Growth adaptation of leaves and internodes of poplar to irradiance, day length and temperature

Tree Physiology 19, 933--942 1999 Heron Publishing----Victoria, Canada Growth adaptation of leaves and internodes of poplar to irradiance, day length and temperature G. A. PIETERS, M. E. VAN DEN NOORT and J. A. VAN NIJKERKEN Department of Plant Physiology, Agricultural University of Wageningen, Wageningen, The Netherlands Received November 13, 1998 Summary The adaptation of absolute growth rate of leaves and internodes of Populus euramericana (Dode) Guinier cv. Robusta to irradiance and day length proceeds by increases in the volume of the apex. Diameter and height of the apex increase linearly with time, resulting in linear increases in rates of leaf initiation and stem height growth that can be described by an acceleration factor. The acceleration factor is proportional to day length. The relationship between the acceleration factor and irradiance is curvilinear and saturates at irradiances above about 300 W m 2. Absolute stem height growth rate is the product of mean relative growth rate in stem height and the length of the growing part of the stem. The temperature-dependent growth pattern of each individual leaf or internode reflects a specific relationship between its relative growth rate and organ age that is independent of irradiance and plant age; however, it is dependent on day length during the primordial phase. The constancy of the growth patterns and the correlation between leaf length and leaf initiation rate indicate that growth of primordia is predetermined in the apex, presumably by the precisely structured vascular system. Keywords: acceleration factors, apex, height growth, internode extension, leaf growth, Populus euramericana Robusta, vascular system. Introduction Poplar is an ideal model growth system because of its large genetic variation and the ease with which it can be propagated vegetatively. If cuttings are taken in summer and grown under optimal nutrient conditions, only irradiance and temperature determine their rate of development. In the macroscopic phase of growth in such plants, relative growth rates of leaf length and internode length at half-mature length (RGR 50 = 0.15 and 0.22 day 1, respectively; see Appendix for symbol definitions) are nearly independent of irradiance and plant height (Pieters 1974, 1986). Thus, although plants grow much faster at high than at low irradiance, mean RGRs of leaves and internodes are almost constant. The slight dependence of RGR on irradiance is the result of temperature increases associated with high irradiances (Pieters 1974, 1975). Similarly, in Populus tremuloides Michx. plants growing in open-top chambers, the relative growth rates at half-mature length, RGR 50, of leaves (0.15 day 1 ) and stem height (0.26 day 1 ) were dependent on temperature but not on irradiance (Pieters 1996). At constant temperature, relative growth rate of the length (L) of an individual leaf (RGR leaf ) or internode (RGR internode ), defined as dl/(ldt), seems to follow an organ-specific pattern with organ age, irrespective of final length of the organ (Pieters 1974, 1986). Because RGR is a measure of the mean absolute growth rate of unit cell length, it is an important physiological property of growing cells. Furthermore, because leaf length is correlated with both leaf width and leaf area (Pieters 1974, 1983, 1984, 1986), the growth of a leaf can be described by the growth pattern of leaf length. Differences in the full-grown length and absolute growth rates among leaves can, thus, be explained by differences in the mass of cells supplied by the apex to the primordia. In many plants, plots of the lengths of successive growing leaves against leaf number (plastochron) yield (descending) slopes ( L/ N) that remain constant at successive measurement times. The constancy of this slope implies that the difference in lengths of successive leaves at about half of their mature length ( L) is constant, irrespective of mature leaf length (L m ) (Pieters 1974). This difference in length can be calculated as: L = 0.5L m (RGR 50 )P, where P is plastochron duration. Because L and RGR 50 are constant, L m /(1/P) must be constant; i.e., mature leaf length is coupled to leaf initiation rate (1/P). The finding that leaf length and leaf initiation rate are correlated is consistent with the observation of a precisely regulated pattern of anatomical development in the apical vascular system, as described by Larson (1975, 1977, 1980). The growing part of the stem, GS, is defined as the axis above an internode (GS i ) or leaf (GS L ) that just reached its final length. The basal diameter of GS L is correlated with final leaf length (Pieters 1974, Pieters and van den Noort 1988). A similar situation is found for stem elongation. The length of the growing part of the stem (GS i ) is directly related to the rate of stem elongation growth (Pieters and van den Noort 1988), indicating that absolute growth rate is determined by the mass of contributing cells, whereas mean RGR of those cells (0.25 day 1 ) remains unchanged. Neither irradiance nor photosynthesis influences the absolute growth rate of cells. The mean internode length (L int ) can be calculated as: L int = (GS i )(RGR internode )P. Because GS i and 1/P are linearly correlated, mean length of an internode is constant, irrespective of

934 PIETERS, VAN DEN NOORT AND VAN NIJKERKEN leaf length. This phenomenon has been observed in many other plants. Adaptation of absolute growth to irradiance proceeds by way of the apex. Because leaves and internodes of poplar grow according to a predetermined pattern, the increasing size of the apex is reflected in the size of GS. Because length and diameter of GS increase linearly with time, final leaf length (L m ) and leaf initiation rate also increase linearly with time (Pieters 1986). If it is possible to determine accurately the rate of growth of GS, it is also possible to predict the moments of initiation of successive leaves and their final lengths. If we then measure the lengths of successive primordia in an apex, we can calculate the age of each primordium in relation to the leaf that has just reached its final length. Correcting the measured length of each primordium for the increase in GS, together with the age data, give the leaf length--leaf age curve throughout the growth of the leaf from initiation to maturation. The relative growth curve of a leaf at a given irradiance can then be calculated from this curve. Because plant temperatures increase with increasing irradiance, these relative growth curves should be corrected for the temperature increase. Neither the difference in length of successive leaves ( L) nor the final length of successive leaves (L m ), nor the final lengths of the internodes, is changed by temperature (Pieters 1974). This indicates that temperature only changes the time scale of the growth process; i.e., it increases leaf initiation rate and mean RGR of leaf and internode growth, and decreases the duration of growth of the individual organs proportionally. We have used published data to examine the effects of irradiance, day length and temperature on the rate and duration of growth of the growing part of the stem (GS) of euramericana poplar (Populus euramericana (Dode) Guinier). Specifically, we studied apex-mediated effects of irradiance on poplar leaf and stem growth on the basis of initial growth parameters and a single irradiance-dependent variable. We also constructed relative growth curves of leaves to evaluate the hypothesis that irradiance does not influence the form of these growth curves. Model development A linear increase in the basal diameter of GS L is accompanied by a linear increase in leaf initiation rate (dn/dt), thus: dn/dt = a N (t N + t), (1) where N = the number of a specific leaf, counting acropetally, a N = leaf acceleration factor (day 2 ), t N = a constant (days), and t = time (days). For each initiated leaf, plant age t is increased by the duration of one plastochron. The relationship between total number of leaves and time is: N(t) = 0.5a N (t N + t) 2, (2) The constant t N describes the developmental stage of the apex with respect to leaf production at the start of the measurements. At t = 0, the rate of leaf production is a N t N and the number of leaves is 0.5a N t N 2. These relationships are also valid for stem height, where the constants a N and t N are replaced by a H and t H. Materials and methods Experiments and plant numbers The experiments reported in this paper are listed in Table 1. Each plant has a code. The first digits indicate the experiment number, the following character indicates the plant material, the digit after this indicates irradiance and the last digit indicates the plant. The code 13R33 means: Experiment 13 with Populus euramericana cv. Robusta, lowest irradiance (in this experiment 15 W m 2 ), plant number 3. During measurements on plants in Experiment 11R, we discovered that the wrong plant material had been taken. This undefined poplar clone is called Clone 2. Growth room All experiments, except where noted otherwise, were done in a 6 4.68 2 m growth room of the phytotron of the Laboratory of Plant Physiological Research, Wageningen, the Netherlands. Irradiance in the growth room was regulated by lamp type, the number of lamps, and by moving vertical screens in the horizontal direction. The plants were irradiated from two sides and from above to ensure uniform irradiance with 7.5, 15, 30 and 60 W m 2 of photosynthetically active radiation (PAR; 32.5, 65, 130 and 260 µmol m 2 at 400--700 nm). Irradiances of 7.5 and 15 W m 2 were provided by Philips TLM(F)-33 65-W fluorescent tubes, and irradiances of 30 and 60 W m 2 were provided by 140-W fluorescent tubes of the same lamp type. The photoperiod was 16 h. Room temperature was maintained at 22 ± 0.5 C and relative humidity was kept between 40 and 60%. For further details see Pieters (1974, 1975). Experiment 15R was done in a growth room of the Department of Plant Physiology. The climate conditions were similar to those of the phytotron growth room, except that irradiance was only from above and temperature was 19.5 C. Maximum Table 1. Summary of experiments discussed in this paper. Details provided include experiment number and year, irradiances used, day length, and the number of plants per treatment. Experiment no. Year Irradiance Photoperiod No. of plants (W m 2 ) (h) 1R 1981 7.5, 15, 30 16 4 5R 1985 7.5, 15, 30 16 4 9R 1992 7.5, 15, 30, 60 16 3 10R 1992 30, 60 16 3 11R 1 1993 7.5, 15, 30, 60 16 3 13R 1993 15, 30, 60 24 3 15R 2 1994 60 16 3 1 Unknown clone designated Clone 2. 2 No lateral illumination. TREE PHYSIOLOGY VOLUME 19, 1999

GROWTH ADAPTATION TO ENVIRONMENTAL CONDITIONS 935 irradiance was about 80 W m 2 supplied by high-frequency 58-W fluorescent tubes (Philips, TLD-50W/HF, color 33). Plant cultivation Populus euramericana cv. Robusta cuttings were cultivated in gravel culture in rectangular polyethylene containers (250 250 300 mm). The plants were subirrigated every 30 min with 50% Hoagland A-Z solution, modified after Steiner (1968). To prevent nitrate deficiency, this concentration was increased to 100% for plants growing at 60 W m 2 with a 16-h photoperiod, and to 150% for plants growing at 60 W m 2 with a 24-h photoperiod. One shoot was allowed to grow on each plant. At the end of the experiment, each shoot consisted of about 50--60 mature leaves, nodes and internodes. For Experiment 15R, in which irradiance was from above only, the tops of the growing plants were maintained about 10 cm under the light ceiling to ensure an irradiance of about 60 W m 2 at the plant tops. Measurements Lengths of successive leaves and internodes (+ node) were measured to the nearest 0.5 mm. Stem diameters at the middle of successive internodes (mid-internode diameter) were measured to the nearest 0.1 mm with calipers held parallel with the base of the attached leaf. Plant height was measured directly or calculated by summing the lengths of internodes (+ nodes). Measurements were made three times a week. Plant age is the number of days after the start of the measurements. Data evaluation Several secondary growth parameters were calculated from the original data. Because the time of the end of leaf growth is difficult to determine, we calculated the moment when each leaf and internode reached 90% of its final length, by linear interpolation between the two closest measurement points. The acceleration factors a N and a H and the constants t N and t H were determined by fitting leaf number (N) to the calculated time of reaching 90% of mature leaf length (t), or successive heights (H) to t with Equation 2. Relationship between RGR leaf and leaf age The acceleration factor was used to calculate the growth pattern of individual primordia and leaves, on the basis of continuous measurements of the visible leaves and destructive measurements of primordia length at the end of the experiment. The time at which a leaf N reaches 90% of its final length was calculated with Equation 2. The age of leaf N + n (n places above leaf N) was then calculated by subtraction of the calculated times. However, because of the increase in apical size, the length of leaf N + n is greater than the length of leaf N at the same age. Because leaf production rate at time t (Equation 1) and final leaf length are correlated, the length of leaf N + n was corrected by dividing it by the ratio of the initiation rates of leaves N and N + n. In this way, a standardized pattern of leaf development was established through the relationship between RGR and leaf age. To minimize variability, successively calculated RGRs were averaged. Effect of temperature on growth pattern Plant temperature was more than 2 C higher at an irradiance of 30 than at 7.5 W m 2 (Pieters, 1975); the temperature difference between plants in 30 W m 2 and plants in the dark was about 2.5 C. A correction for the temperature increase caused by high irradiances was calculated from the relationship between RGR of leaves at half of mature length (RGR 50 ) and temperature (Pieters 1974): RGR 50 = 0.242 0.717 exp T/10.577, (3) where T = temperature ( C). The temperature-induced increase in RGR 50 causes a proportional decrease in growth duration, because neither the full-grown length of successive leaves, nor the difference in length of two successive leaves about half-final length, nor internode length, is changed by temperature (Pieters 1974). This means that, in each individual leaf, the temperature-induced increase in RGR is compensated for by a decrease in the duration of growth. If it is assumed that the relative effect of a temperature change is similar for all growth processes, the relative effect of a temperature change on RGR 50 and other temperature-dependent processes can be calculated with Equation 3. To account for the difference in the duration of irradiance (16-h versus 24-h photoperiod) the relative effect (e) of temperature was approximated as: e = 24e L / (16e L + 8e D ), (4) where e L and e D express the relative temperature effect in light and darkness. Estimation of internode length The model used to estimate internode length was based on stem height growth and leaf initiation. Height growth is the product of the length of GS i and mean RGR internode. Because of periodic leaf initiation, the length of the stem is divided into internodes. When the developmental stages of the apex for leaf production and for height growth are in steady state, the length of an internode is the ratio between the rates of height growth and leaf production. At the start of growth of a plant, however, neither process is at steady state. Under such conditions, the final length of internode N is determined as follows: the time at which leaf N reaches 90% of full-grown length can be calculated with Equation 2 plus the acceleration factor a N and the constant t N of that particular plant. The corresponding internode N reaches 90% of its final length 6.5 days earlier than under steady-state conditions (Pieters 1974). Stem height can be calculated with Equation 2, plus the acceleration factor a H and the constant t H of the same plant and the time internode N reaches 90% of its final length. Rate of height growth at the same moment can be calculated with Equation 1. Because mean RGR stem corresponds with 25% of the length of the stem undergoing extension growth (GS i ), this length can be taken as four times the rate of height growth (Pieters and van den Noort 1988). Subtracting the length of GS i from calculated total plant height gives the length of the full-grown part of the stem at the TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

936 PIETERS, VAN DEN NOORT AND VAN NIJKERKEN end of elongation growth by internode N. The difference between this stem length and the length of the full-grown stem at the end of extension growth of internode N 1, yields the full-grown length of internode N. Destructive measurements of primordial length and primordial stem diameter At harvest, the shoot apex was excised. To prevent drying, it was inserted in an elastic, water filled polyethylene tube that fitted the stem tightly. The length of a primordium and the diameter of the internode were measured with the aid of a binocular stereo-microscope. After measurement, the primordium was carefully removed to get access to the next one. The smallest measured primordium was about 50 µm in length. Leaf weight per unit area At the end of the experiment, the areas of all leaves were measured with a video camera area meter (Pieters 1984). The weight per area ratio of each leaf (WAR) was determined by dividing the fresh weight of each leaf (without petiole) by its area. Results and discussion Leaf initiation and leaf growth Figure 1A shows the time course of leaf number (N) for three plants, grown at three irradiances. The leaf data fit Equation 2 with high correlation coefficients (r 2 ). Because absolute rate of leaf initiation differed among plants of the same age grown at different irradiances, our hypothesis that irradiance primarily affects the rate of increase of the growing part of the stem (GS) (a N t) was fully corroborated. Figure 1B shows that the data on plant height also fit Equation 2 well. Aadaptation of plant growth to irradiance or day length can be described with a limited number of parameters. Thus, t N and t H define the developmental stage of the apex of the plant at the start of the experiment, and the acceleration factors a N and a H define its developmental rate. The acceleration factor a N was linearly related to the weight area ratio (WAR) of the leaves (Figure 2), suggesting that a N and WAR have a similar physiological basis. Dependence of the acceleration factor a N on irradiance and clone The acceleration factor a N was also related to growth irradiance. Figure 3A shows this relationship for Robusta plants and for Clone 2 grown at different irradiances and day lengths. In all cases, the relationship between a N and irradiance was curvilinear. The increase in a N was nearly proportional to day length, indicating that the plants were growing optimally with no limitations in the root environment. Experiment 1R produced a different relationship between a N and irradiance as a result of root constrictions. In this experiment, we reused stubs from a preceding experiment. Consequently, the root system had already filled the pot and new root growth was pressed against the walls of the pot. This limited Figure 1. (A) Examples of measured and simulated production of leaves at 90% of final length versus plant age for individual plants grown at 7.5, 15 or 30 W m 2 in a 16-h photoperiod. Simulated leaf production versus plant age t, N(t), was calculated according to Equation 2: N(t) = 0.5a N (t N + t) 2 in which a N is the acceleration factor for leaf production and t N a time factor, indicating the developmental state of the plant at the start of the experiment. (B) Examples of measured and simulated growth in stem height versus plant age for individual plants grown at 7.5, 15 or 30 W m 2 in a 16-h photoperiod. Simulated height growth versus plant age t, H(t), was calculated according to Equation 2: H(t) = 0.5a H (t H + t) 2 in which a H is the acceleration factor and t H a time factor, indicating the developmental state of the plant at the start of the experiment. growth of the plant because uptake of water or ions was impeded. The only plant that was growing vigorously in Experiment 1R was a new cutting that was included as a replacement for a dead cutting. Note that the acceleration factor of the new cutting (filled circle, in the middle curve in Figure 3A) lies in line with those for other plants in the same photoperiod. Subsequently, fresh plant material was always used for each experiment. The response of a N to irradiance differed for Clone 2, indicating that the relationship between a N and irradiance is also determined by genetic properties of the plant, because there were no limitations in the root environment during experiment 11R. A plot of the acceleration factor against total daily irradiance (Figure 3B) indicated that the effect of day length can be ascribed largely to the increase in the total daily energy supply. The temperature-corrected relationship between a N and irradiance is also shown in Figure 3B. TREE PHYSIOLOGY VOLUME 19, 1999

GROWTH ADAPTATION TO ENVIRONMENTAL CONDITIONS 937 The curvilinear relationship between a N and irradiance (Figures 3A and 3B) supports the idea that the response of a N to irradiance is determined by the photosynthetic production of assimilates. Light saturation occurs at about 300 W m 2 and lies between the light saturation of an individual leaf and a canopy (Monteith 1965, Ceulemans 1990, Ceulemans and Saugier 1990). The value of a N is not a direct measure of the absolute growth response of the plant. Although a larger a N leads to a faster increase in apical size, there will be a delay before the increase in a N causes a substantial increase in absolute growth rate. Based on the study of Doorenstouter et al. (1985), we conclude that the linear relationship between the acceleration factor and weight area ratio of the leaves (WAR in Figure 2) is unlikely to be a result of a direct relationship between photosynthesis and the growth of the apex. Doorenstouter et al. (1985) found that chlorotic, Mg-deficient leaves grown at 7.5 or 30 W m 2 had similar WAR as Mg-sufficient leaves, indicating that WAR is determined by irradiance, not by photosynthesis. However, the relation between a N and WAR depends strongly on temperature (at higher temperature a N increases and WAR decreases), shifting to the left with higher temperature; indicating that the relationship between a N and WAR is indirect. In an experiment with aspen (Populus tremuloides, Pieters 1996), the acceleration factors were negligibly affected by a doubling of the atmospheric CO 2 concentration. This finding provides further circumstantial evidence that photosynthesis does not directly control the growth rate of GS (or the apex); however, there is considerable variation in the growth responses of different tree species to elevated CO 2 (Ceulemans et al. 1992, 1995, 1996). Furthermore, elevated CO 2 influences not only photosynthesis, but also transpiration, and may have other direct or indirect effects on plant functioning (Jarvis 1989). Thus, it is not clear whether photosynthates or hormones direct the growth of GS. A detailed knowledge of the energy balances in the GS and apex may help elucidate whether photosynthates or hormones direct their growth. Figure 2. Linear relationship between the acceleration factor for leaf initiation (a N ) and leaf weight area ratio (WAR) for poplar plants grown at irradiances of 7.5, 15 or 30 W m 2 in a 16-h photoperiod and 15, 30 or 60 W m 2 in a 24-h photoperiod. Relationship between the acceleration factors for leaf initiation and stem elongation The relationship between the acceleration factors for leaf initiation rate and height growth is approximately linear: a N = 0.0026 + 0.352a H, with a correlation coefficient of 0.88. Thus, there is some independence between the factors a N and a H, which differ by a factor of three. This difference arises because they have different dimensions (a N has the dimension leaves day 2, whereas a H has the dimension cm day 2 ) reflecting that each leaf produced corresponds to an increase in stem length (internode length) of about 3 cm. The relationship between t N and t H is less clear than that between a N and a H. The values of t N and t H are mainly determined by leaf number and stem height at the start of the experiment. The variable t N /t H ratio indicates that, at the beginning of the experiment, there exists some variability in the developmental stage of the apex with respect to leaf initiation (the diameter of the apex) and to the rate of stem extension growth (the height of the apex). Independence of the acceleration factor a N from plant age and plant size A doubling of the length of a newly-matured leaf (L m ) roughly corresponds to an eightfold increase in leaf area production per day (Pieters 1974). Also, the total area of growing leaves is correlated with L m 3 (Pieters 1974). The ratio of rate of leaf area production per day and area of growing leaves present along the stem is approximately constant; i.e., at a constant, all-sided irradiance, the ratio between use and production of assimilates for leaf area production remains approximately constant. This Figure 3. (A) Relationship between acceleration factor for leaf initiation (a N ) and irradiance in a 16-h or 24-h photoperiod. (B) Relationship between acceleration factor for leaf production (a N ) and the total daily irradiance both corrected and uncorrected for temperature for Experiments 9R, 10R and 13R. The temperature-corrected values of a N are fitted to a photosynthesis curve with a correlation coefficient of r 2 = 0.94. TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

938 PIETERS, VAN DEN NOORT AND VAN NIJKERKEN may explain why the acceleration factors a N and a H are independent of plant age and, consequently, of plant size and the ever increasing leaf area. The growing shoot seems to be self-sufficient for assimilates. It is known that older leaves do not contribute substantially to the assimilates needed in shoot development in other plant species (Milthorpe and Moorby 1974). In poplar, the shift from acropetal to basipetal transport of assimilates occurs just before the leaf reaches its full length (Larson 1969); however, this observation contrasts with the report that photosynthesis of mature leaves contributes to the development of poplar plants (Ceulemans et al. 1995). Based on an experiment with an irradiance of 60 W m 2 from above only, we calculated a temperature-corrected leaf acceleration factor of a N = 0.016 day 2, which is comparable with a three-sided, uniform irradiance of about 45 W m 2. Because length of the growing part of the shoot (GS) increases from about 5 to 24.0 cm, the average irradiance received by the GS declines with time. Although we do not know how to calculate the average irradiance in this changing light climate, some indication about effective irradiance can be found in the WAR of successive leaves on plants. On plants grown with lateral irradiance, we observed a remarkable constancy of WAR. In contrast, on plants grown with irradiance from above only, WAR declined from 0.03 to 0.025 g cm 2 during the experiment, suggesting that, by the end of the experiment, the effective irradiance had dropped from 60 to below 30 W m 2. This means that the acceleration factor declines with increasing plant size and that, in this situation, a N is not correctly defined by Equations 1 and 2. These results also demonstrate the importance of using a uniform irradiance for growth analysis studies. Growth patterns Temperature-corrected growth patterns for leaves of three plants grown at 30 W m 2 in a 16-h photoperiod are shown in Figure 4A. Average growth patterns for plants grown at 7.5 and 30 W m 2 in a 16-h photoperiod, and at 15, 30 and 60 W m 2 in a 24-h photoperiod are shown in Figure 4B; each graph comprises data for three or four plants. The temperature-corrected growth patterns of plants in the 16-h photoperiod are similar, whereas those of plants in the 24-h photoperiod show some variability in the primordial phase of growth, although no systematic effect of irradiance was evident. Based on the macroscopic phase of growth, we calculated the duration of leaf growth in a 16-h photoperiod as 32 days (Pieters 1986), excluding the primordial phase. The durations of growth before and after temperature correction are given in Table 2. Compared with the RGR in a 16-h photoperiod, a 24-h photoperiod increases the RGR of the primordium in the early phase of growth, but at the same time decreases the duration of growth. These changes in RGR and growth duration probably compensate each other, because the difference in lengths of successive leaves remained similar ( L, Table 3) in both photoperiods. This finding also implies that photosynthesis does not affect final leaf length or internode length in plants grown in a 24-h photoperiod. The increase in RGR with increasing photoperiod is consistent with the absence of an effect of Figure 4. (A) The fixed growth patterns of leaves on four plants grown in a 16-h photoperiod at 30 W m 2. The growth patterns were calculated on the basis of the acceleration factor and the measured lengths of successive primordia and corrected for a temperature of 22 C. The RGR for leaf length is fitted to leaf age with a polynomial equation. One standard deviation is indicated by a bar. (B) The averaged, fixed growth patterns of leaves on plants grown in a 16-h photoperiod at irradiances of 7.5 or 30 W m 2 (n = 4) or in a 24-h photoperiod at irradiances of 15, 30 or 60 W m 2 (n = 3). The growth patterns were corrected for a temperature of 22 C. The standard deviation is indicated for each curve. irradiance, because the ratio of the rate of leaf area production per day and the area of growing leaves present along the stem remained constant, but carbohydrate production per day increased. It is not known why day length increased RGR in the primordial phase of leaf growth but irradiance did not. The constancy of the growth patterns indicates that final leaf length depends on the length of the initiated primordium, but is independent of irradiance or temperature; i.e., extension and division growth are determinate in poplar. Because primordia of similar length are not necessarily at a similar developmental stage, the plastochron index conceived by Erickson and Michelini (1957) does not adequately define the developmental stage of leaves and internodes of poplar. The length of the primordium is already determined before initiation in the apical system, suggesting that the developing vascular system plays an important role in this determination. The constancy of the growth patterns in the various irradiances also indicates that pruning of axillary buds has no effect on growth of the growing part of the shoot (GS). In a 16-h TREE PHYSIOLOGY VOLUME 19, 1999

GROWTH ADAPTATION TO ENVIRONMENTAL CONDITIONS 939 Table 2. Calculated mean duration of leaf growth before and after temperature correction for four plants grown at 7.5 or 30 W m 2 in a 16-h photoperiod and for three plants grown at 15, 30 or 60 W m 2 in a 24-h photoperiod. Day length Irradiance Duration Duration before after correction correction (h) (W m 2 ) (days) (days) 16 7.5 33.5 ± 0.3 35.9 ± 0.1 16 30 31.0 ± 1.1 35.9 ± 0.7 24 15 27.4 ± 0.7 31.2 ± 0.0 24 30 23.5 ± 0.2 31.4 ± 0.0 24 60 21.6 ± 0.6 31.5 ± 0.0 Table 3. Mean values for the difference in length of successive growing leaves at about half mature length ( L; cm), their standard error (SE) and the number of measurements (n) for Experiments 9R (16-h photoperiod), 10R (16-h photoperiod), and 13R (24-h photoperiod). Irradiance Experiment (W m 2 ) 9R (16 h) 10R (16 h) 13R (24 h) photoperiod, no buds started growth at irradiances lower than 30 W m 2 and the acceleration factors were correlated with WAR, which in itself is independent of side branches. Mature leaf length (L m) and leaf initiation rate The acceleration factor can be used to estimate rate of leaf production of a plant at the moment of maturation of a leaf. Calculated leaf production rate, corrected for temperature, is plotted against L m in Figure 5. The correlation coefficient for the relationship between L m and leaf initiation rate is 0.99. Only at the start of cutting growth is L m lower than the expected value, probably because of imbalances between shoot and root growth of the new cutting. Internode formation L SE n L SE n L SE n 7.5 2.06 0.23 51 15 2.25 0.28 44 2.29 0.51 41 30 2.10 0.30 6 1.83 0.11 29 2.43 0.40 37 60 1.92 0.22 52 2.30 0.37 33 Figure 5. Linear relationship between temperature-corrected leaf initiation rates (1/P) and full-grown leaf lengths (L m ). The leaf initiation rate for leaf N is calculated from Equation 1: dn/dt = a N (t N + t), where a N is the acceleration factor for leaf production, t N a time factor indicating the developmental state of the plant at the start of the experiment, and t is the time at which leaf N reached 90 % of its final length. The calculated leaf initiation rates were corrected for a temperature of 22 C. Internode formation is principally dependent on rates of stem elongation and leaf initiation. The rate of height growth divided by the leaf production rate determines internode length, which is usually about 30 mm. At the start of cutting growth, short internodes are formed. As in leaves, this is probably a result of an imbalance between shoot and root growth. (The first internodes of a side branch, growing on a vigorously growing main shoot, are normally about 30 mm long.) Internode length gradually increases to a more or less constant length (steady state). The lengths of the successive internodes during this period of adaptation can be calculated as the ratio of actual stem elongation to leaf initiation rate (see Materials and methods). A comparison of such a calculation with measured internode lengths is presented in Figure 6A. There was agreement between the measured and modeled data during the adaptation period; however, internode lengths in the steady state were somewhat underestimated, because calculated leaf production rate was accurate, but calculated height growth was lower than actual height growth. This deviation occurred in all of the plants in this experiment. Relationship between RGR internode and internode age We analyzed the relation between RGR internode and internode age (M) for the period where it could be measured nondestructively. We set RGR internode at half of mature length to Day 20, which was the mean date on which this occurred. Linear relationships between RGR internode and internode age are presented in Figure 6B, and the slope of the relationship and the standard deviation of the mean are given in Table 4 for the different irradiances. Values both corrected for temperature and uncorrected for temperature are given. Values of M depended only slightly on irradiance. Mean RGR stem of the growing stem part is 0.25 day 1 (Pieters and van den Noort 1988). On the basis of this value, mean RGR in the primordial phase was calculated as the maximum RGR internode. Values are listed in Table 4 and again show little dependence on irradiance. Primordium length, initiation rate and vascular system development The finding that leaf length and leaf initiation rate are correlated indicates that the apex of GS is precisely structured. Remarkable parallels exist between the increasing size of the growing part of the stem and the change in architecture of the vascular system in the apex (Larson 1975, 1977 and 1980). The number of vascular traces and the diameter of GS gradually TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

940 PIETERS, VAN DEN NOORT AND VAN NIJKERKEN Figure 6. (A) Comparison of measured ( ) and modeled ( ) internode lengths. The first internodes are short, but successive internodes gradually increase in length to between 20 and 30 mm. The length of GS i (-- --) is plotted against internode number as a measure of plant age. Internode length is underestimated in the period after adaptation, because the actual rate of height growth was greater than the calculated mean rate. (B) Example of the fixed growth pattern of all internodes of one plant during the macroscopic phase of growth ( ). Table 4. Estimated slope of the relationship between RGR internode and internode age in the macroscopic phase (M) of all growing internodes (see Figure 6B). The calculated maximum RGR internode in the primordial phase and the duration of growth are also given. Values are means ± standard error. Irradiance (W m 2 ) 7.5 15 30 Slope uncorrected for temperature 0.034 ± 0.003 0.037 ± 0.002 0.045 ± 0.005 Maximum RGR (day 1 ) 0.304 ± 0.006 0.297 ± 0.003 0.288 ± 0.005 Growth duration (day) 25.4 ± 0.14 25.2 ± 0.18 24.7 ± 0.31 Slope corrected for temperature 0.033 ± 0.003 0.035 ± 0.002 0.040 ± 0.005 Maximum RGR (day 1 ) 0.295 ± 0.006 0.279 ± 0.002 0.256 ± 0.004 Growth duration (day) 24.6 ± 0.1 23.7 ± 0.2 21.9 ± 0.3 Total no. of observations 191 264 142 No. of internodes measured 41 72 49 No. of plants measured 3 3 2 increase during the development of the plant. Also, the lengths of the procambial traces increase proportionally, as well as the length of GS. By increasing the number of traces from two to thirteen, the phyllotactic order increases from 1/2, 1/3, 2/5, 3/8 to 5/13, as is also evident in in the enlarging GS. Because the procambial traces develop acropetally long before the primordium that they will feed is initiated (Larson 1975), the change in vascular architecture anticipates the change in GS. In poplar clone Robusta, the time-span between the initiation of two successive primordia on the same vessel (one orthostichy) was about 13 days at 22 C. If the number of traces is five, leaf production is about one leaf per 13/5 = 2.8 days, if the number of traces is 13 about one leaf per 13/13 = 1 day is produced by GS. These estimates compare well with our observations on leaf production and phyllotactic order (data not shown). Growth of the apex, development of final leaf length and of rate of leaf initiation or height growth proceed linearly with time. Leaf production and height growth are both quadratic functions of time. Total leaf production is a cubic function of the length of the youngest matured leaf. In contrast, total biomass production of a shoot is not an exponential function of time and shoot RGR is a physiologically meaningless parameter. Relative growth rate is a meaningful physiological parameter only for the growth capacity of cells during primary extension growth of leaves, internodes or roots. It cannot be used for whole plants because the increasing proportion of plant mass that does not participate in growth causes a continuous decline in RGR, which is called ontogenetic drift (Lord et al. 1993). To calculate absolute growth, it is necessary to know at least mean RGR and the cell capital participating in growth (Lambers 1987). For a plant that can branch freely, the use of RGR may have significance when related to the number of developing buds over the years. Relative growth rate Conclusions Because leaves and internodes follow characteristic relative growth patterns, constant gradients of RGR internode and RGR leaf TREE PHYSIOLOGY VOLUME 19, 1999

GROWTH ADAPTATION TO ENVIRONMENTAL CONDITIONS 941 exist along the growing part of the stem that are related to the relative distance from the shoot apex. The developmental characteristics of the growing cells are coupled through their age to a specific location on the GS. Our model for leaf and internode growth is based exclusively on the age of each individual organ. Growth response to irradiance occurs by simultaneous increases in leaf length and leaf initiation rate, according to a growth pattern predetermined in the apex. Constant internode lengths, together with increasing leaf lengths, are often observed in other plant species, indicating that their growth is probably regulated by mechanisms similar to those in poplar. Future research should concentrate on how physiological cell age regulates organ development. We also need to determine whether the architecture of the vascular system plays a fundamental role in regulating cell numbers in the shoot apex, leading to simultaneous increases in rates of leaf elongation and initiation. References Ceulemans, R. 1990. Genetic variation in functional and structural productivity determinants in poplar. Thesis Publishers, Amsterdam, 99 p. Ceulemans, R. and B. Saugier. 1990. Photosynthesis. In Physiology of Trees. Ed. A.S. Raghavendra. J.Wiley and Sons Inc., New York, London, 509 p. Ceulemans, R., G. Scarascia-Mugnozza, B.M. Wiard, J.H. Braatne, T.M. Hinckley, R.F. Stettler, J.G. Isebrands and P.E. Heilman. 1992. Production, physiology and morphology of Populus species and their hybrids grown under short rotation. I. Clonal comparisons of 4 year growth and phenology. Can. J. For. Res. 22:1937--1948. Ceulemans, R, X.N. Jiang and B.Y. Shao. 1995. Growth and physiology of one-year-old poplar (Populus) under elevated atmospheric CO 2 levels. Ann. Bot. 75:609--617. Ceulemans, R., A.J.S. McDonald and J.S. Pereira. 1996. A comparison among eucalypt, poplar and willow characteristics with particular reference to a coppice, growth-modelling approach. Biomass Bioenergy 11:215--231. Doorenstouter, H., G.A. Pieters and G.R. Findenegg. 1985. Distribution of magnesium between chlorophyll and other photosynthetic functions in magnesium-deficient sun and shade leaves of poplar. J. Plant Nutr. 8:1089--1101. Erickson, R.O. and F.J. Michelini. 1957. The plastochron index. Am. J. Bot. 44:297--305. Jarvis, P.G. 1989. Atmospheric carbon dioxide and forests. Phil. Trans. R. Soc., London. B 324, 369--392. Lambers, H. 1987. Does variation in photosynthetic rate explain variation in growth rate and yield? Neth. J. Agr. Sci. 35:505--519. Larson, P.R. 1969. Leaf development, photosynthesis, and 14 C distribution in Populus deltoides seedlings. Am. J. Bot. 56:1058--1066. Larson, P.R. 1975. Development and organization of the primary vascular system in Populus deltoides according to phyllotaxy. Am. J. Bot. 62:1084--1099. Larson, P.R. 1977. Phyllotactic transitions in the vascular system of Populus deltoides Bartr. as determined by 14 C labeling. Planta 134:241--249. Larson, P.R. 1980. Interrelations between phyllotaxis, leaf development and primary-secondary vascular transition in Populus deltoides. Ann. Bot. 46:757--769. Lord, D., S. Morissette and J. Allaire. 1993. Influence of light intensity, night air temperature and CO 2 concentration on the growth of containerized black spruce seedlings grown in the greenhouse. Can. J. For. Res. 23:101--110. Milthorpe, F.L. and J. Moorby. 1974. An introduction to crop physiology. University Press, London, 202 p. Monteith, J.L. 1965. Light distribution and photosynthesis in field crops. Ann. Bot. 29:17--37. Pieters, G.A. 1974. The growth of sun and shade leaves of Populus euramericana Robusta in relation to age, light intensity and temperature. Meded. Landb. 74:106. Pieters, G.A. 1975. Thermography and plant physiology. In Thermography. Proc. 1st European Congress on Thermography. Ed. S. Karger, pp 210--218. Pieters, G.A. 1983. Growth of Populus euramericana. Physiol. Plant. 57:455--462. Pieters, G.A. 1984. A television area meter. Photosynthetica 18:454-- 458. Pieters, G.A. 1986. Dimensions of the growing shoot and the absolute growth rate of a poplar shoot. Tree Physiol. 2:283--288. Pieters, G.A. and M.E. van den Noort. 1988. Effect of irradiance and plant age on the dimensions of the growing shoot of poplar. Physiol. Plant. 74:467--472. Pieters, G.A. 1996. The growth of aspen, exposed to ozone and CO 2 in open top chambers. Internal report on a half year stage at the US Forest Service, North Central Forest Experiment Station, Rhinelander, WI, 61 p. Steiner, A.A. 1968. Soilless culture. In Proc. 6th Coll. Intern. Potash Inst., Florence, Italy. International Potash Institute, Berne, Switzerland, pp 324--341. TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

942 PIETERS, VAN DEN NOORT AND VAN NIJKERKEN Appendix Symbol Definition and units a N Acceleration factor for leaf initiation (proportional to the growth rate of apical diameter) in N day 2 a H Acceleration factor for height growth (proportional to the growth rate of apical length) in cm day 2 dn/dt Leaf production rate in leaves day 1 L Difference in length (cm) of two successive leaves at half final length e Relative temperature effect e D Relative temperature effect in the dark e L Relative temperature effect in the light GS The growing part of the shoot GS i Part of the stem with growing internodes (cm) GS L Part of the stem with elongating leaves (cm) H Shoot height (cm) L int Final internode length (cm) L m Final leaf length (cm) M Slope of the relation between RGR internode and internode age during macroscopic growth N Number of a leaf or internode, counting acropetally n Number of a leaf or internode, counted from the youngest mature leaf P Plastochron duration (days) RGR x Relative growth rate of the indicated organ (dl/(ldt)) (day 1 ) RGR 50 Relative growth rate at half of final organ length (day 1 ) t Plant age (days) t H Developmental stage of height growth or of apical height (days at the start of the experiment). t N Developmental stage of leaf initiation or of apical diameter (days at the start of the experiment). T Temperature ( C) WAR Leaf weight to leaf area ratio (g cm 2 ) TREE PHYSIOLOGY VOLUME 19, 1999