table 2). In tall plants the inter- nodes which possessed low GA~ levels were relatively short, and were capable of two- to three-fold increases in length in response to applied GA3, indicating that the endogenous G A 1 levels in these tissues were limiting for growth. These observations, considered together, argue that the level of GA~ partially regulates the onto- genetic variation in internode length in wild-type peas. However, during ontogeny there are also substantial changes in the ability of individual internodes to elongate in response to biologically active GAs. For example, even after t reatment with a saturating dose of GA3, in- ternode 1-2 was still only 20% the length of internode 16-17 (Fig. 4). Thus internode 1-2 is short not only be- cause it possesses relatively little G A t during elongation, but also because of its relative inability to respond to G A l . Clearly in tall peas some other factor or factors in addition to GA1 exert a major effect on the potential for elongation of different internodes.

The results provide additional evidence that GAzo possesses at most very little activity per se in controlling internode length in peas (Ingram et al. 1986). For exam- ple, when the plants had seven to nine expanded leaves, Le plants contained approx. 10 times more GA~ than did le plants. In the same tissue GA2o accumulated to very high levels in le plants (over 5 times that present in genotype Le, Table 2), but this did not offset the large (over 2.5-fold) reduction in internode length caused by the lower GA1 level in the mutant.

While there is little doubt that applied GA3 possesses activity in promot ing elongation in Pisum sativum (and in other species) an important endogenous role for this G A in peas appears to be precluded by its extremely low level (or absence), compared to that of GA~. In con- trast, in certain other herbaceous species, G A 3 may be impor tant in controlling elongation since the ratio of G A 3 to GA~ appears to be much higher than in peas. For example, in immature internodes and leaf sheaths of maize, this ratio may be greater than 0.3 (Phinney et al. 1990) while in Brassica rapa the ratio may exceed 1 (Rood et al. 1989).

While previous reports (Reid and Potts 1986; Ross et al. 1989) have established a relationship between GA~ levels and internode length across a range of Pisum geno-

We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuter- ated GA standards and the Australian Research Council for finan- cial assistance.

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