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Stem Growth Habit and Starch Statolith Content of the creep Pea Mutant
/.PlantPhysiol. Vol. 133.pp. l03-106(1988}
Stem Growth Habit and Starch Statolith Content of the creep Pea Mutant IAN
M.
SCOTT
Department of Botany and Microbiology, University College of Wales, Aberystwyth, Dyfed, SY23 3DA, U.K. Received November 30, 1987 . Accepted March 15, 1988
Summary Pea seedlings homozygous for the mutant creep gene initially grew in an upright but significantly offvertical orientation which became progressively more horizontal as growth continued. When the weak lower internodes were supported by staking in a vertical orientation, the subsequently produced internodes of creep plants rapidly became prostrate, in contrast to the internodes of normal plants, which continued in vertical growth. Etiolated creep seedlings, however, showed normal vertical growth. Microscopic examination revealed that stems of the creep mutant possessed a starch sheath with a normal content of amyloplasts sedimented according to the direction of gravity.
Key words: Pisum sativum, amyloplasts, creep mutant, gravitropism, statoliths.
Introduction The mechanisms underlying plant responses to gravity remain uncertain (Halstead and Scott 1984). An increasingly favoured approach to this problem is the characterization of genes involved in gravity responses, and tropic mutants have now been described in several species (Roberts 1987). Such mutants have in some cases proved informative. For example, the theory that sedimenting amyloplasts act as gravity-sensing statoliths has received support from studies on certain abnormally gravitropic mutants, such as the lazy-1 tomato (Roberts 1984), the amylomaize mutant (Hertel et al. 1969), and the aux-1 mutant of Arabidopsis (Olsen et a1. 1984), in which statoliths are either absent, smaller than normal, or exhibit reduced sedimentation. One of the first discoveries of a mutation affecting gravitropism was that of the age gene of Pisum sativum, which causes a loss of both root and shoot gravitropism in addition to weak germination and growth (Scholdeen and Burstr6m 1960, Olsen and Iversen 1980). Another interesting mutant pea gene, creep, was induced by Sidorova (1975), but has as yet been little investigated by physiologists. The present paper describes some features of the creep mutant. © 1988 by Gustav Fischer Verlag, Stuttgart
Materials and Methods Plant material
Pure lines of Pisum sativum L. were kindly supplied by the Nordic Gene Bank, Alnarp, Sweden. The creep mutant was line 5859 of this institute, while the isogenic parent Torsdag (line 2157) was used as the normal control. The age mutant was line 5102.
Growth habit measurements
Plants were grown in 7.6cm diameter pots of John Innes No.2 potting compost in a glasshouse heated to 18°C minimum and with a minimum 16 h photoperiod extended when necessary by mercury vapour lamps. Stem orientation was measured using a protractor. For the initial growth observations (Fig. 2), random samples (n = 10) were taken from a population and discarded after measuring, in order to avoid handling effects. In the experiment on vertically supported plants (Fig. 3), plants were lightly tied with a single loop to a vertical stake. Fifteen specimens of each genotype were selected for initial uniformity and the orientation of this single population was monitored without handling except to prevent attachment of tendrils. Significance of data was tested using a one-way analysis of variance at the p = 0.01 level.
104
IAN
M.
SCOTT
Microscopy For observations on amyloplasts, fresh tissue sections were stained briefly with a solution containing KI (20 gl-') and h (2 g 1- ') prior to examination at x 100 and x 400 magnification on a light microscope fitted with a calibrated eyepiece graticule.
90 NORMAL C/)
w w
a:
Results
Growth Habit
No significant differences were observed between plants homozygous for the creep gene and the isogenic parent cultivar T orsdag so far as germination, shoot growth-rate or morphology, or root gravitropism were concerned. The creep gene, however, exerted a marked effect on stem orientation, conferring a much increased tendency to prostrate growth in light-grown plants (Fig. 1). In the early stages, creep seedlings grew in an upright fashion, but the mean stem angle of populations of creep seedlings was significantly (p = 0.01) further than normal from a true vertical alignment at all stages measured (Fig. 2). The mutant plants showed an increased tendency away from the vertical as they grew, until a relatively sharp reorientation towards the horizontal was observed (Fig. 2), generally following the expansion of internode 5 - 6. The rate of progression to the prostrate orientation over this period varied considerably between individual plants. Bending occurred near the base of the stem in one of the first three short internodes. The apical region of the prostrate creep plants retained a tendency towards the vertical (Fig. 1), a feature which was retained in plants transferred for 4 days to a dark room, implying that gravitropism was involved. It is a familiar observation that the weak lowest internodes of pea stems fail to support tall plants in the absence of external support; in these experiments the normal plants all eventually became prostrate like the mutants. The persistence of an altered response to gravity in the creep mutants beyond this stage was demonstrated by vertically staking at internode 5 - 6 plants in which internode 7 - 8 was beginning to expand. As seen in Fig. 3, the subsequently produced
(!)
w 60
0
W
-I
(!)
Z
« ::E
w
I-
30
C/)
CREEP
o~
8
____~__~____~__~__~__~ 12
10
14
16
18
AGE (DAYS) Fig. 2: Change in stem orientation with age in normal (closed circles) and creep (open circles) plants. The angle from the horizontal (i.e. vertical = 90°) of internode 3 - 4 was measured except for the day 8 values which represent internode 2-3. Bars indicate SE.
90
NORMAL C/)
w w
a: 60 (!) w
0
W
-I
(!)
Z
«
::E 30
CREEP
w
I-
C/)
o~
______~____~______~____~~
17
19
21
23
25
AGE (DAYS) Fig. 3: Change in stem orientation with time following staking of normal (closed circles) and creep (open circles) plants. 17 day old plants with internode 7 - 8 beginning to expand were lightly tied to a vertical stake at internode 5-6. The angle from the horizontal (i.e. vertical = 90°) of internode 6-7 was measured. Bars indicate SE. Fig. 1: Growth habit of the creep pea mutant (right) compared with an isogenic normal plant (cv. Torsdag) of the same age.
internodes of the staked normal plants rigorously found and persisted in vertical growth, even though the tendrils were
The creep mutant of pea Table 1: Mean angle from the horizontal of the youngest internodes of pea seedlings (n = 8) grown for 6 days in darkness at 21 0c. Genotype Normal
creep age
LSD (p
=
0.01)
Stem angle 81.1 0 84.10 48.4 0 11.6 0
not allowed to form attachments, whereas the subsequently produced internodes of the creep plants variably but progressively lost the vertical orientation, passing through the horizontal and eventually resting on the glasshouse bench. Again, the apical one or two internodes always tended to seek the vertical. The same pattern was observed if the plants were staked at internode 11-12. Etiolated creep seedlings, however, showed no abnormal orientation. This contrasted with the agravitropism exhibited by etiolated seedlings of the age mutant (Table 1).
Starch Statolith Content Fresh, iodine-stained longitudinal and transverse sections were examined from various points along the lengths of all the internodes of mutant and normal plants of various ages. The expanded internodes of both genotypes possessed a distinctive starch sheath layer, comprising a continuous single layer of endodermal cells surrounding the vascular tissue, which contained numerous amyloplasts ranging in size from 2 -7.5 /lm diameter. The amyloplasts of both genotypes were sedimented according to the direction of gravity, as predicted by the statolith theory. Thus, the horizontally orientated internodes of creep plants contained numerous amyloplasts almost entirely sedimented to the lower side walls of the endodermal cells (Fig. 4). The amyloplast content of the starch sheath declined in the lower internodes in a variable fashion, sedimented starch tending to be absent from the first or second internodes of young plants with 5 or 6 internodes, or from approximately
Fig.4: Longitudinal section of the endoderm is of a horizontally orientated internode 8- 9 of a creep plant with 11 expanded internodes. Amyloplasts are sedimented on the lower side walls of the cells. Bar = 50/Lm. Arrow indicates direction of gravity.
105
the lower half of the stem of older plants with 17 or 18 internodes, although aggregations of storage amyloplasts were often present in the pith and vascular tissues of the oldest internodes. No differences were apparent between creep and normal plants in these respects. Formation of the starch sheath occurred in the subapical internodes protected by the folded apical stipules. A starch sheath containing amyloplasts of 2 - 5 /lm diameter could be detected in such internodes, but sedimentation was not apparent. Smaller (0.5 - 3 /lm) non-sedimented amyloplasts were also distributed through the cortex in these internodes. Again, no differences between creep and normal plants were apparent.
Discussion An effect of the creep gene on the stem growth habit of P. sativum could be clearly determined, but the mutant phenotype is more subtle than that of the better known age mutant, in which germination and growth-rate are affected and which exhibits a higher degree of gravitropic abnormality (Scholdeen and Burstr6m 1960, Olsen and Iverson 1980). The growth of the creep mutant appeared quite normal apart from the enhanced tendency to prostrate growth in lightgrown plants. The basis of this phenomenon requires further investigation. The mutant plants appeared to have a perfectly normal content of amyloplasts in the starch sheath, which sedimented as expected in relation to gravity. An interesting feature of the creep gene is that, unlike age, it has no apparent effect on the stem orientation of etiolated seedlings. Etiolated shoots of the age mutant, on the other hand, have been reported to become gravitropic upon illumination Gaffe et al. 1985}. This implies some difference in the mechanism or genetic control of gravitropism in lightand dark-grown pea plants. Little information exists in this area. There are marked differences in the circumnutation behaviour of light- and dark-grown pea seedlings, although the relationship between this phenomenon and gravitropism is not clear (Galston et al. 1964, Britz and Galston 1982). There are instances in other species of the modification by light of the pattern of gravitropism. Roberts (1987) has recently reported that the stem of the lazy-2 mutant of tomato is positively gravitropic in the light but responds normally to gravity in the dark. Also, it has long been known that the prostrate stems, runners or stolons of many plant species become erect in the shade or in darkness (Palmer 1956, Ziv et al. 1973). Illumination can also affect root gravitropism in some cultivars of maize (Feldman 1985). The growth of the creep pea mutant is reminiscent of the prostrate habit which represents an ecological adaptation of very many species found in the natural environment (Lovell and Lovell 1985). Prostrate species are often closely related to species with upright stems, and the marked enhancement of prostrate growth by a single gene mutation in pea suggests that in genetic terms the evolutionary conversion from the one growth habit to the other may not be complex. The product of the creep gene therefore could be of widespread relevance.
106
IAN M. SCOTT
References BRITZ, S. J. and A. W. GALSTON: Plant Physiol. 70, 264-271 (1982). FELDMAN, L. J.: Physiol. Plant. 65, 341- 344 (1985). GALSTON, A. W. A. A. TUTTLE, and P. J. PENNY: Amer. Jour. Bot. 51,853-858 (1964).
HALSTEAD, T. W. and T. K. SCOTT (eds.): Plant Gravitational and Space Research. American Society of Plant Physiologists, Rockville, MD, USA, 1984. HERTEL, R., R. K. DE LA FUENTE, and A. C. LEOPOLD: Planta 88, 204-214 (1969).
JAFFE, M. J., H. TAKAHASHI, and R. L. BIRO: Science 230, 445-447 (1985).
LOVELL, P. H. and P. J. LOVELL: The importance of plant form as a determining factor in competition and habitat exploitation. In:
WHITE, J. (ed.): Studies on Plant Demography, pp. 209-221. Academic Press, London, 1985. OLSEN, G. M. and T.-H. IVERSEN: Physiol. Plant. 50, 269-274 (1980). OLSEN, G. M., ]. I. MIRZA, P. MAHER, and T.-H. IVERSEN: Physiol. Plant. 60, 523-531 (1984). PALMER,]. H.: New Phytol. 55, 346-355 (1956). ROBERTS,]. A.: Plant, Cell Environ. 7, 515-520 (1984). - Mutants and gravitropism. In: THOMAS, H. and D. GRIERSON (eds.): Developmental Mutants in Higher Plants, pp. 135-153. Cambridge University Press, Cambridge, UK, 1987. SCHOLDEEN, C. and H. BURSTROM: Physiol. Plant. 13, 831- 838 (1960). SIDOROVA, K. K.: Pisum News Letter 7, 57 -58 (1975). ZIV, M., A. H. HALEVY, and A. ASHRl: Plant Cell Physiol. 14, 727 -735 (1973).
Stem Growth Habit and Starch Statolith Content of the creep Pea Mutant IAN
M.
SCOTT
Department of Botany and Microbiology, University College of Wales, Aberystwyth, Dyfed, SY23 3DA, U.K. Received November 30, 1987 . Accepted March 15, 1988
Summary Pea seedlings homozygous for the mutant creep gene initially grew in an upright but significantly offvertical orientation which became progressively more horizontal as growth continued. When the weak lower internodes were supported by staking in a vertical orientation, the subsequently produced internodes of creep plants rapidly became prostrate, in contrast to the internodes of normal plants, which continued in vertical growth. Etiolated creep seedlings, however, showed normal vertical growth. Microscopic examination revealed that stems of the creep mutant possessed a starch sheath with a normal content of amyloplasts sedimented according to the direction of gravity.
Key words: Pisum sativum, amyloplasts, creep mutant, gravitropism, statoliths.
Introduction The mechanisms underlying plant responses to gravity remain uncertain (Halstead and Scott 1984). An increasingly favoured approach to this problem is the characterization of genes involved in gravity responses, and tropic mutants have now been described in several species (Roberts 1987). Such mutants have in some cases proved informative. For example, the theory that sedimenting amyloplasts act as gravity-sensing statoliths has received support from studies on certain abnormally gravitropic mutants, such as the lazy-1 tomato (Roberts 1984), the amylomaize mutant (Hertel et al. 1969), and the aux-1 mutant of Arabidopsis (Olsen et a1. 1984), in which statoliths are either absent, smaller than normal, or exhibit reduced sedimentation. One of the first discoveries of a mutation affecting gravitropism was that of the age gene of Pisum sativum, which causes a loss of both root and shoot gravitropism in addition to weak germination and growth (Scholdeen and Burstr6m 1960, Olsen and Iversen 1980). Another interesting mutant pea gene, creep, was induced by Sidorova (1975), but has as yet been little investigated by physiologists. The present paper describes some features of the creep mutant. © 1988 by Gustav Fischer Verlag, Stuttgart
Materials and Methods Plant material
Pure lines of Pisum sativum L. were kindly supplied by the Nordic Gene Bank, Alnarp, Sweden. The creep mutant was line 5859 of this institute, while the isogenic parent Torsdag (line 2157) was used as the normal control. The age mutant was line 5102.
Growth habit measurements
Plants were grown in 7.6cm diameter pots of John Innes No.2 potting compost in a glasshouse heated to 18°C minimum and with a minimum 16 h photoperiod extended when necessary by mercury vapour lamps. Stem orientation was measured using a protractor. For the initial growth observations (Fig. 2), random samples (n = 10) were taken from a population and discarded after measuring, in order to avoid handling effects. In the experiment on vertically supported plants (Fig. 3), plants were lightly tied with a single loop to a vertical stake. Fifteen specimens of each genotype were selected for initial uniformity and the orientation of this single population was monitored without handling except to prevent attachment of tendrils. Significance of data was tested using a one-way analysis of variance at the p = 0.01 level.
104
IAN
M.
SCOTT
Microscopy For observations on amyloplasts, fresh tissue sections were stained briefly with a solution containing KI (20 gl-') and h (2 g 1- ') prior to examination at x 100 and x 400 magnification on a light microscope fitted with a calibrated eyepiece graticule.
90 NORMAL C/)
w w
a:
Results
Growth Habit
No significant differences were observed between plants homozygous for the creep gene and the isogenic parent cultivar T orsdag so far as germination, shoot growth-rate or morphology, or root gravitropism were concerned. The creep gene, however, exerted a marked effect on stem orientation, conferring a much increased tendency to prostrate growth in light-grown plants (Fig. 1). In the early stages, creep seedlings grew in an upright fashion, but the mean stem angle of populations of creep seedlings was significantly (p = 0.01) further than normal from a true vertical alignment at all stages measured (Fig. 2). The mutant plants showed an increased tendency away from the vertical as they grew, until a relatively sharp reorientation towards the horizontal was observed (Fig. 2), generally following the expansion of internode 5 - 6. The rate of progression to the prostrate orientation over this period varied considerably between individual plants. Bending occurred near the base of the stem in one of the first three short internodes. The apical region of the prostrate creep plants retained a tendency towards the vertical (Fig. 1), a feature which was retained in plants transferred for 4 days to a dark room, implying that gravitropism was involved. It is a familiar observation that the weak lowest internodes of pea stems fail to support tall plants in the absence of external support; in these experiments the normal plants all eventually became prostrate like the mutants. The persistence of an altered response to gravity in the creep mutants beyond this stage was demonstrated by vertically staking at internode 5 - 6 plants in which internode 7 - 8 was beginning to expand. As seen in Fig. 3, the subsequently produced
(!)
w 60
0
W
-I
(!)
Z
« ::E
w
I-
30
C/)
CREEP
o~
8
____~__~____~__~__~__~ 12
10
14
16
18
AGE (DAYS) Fig. 2: Change in stem orientation with age in normal (closed circles) and creep (open circles) plants. The angle from the horizontal (i.e. vertical = 90°) of internode 3 - 4 was measured except for the day 8 values which represent internode 2-3. Bars indicate SE.
90
NORMAL C/)
w w
a: 60 (!) w
0
W
-I
(!)
Z
«
::E 30
CREEP
w
I-
C/)
o~
______~____~______~____~~
17
19
21
23
25
AGE (DAYS) Fig. 3: Change in stem orientation with time following staking of normal (closed circles) and creep (open circles) plants. 17 day old plants with internode 7 - 8 beginning to expand were lightly tied to a vertical stake at internode 5-6. The angle from the horizontal (i.e. vertical = 90°) of internode 6-7 was measured. Bars indicate SE. Fig. 1: Growth habit of the creep pea mutant (right) compared with an isogenic normal plant (cv. Torsdag) of the same age.
internodes of the staked normal plants rigorously found and persisted in vertical growth, even though the tendrils were
The creep mutant of pea Table 1: Mean angle from the horizontal of the youngest internodes of pea seedlings (n = 8) grown for 6 days in darkness at 21 0c. Genotype Normal
creep age
LSD (p
=
0.01)
Stem angle 81.1 0 84.10 48.4 0 11.6 0
not allowed to form attachments, whereas the subsequently produced internodes of the creep plants variably but progressively lost the vertical orientation, passing through the horizontal and eventually resting on the glasshouse bench. Again, the apical one or two internodes always tended to seek the vertical. The same pattern was observed if the plants were staked at internode 11-12. Etiolated creep seedlings, however, showed no abnormal orientation. This contrasted with the agravitropism exhibited by etiolated seedlings of the age mutant (Table 1).
Starch Statolith Content Fresh, iodine-stained longitudinal and transverse sections were examined from various points along the lengths of all the internodes of mutant and normal plants of various ages. The expanded internodes of both genotypes possessed a distinctive starch sheath layer, comprising a continuous single layer of endodermal cells surrounding the vascular tissue, which contained numerous amyloplasts ranging in size from 2 -7.5 /lm diameter. The amyloplasts of both genotypes were sedimented according to the direction of gravity, as predicted by the statolith theory. Thus, the horizontally orientated internodes of creep plants contained numerous amyloplasts almost entirely sedimented to the lower side walls of the endodermal cells (Fig. 4). The amyloplast content of the starch sheath declined in the lower internodes in a variable fashion, sedimented starch tending to be absent from the first or second internodes of young plants with 5 or 6 internodes, or from approximately
Fig.4: Longitudinal section of the endoderm is of a horizontally orientated internode 8- 9 of a creep plant with 11 expanded internodes. Amyloplasts are sedimented on the lower side walls of the cells. Bar = 50/Lm. Arrow indicates direction of gravity.
105
the lower half of the stem of older plants with 17 or 18 internodes, although aggregations of storage amyloplasts were often present in the pith and vascular tissues of the oldest internodes. No differences were apparent between creep and normal plants in these respects. Formation of the starch sheath occurred in the subapical internodes protected by the folded apical stipules. A starch sheath containing amyloplasts of 2 - 5 /lm diameter could be detected in such internodes, but sedimentation was not apparent. Smaller (0.5 - 3 /lm) non-sedimented amyloplasts were also distributed through the cortex in these internodes. Again, no differences between creep and normal plants were apparent.
Discussion An effect of the creep gene on the stem growth habit of P. sativum could be clearly determined, but the mutant phenotype is more subtle than that of the better known age mutant, in which germination and growth-rate are affected and which exhibits a higher degree of gravitropic abnormality (Scholdeen and Burstr6m 1960, Olsen and Iverson 1980). The growth of the creep mutant appeared quite normal apart from the enhanced tendency to prostrate growth in lightgrown plants. The basis of this phenomenon requires further investigation. The mutant plants appeared to have a perfectly normal content of amyloplasts in the starch sheath, which sedimented as expected in relation to gravity. An interesting feature of the creep gene is that, unlike age, it has no apparent effect on the stem orientation of etiolated seedlings. Etiolated shoots of the age mutant, on the other hand, have been reported to become gravitropic upon illumination Gaffe et al. 1985}. This implies some difference in the mechanism or genetic control of gravitropism in lightand dark-grown pea plants. Little information exists in this area. There are marked differences in the circumnutation behaviour of light- and dark-grown pea seedlings, although the relationship between this phenomenon and gravitropism is not clear (Galston et al. 1964, Britz and Galston 1982). There are instances in other species of the modification by light of the pattern of gravitropism. Roberts (1987) has recently reported that the stem of the lazy-2 mutant of tomato is positively gravitropic in the light but responds normally to gravity in the dark. Also, it has long been known that the prostrate stems, runners or stolons of many plant species become erect in the shade or in darkness (Palmer 1956, Ziv et al. 1973). Illumination can also affect root gravitropism in some cultivars of maize (Feldman 1985). The growth of the creep pea mutant is reminiscent of the prostrate habit which represents an ecological adaptation of very many species found in the natural environment (Lovell and Lovell 1985). Prostrate species are often closely related to species with upright stems, and the marked enhancement of prostrate growth by a single gene mutation in pea suggests that in genetic terms the evolutionary conversion from the one growth habit to the other may not be complex. The product of the creep gene therefore could be of widespread relevance.
106
IAN M. SCOTT
References BRITZ, S. J. and A. W. GALSTON: Plant Physiol. 70, 264-271 (1982). FELDMAN, L. J.: Physiol. Plant. 65, 341- 344 (1985). GALSTON, A. W. A. A. TUTTLE, and P. J. PENNY: Amer. Jour. Bot. 51,853-858 (1964).
HALSTEAD, T. W. and T. K. SCOTT (eds.): Plant Gravitational and Space Research. American Society of Plant Physiologists, Rockville, MD, USA, 1984. HERTEL, R., R. K. DE LA FUENTE, and A. C. LEOPOLD: Planta 88, 204-214 (1969).
JAFFE, M. J., H. TAKAHASHI, and R. L. BIRO: Science 230, 445-447 (1985).
LOVELL, P. H. and P. J. LOVELL: The importance of plant form as a determining factor in competition and habitat exploitation. In:
WHITE, J. (ed.): Studies on Plant Demography, pp. 209-221. Academic Press, London, 1985. OLSEN, G. M. and T.-H. IVERSEN: Physiol. Plant. 50, 269-274 (1980). OLSEN, G. M., ]. I. MIRZA, P. MAHER, and T.-H. IVERSEN: Physiol. Plant. 60, 523-531 (1984). PALMER,]. H.: New Phytol. 55, 346-355 (1956). ROBERTS,]. A.: Plant, Cell Environ. 7, 515-520 (1984). - Mutants and gravitropism. In: THOMAS, H. and D. GRIERSON (eds.): Developmental Mutants in Higher Plants, pp. 135-153. Cambridge University Press, Cambridge, UK, 1987. SCHOLDEEN, C. and H. BURSTROM: Physiol. Plant. 13, 831- 838 (1960). SIDOROVA, K. K.: Pisum News Letter 7, 57 -58 (1975). ZIV, M., A. H. HALEVY, and A. ASHRl: Plant Cell Physiol. 14, 727 -735 (1973).