Age and Rhythm Patterns1 - NCBI

Plant Physiol. (1992) 98, 1381-1385

Received for publication July 29, 1991 Accepted November 27, 1991

0032-0889/92/98/1381 /05/$01 .00/0

Phase Shift in Leaf Movements of Xanthium Attributed to Age and Rhythm Patterns1 Willard L. Koukkari, LeAnne Carlson Hobbs, and Frank B. Salisbury* Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 (W.L.K., L.C.H.); and Department of Plants, Soils, and Biometeorology, Utah State University, Logan, Utah 84322-4820 (F.B.S.) ABSTRACT

Preliminary experiments showed that it was primarily the young Xanthium leaves that were in a more upward position during darkness. If movements of older leaves differ from those of younger leaves, then the leaves ofXanthium undergo a large phase shift that follows the developmental and morphological changes associated with leaves during the process of maturation. It is rare for a phase shift of 1800 or even 900 to occur in a synchronized circadian rhythm without a prior change in the phase of a synchronizer (e.g. an LD cycle). In this paper we report how an apparent paradox in the rhythm of Xanthium leaf movements is related to the development of the plant and the pattern of the rhythm and how the rhythm characteristics are mathematically analyzed.

Leaves of cockelbur (Xanthium strumarium L.) have been reported to be in either an upright or downward position during the dark span (night) of a 24-hour cycle. Results from our studies clearly indicate that such differences in leaf position are not related to differences in ecotypes but can be attributed to age of the leaf, pattem of the waveform of the rhythm at various stages of the light-dark synchronizer regimen, and the statistical model used for the analysis of the waveform. Younger leaves reached a maximum upright position closer to the middle of the dark span, whereas older leaves reached this position closer to the end of the dark span. A phase shift of up to 6 to 10 hours may occur as the leaf ages. Results from the examination of the pattem of the waveform at four different times showed that the pattem of a younger leaf was different from that of an older leaf during the middle of the dark span, during the light-to-dark transition, and during the middle of the light span, but not during the dark-tolight transition. Linear regression, statistical analyses, and the fitting of harmonics clearly indicate that it is the trough, more than the peak, that differs with the age of the leaf.

MATERIALS AND METHODS Plant Material

Common cocklebur (Xanthium strumarium L.) plants were raised from seeds of burs obtained from either Minnesota (1) or Michigan (Chicago strain ofXanthium supplied by Professor Jan A.D. Zeevaart, Michigan State University). Chicago strain seeds were used in most of the experiments. For each experiment, the burs were placed in distilled water and maintained in a small controlled environment chamber (6) under a LD 15:9 regimen (15 h of light followed by 9 h of darkness) at about 23°C. After 2 d, the burs were distributed on top of 4.0 cm of vermiculite in 26.5- x 26.5-cm flats, covered with approximately 0.7 cm of sand, moistened with distilled water, and transferred to controlled environment chambers (2325°C and 38-61% RH) with light sources similar to those described elsewhere (12) and providing about 130 gmol m-2 s-' (PPF).The regimens most often used were LD 15:9, LD 20:4, LD 12:12, and a regimen in which the middle of the 12h dark span was interrupted with 15 min of light. During the 1 5-min interruption of darkness, only eight of the 16 fluorescent lamps and none of the incandescent lamps were used (for specifications of lamps, see ref. 12). When the plants reached the two-leaf stage (7-9 d later), individual seedlings were transplanted to 10.5-cm2 plastic pots containing a mixture of two parts soil, one part sand, and one part vermiculite. Generally, 14-d-old plants were selected for uniformity and used to monitor leaf movements.

Going back at least to the Darwins in 1881 (5), the study of leaf movements has been a classic area of plant physiology (4, 16, 23, 24). Although much of the work has centered on the circadian rhythms of legumes (Phaseolus and Glycine species), the leaves of many other plants, including cocklebur (Xanthium strumarium), also display rhythms (1, 20). Andersen and Koukkari (1) reported that leaves ofXanthium moved upward to a night position and downward to a day position. This night position of Xanthium leaves was different from that of bean plants (6), and, as we discovered later, the position was exactly opposite to that illustrated for Xanthium in the plant physiology textbook by Salisbury and Ross (21). During the dark span, Xanthium leaves were reported to be either up (1) or down (21). The opposite responses in these two studies could have been caused by genetic differences in different ecotypes of X. strumarium. Although different cultivars of soybean (Glycine) exhibited changes in the amplitude of their rhythms, they still maintained a close phase relationship (3). A discrepancy in such a basic physiological phenomenon as the rhythmic movements of leaves mandated that we reinvestigate the leaf movements of Xanthium.

Monitoring Leaf Movements Positions of leaves at various times were determined by either manual or automated procedures. The manual proce-

'This is paper No. 17,149 in the Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, MN 55108.

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1800 140

UL) 0L) 0 0

Figure 1. Chronogram illustrating leaf movements of two ecotypes of X. strumarium L. (A, Chicago strain; B, Minnesota strain) maintained on LD 15:9 conditions. Each waveform represents the movement of a single leaf measured manually every 3 h. The orientation reference for leaf position (O°, 900, and 1800) is illustrated in the top left corner.

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dure required the use of a rectangular paper card that was divided into degrees. A weighted string suspended from the central vertex of the card served as a vertical reference (13). The position of the leaf was determined by placing the card over the midrib of the leaf and noting the degree mark closest to the suspended string. If the tip of a leaf were to point directly upward (as illustrated in Fig. 1) and be parallel to the stem, the angle would be 180°. The automated procedure, as well as the specifications of the leaf-movement device, have been described in detail elsewhere (7). Briefly, the vertical movements of a leaf are transformed to a rotational movement by a lever, the tip of which is connected to a leaf by a string. The lever is free to move up and down on a fulcrum. A magnetic cube, which is attached on the lever rotation axis (fulcrum), is positioned above a "Hall effect" sensor. Voltage from the sensor is marked on a strip chart recorder for a permanent record. Leaf positions, as indicated by voltage,

were recorded and plotted as relative elevations in chronograms (Fig. 2). Analyzing Data

Chronograms representing plots of leaf position (ordinate) against time (abscissa) provided a visual means for comparing variations in rhythm patterns (13). The data were further subjected to cosinor analyses by least squares fit of cosine functions (8, 1 1) to quantify the amplitudes and peaks of the fitted curves. Because of the sharp departure of the waveform (pattern of the actual rhythm) from a fitted cosine curve, several approaches were used for the mathematical comparison of rhythm patterns. With one approach, we compared the relative leaf position at fixed stages of the synchronized schedule: beginning of light span, middle of light span, beginning of

100

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Figure 2. Chronogram illustrating the leaf movement patterns of plants subjected to LD conditions with or without a 1 5-min light break during the middle of the dark span. The bottom leaf was monitored on each plant, and all leaves except the bottom two were removed as they developed on the shoot.

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LEAF MOVEMENTS OF XANTHIUM: AGE AND RHYTHM PATTERNS

dark span, and middle of dark span (Table I). Another approach was to determine given features of the waveform such as the time of the sharp rise from the down to the up position. A relative phase difference was then determined for each feature (Table II). To render the approach more rigorous, the analyses were implemented for each cycle separately so that a few simple statistical tests such as Student's t test could be applied. Finally, a multiple component model involving as many as six harmonics (24.0, 12.0, 8.0, 6.0, 4.8, and 4.0 h) was fitted by linear least squares to determine the timing of the high and low values and magnitude of the composite curve (25). This approach attempts to quantify some of the characteristics of the waveform for a nonsinusoidal rhythm and how they change with time. In the present study, the model was used to determine whether either peaks or troughs of the curve for the younger leaf were different from those of the older leaf (Table III). The values were then subjected to the Student's t test.

RESULTS AND DISCUSSION

Two Ecotypes Regardless of the source of seed, the pattern of the leaf movement rhythm was about the same for both ecotypes of X. strumarium (Fig. 1). Thus, the apparent disagreement reported in the literature regarding the position of the leaves during the dark span was not related to differences between the ecotype used by Andersen and Koukkari (1) and the ecotype used by Salisbury and Ross (21). Originally, the plants used in the Andersen and Koukkari (1) study were listed as a different species (Xanthium pensylvanicum Wallr.), and even though two groups can be distinguished from each other by visual observation when grown together, they are both considered to be the species X. strumarium (2, 15). The Chicago strain was used in most of the remaining experiments because it displayed a slightly larger amplitude (Fig. 1), showed more uniformity, and represented the classical strain that has contributed so much to our knowledge of plant physiology. Photoperiodic Restraints X. strumarium L. (Chicago strain) is a highly sensitive photoperiodic species (cf 17, 18, 22). For the seedlings to Table I. Data Based on the Waveforms of the Movements of a Single Leaf at Two Ages Each value is the difference in relative elevation from the older to the younger stage. Calculations were done at four fixed time points: beginning of the dark span (L to D), middle of the dark span (1/2 D), beginning of the light span (D to L), and middle of the light span (1/2 L). Values for individual cycles, as well as the mean ± SE, are listed. 1/2 D D to L Lto D 1/2 L Cycle -4 -32 -21 59 4 -47 2 -27 89 0 -64 -40 100 3 4 92 -82 0 -51 Mean±sE

-35±7

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Table II. Time when the Sharp Rise in Leaf Position Occurs Analysis based upon data for a single leaf at a younger and older stage (see Fig. 3). Time determined by the intercept of regression line fitted by least squares to data on the linear portion of the up slope of each cycle. Note: Mean rise of leaf at younger stage occurs about 5.1 h earlier than rise at older stage, as determined by difference in time of intercept between the two stages. Cycle

Younger Stage

Older Stage

Difference in Time

1 2 3 4

4.6 4.6 4.1 5.2

9.2 9.5 10.1 10.1

4.6 4.9 6.0 4.9

Mean ± SE

4.6 ± 0.2

9.7 ± 0.2

5.1 ± 0.3

remain in a vegetative stage of development, they must be maintained on cycles having short dark spans. Although such conditions (e.g. LD 20:4) were noninductive for flowering, they were too biased toward the light span and did not represent a true evaluation of leaf movements during the critical dark span (data not shown). On the other hand, a brief interruption of a longer dark span, which is well known to inhibit floral induction, did not greatly influence the rhythmic pattern of leaf movements (Fig. 2). Because floral induction and leaf movement rhythms are not coupled in Xanthium (19, 20), the procedure could be used to examine the leaf movements under cycles having light and dark spans of approximately equal length. Five experiments involving various light-dark regimens (see "Materials and Methods"), with and without some leaves being removed, were conducted. Depending upon the experiment, either manual or automated procedures were used. Leaf Age The pattern of the leaf movement rhythm of the newly developed leaf was found to be distinctly different from that Table Ill. Timing of the High and Low Values of the Best Fitting Curve Combining Six Harmonic Components Each value represents the timing in minutes after the beginning of the dark span for individual cycles of the actual curves. -, The high values of cycles 3 and 4 of the younger leaves were not analyzed because the upper values were not available from the strip chart due to the limits of instrumentation (see Fig. 3). Younger Stage

Older Stage

Difference in Time

636 660

36 24

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672 684 720 728

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701 ±14

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36 152 144 204

284 436 488 488

248 284 344 284

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423 ± 48

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Figure 3. Chronogram comparing the movements of a single leaf during two stages of development. The older stage is approximately 2 weeks older than the younger stage. Data were normalized for each cycle individually so that the maximum upward position was 1000 and the minimum was 00. Because of the magnitude of leaf movement and the limited range of our instrumentation, the third and fourth cycles for the younger leaf appear to be clipped. (Analyses of data are presented in Tables I, II, and 111.)

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of the same leaf 2 weeks later (Fig. 3). This could explain how the leaves of Xanthium may be viewed as being either up or down during the dark span. Results based upon the cosinor analyses of data (I 1) from many experiments involving various LD cycles and numerous plants, in which the differences in the synchronized leaf movement rhythms of young leaves were compared with those of old leaves, revealed a phase difference of 6.5 to 10.3 h. Although a cosinor model does not appear to fit the pattern of Xanthium leaf movement well, these phase shifts are in accord with those that can be visually observed in most instances. The difference in the waveform pattern between young and old leaves was evident when we focused on the timing of fixed stages of the synchronized regimen. For example, as illustrated in Figure 3, when a leaf is younger it reaches a maximum upright position closer to the middle of the dark span, confirming the report of Andersen and Koukkari (1). As is clear in Figure 20-1 of Salisbury and Ross (21), the younger leaves begin to rise just before the subjective night (also see refs. 19 and 20). For an older leaf, the maximum upright position occurs near the end of the dark span, supporting the description of Salisbury and Ross (21). In addition, the leaf remains more upright during most of the remaining light span. Results from the examination of leaf position at four fixed stages of the synchronized schedule showed that the pattern of the waveform of a younger leaf was different from that of an older leaf during the light-to-dark transition, the middle of the dark span, and the middle of the light span. The pattern was not different during the dark-to-light transition (Table I; Fig. 3). The linear regression of the rise in the curve (Table II) also differed significantly (t test) in relation to the age of the leaf. The fitting of six harmonics of the 24-h cycle in the statistical model of Tong et al. (25) supports what the chronogram illustrates (Table III; Fig. 3): The trough (minimum leaf position), rather than the peak (maximum leaf position), differs with the age of the leaf. The localization of the change in waveform occurring during one primary phase of a rhythm (Table 3), as well as during a given span of an environmental cycle, can have great implications in physiology and pathology. For example, higher frequency oscillations of a fundamental nature may become evident during certain phases (7, 10). The response of a plant to environmental agents and the regulation of growth and development are also temporally organized relative to phase (9, 14).

In the present study, the paradox regarding the night position of Xanthium leaves has been resolved. The detection of a phase shift of up to 1540 (10.3 h in a 24-h cycle) under synchronized conditions is relatively uncommon among plants, and perhaps other organisms as well, and in this case indicates a temporal change associated with the development of an organ of a plant. ACKNOWLEDGMENTS We gratefully acknowledge the counsel and suggestions of Dr. Germaine Cornelissen during the analyses of the waveform and rhythm characteristics. The comments of Dr. Christopher Bingham and Dr. Thomas K. Soulen are greatly appreciated.

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833-1021 3. Bunning E (1979) Circadian rhythms, light, and photoperiodism: a reevaluation. Bot Mag Tokyo 92: 89-103 4. Bunning E, Chandrashekaran MK (1975) Pfeffer's view on rhythms. Chronobiology 2: 160-167 5. Darwin C, Darwin F (1881) The Power of Movement in Plants. Appleton and Co., New York 6. Guillaume FM, Kennedy BW, Carlson L, Koukkari WL (1986) Leaf movement alterations on bean plants with common bacterial blight. Phytopathology 76: 270-272 7. Guillaume FM, Koukkari WL (1987) Two types of high frequency oscillations in Glycine max (L.) Merr. In JE Pauly, LE Scheving, eds, Advances in Chronobiology, Part A. Alan R. Liss, New York, pp 47-57 8. Halberg F, Tong YL, Johnson EA (1967) Circadian system phase-an aspect of temporal morphology: procedures and illustrative examples. In H von Mayersbach, ed, The Cellular Aspects of Biorhythms. Proceedings of the International Congress of Anatomists. Springer-Verlag, Berlin, pp 20-48 9. Koukkari WL (1988) The broad spectrum of plant rhythms. In WTJM Hekkens, GA Kerkhof, WJ Rietveld, eds, Advances in the Biosciences: Trends in Chronobiology, Vol 73. Pergamon Press, Oxford, United Kingdom, pp 31-41 10. Koukkari WL, Bingham C, Duke SH (1987) A special group of ultradian rhythms. In JE Pauly, LE Scheving, eds, Advances in Chronobiology, Part A. Alan R. Liss, New York, pp 29-33 11. Koukkari WL, Halberg F, Gordon SA (1973) Quantifying rhythmic movements of Albizzia julibrissin pinnules. Plant Physiol 51: 1084-1088 12. Koukkari WL, Johnson MA (1979) Oscillations of leaves of

LEAF MOVEMENTS OF XANTHIUM: AGE AND RHYTHM PATTERNS

13. 14.

15. 16. 17. 18.

Abutilon theophrasti (Velvetleaf) and their sensitivity to bentazon in relation to low and high humidity. Physiol Plant 47: 158-162 Koukkari WL, Tate JL, Warde SB (1987) Chronobiology projects and student exercises. Chronobiology 14: 405-442 Koukkari WL, Warde SB (1985) Rhythms and their relations to hormones. In RP Pharis, DM Reid, eds, Encyclopedia of Plant Physiology, New Series, Vol 11. Springer-Verlag, Berlin, pp 35-77 Uve D, Dansereau P (1959) Biosystematic studies on Xanthium: taxonomic appraisal and ecological status. Can J Bot 37: 173-208 Pfeffer W ( 1906) The Physiology of Plants. A Treatise upon the Metabolism and Sources of Energy in Plants, Vol III. Oxford Press, Clarendon, UK Salisbury FB (1985) Xanthium strumarium. In A Halavy, ed, CRC Handbook of Flowering. CRC Press, Boca Raton, FL, pp 473-522 Salisbury FB (1990) The use of Xanthium in flowering research. In R Maksymowych, principal author, Analysis of Growth and Development of Xanthium. Developmental and Cell Biology Series. Cambridge University Press, Cambridge, UK, pp 153-194

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19. Salisbury FB, Denney A (1971) Separate clocks for leaf movements and photoperiodic flowering in Xanthium strumarium L. In M Menaker, ed, Biochronometry. National Academy of Sciences, Washington, DC, pp 292-311 20. Salisbury FB, Denney A (1974) Noncorrelation of leaf movements and photoperiodic clocks in Xanthium strumarium L. In LE Scheving, F Halberg, JE Pauly, eds, Chronobiology. Iqaku Shoin, Tokyo, Japan, pp 679-686 21. Salisbury FB, Ross CW (1985) Plant Physiology, Ed 3. Wadsworth Publishing Co., Belmont, CA 22. Salisbury FB, Ross CW (1991) Plant Physiology, Ed 4. Wadsworth Publishing Co., Belmont, CA 23. Sweeney BM (1979) Endogenous rhythms in the movement of plants. In W Haupt, ME Feinleib, eds, Encyclopedia of Plant Physiology, New Series, Vol. 7. Springer-Verlag, Berlin, pp 71-93 24. Sweeney BM (1987) Rhythmic Phenomena in Plants, Ed 2. Academic Press, San Diego, CA 25. Tong YL, Nelson WL, Sothern RB, Halberg, F (1977) Estimation of the orthophase (timing of high values) on a non-sinusoidal rhythm, illustrated by the best timing for experimental cancer chronotherapy. In Proceedings, XII International Conference, International Society for Chronobiology. Washington D. C. Publishing House 'II Ponte,' Milan, Italy, pp 765-769