Effects of Constant and Variable Nitrogen Supply on Sunflower (Helianthus annuus L.) Leaf Cell Number and Size

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1 nnals of otany 84: , 1999 rticle No. anbo , available online at http: on Effects of Constant and Variable Nitrogen Supply on Sunflower (Helianthus annuus L.) Leaf Cell Number and Size NOR TR PNI*, NTONIO J. HLL and MRC WEER IFEV, Departamento de Ecologı a, Facultad de gronomı a, Uni ersidad de uenos ires, da. San Martı n 4453, 1417 uenos ires, rgentina and Hohenheim Uni ersity, Stuttgart, Germany Received: 1 February 1999 Returned for revision: 1 June 1999 ccepted: 9 July 1999 The effects of nitrogen (N) availability on cell number and cell size, and the contribution of these determinants to the final area of fully expanded leaves of sunflower (Helianthus annuus L.) were investigated in glasshouse experiments. Plants were given a high (N 315 ppm) or low (N 21 ppm) N supply and were transferred between N levels at different developmental stages (5 to 6% of final size) of target leaves. The dynamics of cell number in unemerged ( 1 m in length) leaves of plants growing at high and low levels of N supply were also followed. Maximum leaf area (L max ) was strongly (up to two-fold) and significantly modified by N availability and the timing of transfer between N supplies, through effects on leaf expansion rate. Rate of cell production was significantly (P 5) reduced in unemerged target leaves under N stress, but there was no evidence of a change in primordium size or in the duration of the leaf differentiation emergence phase. In fully expanded leaves, number of cells per leaf (N cell ), leaf area per cell (L cell ) and cell area ( cell ) were significantly reduced by N stress. While L cell and cell responded to changeover treatments irrespective of leaf size, significant (P 5) changes in N cell only occurred when the changeover occurred before the leaf reached approx. 1% of L max. There were no differential effects of N on numbers of epidermal s. mesophyll cells. The results show that the effects of N on leaf size are largely due to effects on cell production in the unemerged leaf and on both cell production and expansion during the first phase of expansion of the emerged leaf. During the rest of the expansion period N mainly affects the expansion of existing cells. Cell area plasticity permitted a response to changes in N supply even at advanced stages of leaf expansion. Increased cell expansion can compensate for low N cell if N stress is relieved early in the expansion of emerged leaves, but in later phases N cell sets a limit to this response nnals of otany Company Key words: Helianthus annuus, leaf expansion, leaf cell number, leaf cell size, nitrogen, leaf growth, sunflower. INTRODUCTION The central role of nitrogen on leaf growth has long been recognized (e.g. Trewavas, 1985). Thus, the effects of nitrogen supply on sunflower leaf area have been investigated in a number of studies (e.g. Connor, Hall and Sadras, 1993; Palmer et al., 1996; Tra pani and Hall, 1996; Sadras and Tra pani, 1999 and references therein). Leaf area can be viewed as the result of the rate and duration of leaf expansion. Leaf expansion rate (LER) is very responsive to nitrogen supply both under controlled and field conditions, while the duration of expansion is less affected, especially under field conditions (Steer and Hocking, 1983; Steer et al., 1986; Connor et al., 1993). t the cellular level, the processes that determine leaf area in dicotyledons are cell production, mainly occurring during the early phase of leaf development, followed by cell expansion in later phases, with some overlap in the intermediate phase (e.g. Pisum sati um: Lecoeur et al., 1995). In species other than sunflower (e.g. sugarbeet: Morton and Watson, 1948; Festuca arundinacea: Macdam, Volenec and Nelson, 1989) nitrogen stress affected leaf size through both cell production and cell expansion. Thus, in * For correspondence. Fax , trapani ifeva. edu.ar $3. N-stressed sugarbeet there was a reduction of approx. 3% in both cell number and size, as measured in the completely expanded leaf 1 (Morton and Watson, 1948). Division of mesophyll cells in monocotyledon leaves was reduced by N stress as was the expansion rate of epidermal cells (Macdam et al., 1989). In sunflower, data from Sunderland (196) on the dynamics of total leaf cell number and those from Garnier and Tardieu (1998) on leaf epidermal cells, indicate that cellular divisions take place throughout most of the period of leaf expansion. In contrast, other results for non-stressed sunflower (Yegappan et al., 198) and pea (Lecoeur et al., 1995) indicate that cell division finishes early during leaf development. lthough some of the discrepancies between these results might be partly explained by the experimental conditions used (e.g. irradiance levels, leaf insertion levels), leaf processes at the cellular level in dicotyledonous plants deserve further attention. Much of the previous work regarding nitrogen was conducted under conditions of fairly constant supply levels. However, in the field many crops face conditions of transient or gradually increasing N stress. better understanding of leaf responses to these conditions and the part that cellular determinants of leaf area play is required. Ma, Longnecker and Dracup (1997) investigated responses to transient N deficiency of leaf area in terms of individual leaf area, but did not explore N effects on the underlying cell processes 1999 nnals of otany Company

2 6 Tra pani et al. Sunflower Leaf Cell Number and Size nor did they determine the developmental windows for cell division and expansion responses to nitrogen. The purpose of this work is to advance the present understanding of responses to nitrogen of leaf expansion in sunflower by focusing on the determinants of leaf area at the cellular level. Experiments involving high and low nitrogen supplies, covering the range of N supply capable of affecting leaf expansion but not leaf number per plant (Tra pani and Hall, 1996), and transfer between these N levels at different developmental stages of target leaves were used to investigate effects on leaf cell number and size of the fully expanded leaf. Effects of nitrogen supply on the dynamics of cell number of unemerged leaves were also studied. To accomplish these objectives, sunflower plants were grown in nutrient solutions in the glasshouse, under controlled temperature to minimize the effects of temperature variations on leaf growth. MTERILS ND METHODS Plant material and conditions Pre-germinated seeds (radicle 5 mm), Helianthus annuus G1 (Dekalb, ragado, rgentina) were sown in sand on 21 Sep (expt 1), 22 Feb (expt 2), 26 Mar (expt 3) and 4 Mar (expt 4). When the cotyledons were fully expanded (3 d later), they were transferred to 2-l plastic pots with aerated nutrient solution (Moore, 1981) in which two levels of N were supplied: high (H, 315 ppm) and low (L, 21 ppm). The pots were placed in a glasshouse at the Facultad de gronomı a, Universidad de uenos ires (34 35 S, W). Plants were spaced 3 m apart following a completely randomized design with four (expts 1 3) and three (expt 4) replications. Glasshouse conditions Glasshouse mean temperature was maintained at C throughout the experiments. Mean irradiance was 16, 15, 15 and 14 MJ PR m d in expts 1 to 4, respectively, 33% lower than the external value. Treatments In expts 1 to 3, nitrogen nutrition was manipulated during the expansion period of target leaves. The insertion level ( cotyledons) of target leaves was 8 (L8, expt 1), 1 (L1, expt 2) and 1 and 11 (L1 and L11, expt 3). Plants were transferred from high to low N supply (HL plants) and ice ersa (LH plants) when target leaves were approx. 1, 2 and 5% of the maximum area (L max ) achieved by L8 and L1 in preliminary experiments (for actual fractional leaf area at transfer, see Table 1). Control plants were maintained in each of the two conditions (CH and CL plants). Nutrient solutions were renewed every 3 d. Combination of N supply and stages of leaf expansion at transfer generated eight treatments: CH, HL1, HL2, HL5, CL, LH1, LH2 and LH5. Plants were harvested when the target leaves reached L max. In expt 4, which focused on cell number of unemerged leaves, only the CH and CL treatments were applied. Measurements on emerged lea es Leaf area. Size of the target leaves was recorded at approximately daily intervals (expts 1 and 2) and three times a week (expt 3) between appearance (day on which lamina length exceeded 1 mm) and achievement of L max. In all treatments width (w) and length (l) of leaf laminae were measured and leaf area (L) was estimated (L w l 73; Takami, Turner and Rawson, 1981). The Richards function (Hunt, 1982) was fitted to L s. time data from individual target leaves to obtain estimates of leaf expansion rate and duration of expansion (Tra pani and Hall, 1996). fter harvest, the target leaves were divided into four sections of equivalent area (i.e. apical, ; middle 1, M1; middle 2, M2; and basal, ) from each of which three disc samples were taken. Copies of the leaf sections were made on paper and their area determined using an electronic area meter (Li-35a, LiCor Inc., Lincoln, N, US). In expt 3, specific leaf area was calculated from data of leaf area and dry weight. Leaf nitrogen concentration was determined by the microkjeldahl technique on dried (48 h at 6 C), weighed, and milled samples of the companion leaf 9. This was done to assess nitrogen leaf levels in these experiments to allow comparison with results of a previous experiment (Tra pani and Hall, 1996). Cell number and areas of mesophyll cells and intercellular spaces. Three discs ( 1 m diameter) from the centre, middle and border of target leaves were extracted from sections, M1, M2 and, and preserved in 7% alcohol until clearing (Dizeo de Strittmatter, 1973) prior to cell counting. riefly, leaf discs were cleared by boiling in 96% ethyl alcohol for 3 min followed by boiling in a solution of equal parts of 96% alcohol and 5% KOH for 1 min. oiled discs were rinsed in distilled water, decoloured in 5% sodium hypochlorite for 5 min, and finally rinsed in distilled water. Cleared discs were kept in 25% chloral hydrate until cell counting. lterations in disc diameter due to clearing were less than 3 mm (error in area 5%). Leaf discs were laid on microscope slides with the adaxial face upwards. baxial epidermal cells and mesophyll cells in the plane of the minor veins were counted using a camera-lucida, at a magnification of 2 and 4 for epidermal and mesophyll cells, respectively. The number of abaxial epidermal cells was determined on CH and CL (expt 2) and in all treatments (expt 3), while the number of mesophyll cells was determined in all three experiments. oth epidermal and mesophyll cells were studied to detect possible differential N effects on these tissues. This approach allowed us to compare our results with previous ones obtained for mesophyll cells of unstressed sunflower leaves (Yeggapan et al., 198) and with results on epidermal and mesophyll cells of monocotyledon leaves under N stress (Macdam et al., 1989). Transverse sections of leaves showed that minor veins were located under two layers of palisade parenchyma cells. In this plane mesophyll cells have circular sections, allowing for measurement of cell diameter using the microscope

3 Tra pani et al. Sunflower Leaf Cell Number and Size 61 ocular rule. Three fields (area 64 mm, encompassing an average of 2 mesophyll cells and six epidermal cells) within each disc were evaluated to determine cell number per field (abaxial epidermal or mesophyll cells). Leaf area per mesophyll cell (L cell, i.e. cell area plus associated intercellular spaces) was derived by relating cell number per field to field area. Mesophyll cell number per leaf (N cell )in the vascular tissue plane was obtained relating the area of the leaf sections (i.e., M1, M2, and ) and the number of cells per field. Direct measurement of mesophyll cell diameter was performed in target leaves of expt 3, under a magnification of 6. Cell area ( cell, i.e. without associated intercellular spaces) was calculated as π r. rea of intercellular spaces in the same plane was estimated as the difference between L cell and cell. Measurements on unemerged lea es Har ests. In expt 4, plants were periodically harvested (six harvests) and dissected under a binocular microscope (magnification 1 to 7 ) to sample unemerged target leaves 9 and 1 (L9 and L1) of different ages. t the first harvest, leaves were approx. 1 5 mm long. Unemerged leaves were preserved in 7% alcohol until cell number was determined. The date of appearance of L9 and L1 (1 2 mm of leaves visible) was recorded. Cell number. The procedure used was adapted from that developed by urrus et al. (1991) to obtain sunflower protoplasts. Preserved unemerged leaves were rinsed with distilled water to eliminate alcohol; they were shredded and incubated at 25 C in 5 µl of S medium (urrus et al., 1991) containing CaCl 2H O(2gl ); KCl (25 g l );MES ( 7 gl ), ph 5 6 and 2% of Driselase (Sigma). Maximum cell number was obtained after incubation during 2 3 h. liquots of suspended leaf cells were counted on a haemocytometer under a magnification of 2 and used to estimate number of cells per leaf. Data analysis nalysis of variance was used to establish significance (P 5) of differences among treatments, and regression analysis was used to establish significant (P 5) associations between variables. RESULTS Responses of leaf area to N and timing of changeo er The maximum area of target leaves was significantly (P 5) different between CH and CL treatments in all experiments (Fig. 1; see Table 3) and the general response of area dynamics to N treatments is exemplified in Fig. 1. Fractional leaf area at the time of transfer between N levels varied slightly between experiments, target leaves and treatments (Table 1), as shown by subsequent analysis of leaf area evolution. Taking the three experiments together, the range of fractional leaf expansion at transfer (5 to 61% of L max ) allowed the analysis of responses from very early to late stages of emerged leaf growth (Table 1). Leaf area (cm 2 ) Leaf area (cm 2 ) Leaf area (cm 2 ) C 1 2 Days after leaf appearance 1 2 Days after leaf appearance 1 2 Days after leaf appearance CH CH HL LH CL CH HL LH FIG. 1. Leaf area dynamics of leaf 11 (L11, expt 3) in sunflower plants grown hydroponically with high (CH, 316 ppm N, ) or low (CL, 21 ppm N, ) nitrogen levels. Treatment plants were transferred to the opposite N level (HL, ; LH, ) at defined stages of L11 expansion: nominally 1%, (); 2%, (); 5%, (C) of maximum leaf area (L max ). rrows indicate the day of changeover. ars show 1 s.e.m. (n 4) and are drawn only when larger than symbols. In expt 3, as in the remaining experiments (data not shown), expansion of L11 was complete in all treatments by day 2 after leaf appearance (Fig. 1). Maximum area (L max ) of L11 did not differ significantly (P 5) from that of L1 in expt 3. Leaf 11 L max showed a significant (P 5) response to N level (i.e. CL s. CH) and to changes in N availability (i.e. HL1, HL2, HL5 s. CH, and LH1, LH2, LH5 s. CL) at all transfer stages (Fig. 1). LH HL CL CL 3 3 3

4 62 Tra pani et al. Sunflower Leaf Cell Number and Size TLE 1. ctual fractional leaf area, expressed as percent of maximum leaf area of target lea es on control plants (CH or CL), for treatments applied at nominal fractional leaf areas of 1, 2 and 5% Percent of maximum leaf area at changeover Treatments Experiment Target leaf HL1 HL2 HL5 1 L L L L LH1 LH2 LH5 1 L L L L TLE 2. Specific leaf area (SL, cm g ) and specific leaf nitrogen (SLN, gnm ) in completely expanded leaf 9 of plants grown hydroponically under high (CH 315 ppm N) or low (CL 21 ppm N) nitrogen supply and changed to the opposite N le el (LH and HL treatments) when achie ing 1, 2 and 5% of maximum leaf area; expt 3 Treatments SL SLN CH HL HL HL CL LH LH LH Data are means s.e. Treatments are changeovers from a high (CH 315 ppm N) to a low (CL 21 ppmn) nitrogen supply (Treatments HL1, HL2, HL5) and ice ersa (Treatments LH1, LH2, LH5). L max (cm 2 ) LER max (cm 2 d 1 ) CH CL HL1 HL2 HL5 LH1 LH2 LH5 FIG. 2. Relationship between maximum leaf area (L max ) and maximum leaf expansion rate (LER max ) for leaves 1 and 11 (L1 and L11, expt 3) of plants grown hydroponically with continuously high (CH, 315 ppm N) or low (CL, 21 ppm N) nitrogen levels or transferred to the opposite N level at defined stages of L1 and L11 development (see Table 1 for details of L1 and L11 fractional leaf area at changeover). Regression line [L max ( ) (7 7 65) LER max, R 7, n 64, P 5] was fitted to individual data. In the interest of clarity only means are shown in the figure. Similar results were obtained for other target leaves in all experiments (see Table 3 and Fig. 4). Effects of N availability and the leaf expansion stage at transfer on the duration of leaf expansion were nonsignificant in all experiments (range of average duration 12 7 to14 6 d), effects of N on L max being due to changes in leaf expansion rate, as exemplified in Fig. 2 for expt 3. This indicates the determinant role of LER in 3 response to transfer between N levels during the expansion of the leaves and, in the case of CH and CL leaves, is consistent with previous work on sunflower LER responses to fixed N availability levels (Steer et al., 1986; Connor et al., 1993; Tra pani and Hall, 1996). Values of L max obtained in CH and CL leaves in the three experiments were comparable to those for isolated field-grown plants of the same cultivar in the work of Tra pani and Hall (1996), suggesting that leaf expansion was not limited by glasshouse conditions. However, and as commonly found in glasshouse plants, specific leaf area values (SL, cm g ) (Table 2) were higher than those measured in field-grown plants of the same cultivar under different levels of nitrogen supply (e.g. SL 157 cm g, Tra pani and Hall, 1996). There were no significant differences in SL among treatments in expt 3 (Table 2) and limited data from expts 1 and 2 were consistent with this result. This suggests that the number of leaf cell layers was conserved in the face of different levels of N availability and transfer between N levels. Specific leaf nitrogen (SLN, g N m ) at L max was significantly higher (P 5) in CH than in CL treatments (Table 2). Moreover SLN showed a strong capacity to respond to changes in N availability: i.e. SLN of fully expanded leaves in LH and HL treatments did not differ significantly (P 5) from the CH and CL values, respectively (Table 2). SLN results were lower in glasshouse leaves (Table 2) compared to field-grown leaves, as a consequence of their lower SL. Nitrogen concentration at L max was 48 and 34 mg N g d. wt for CH and CL treatments, respectively, the rest of the treatments being within this range. These values compare well with those obtained for leaf 12 of the same cultivar in the previous work of Tra pani and Hall (1996); in that case N concentration was approx. 5 and 3 mg N g d. wt for treatment N5 (N supply g N per plant) and N2 (N supply 95 g N per plant), respectively. In Tra pani and Hall s (1996) work, levels N2 to N5 covered the range of conditions in which N effects were limited to changes in individual leaf area with leaf number per plant being unaffected.

5 Tra pani et al. Sunflower Leaf Cell Number and Size 63 Cell area (µm 2 ) M1 M2 Cell area (µm 2 ) M1 M2 CH HL1 CL LH1 CH HL1 CL LH C 5 45 D Cell area (µm 2 ) Cell area (µm 2 ) M1 M2 Leaf sections 1 M1 M2 Leaf sections CH HL2 CL LH2 CH HL5 CL LH5 FIG. 3. rea of cells measured in the vascular tissue plane for the apical (), middle 1 (M1), middle 2 (M2) and basal () sections of completely expanded sunflower leaf 1 or leaf 11 from expt 3. D, Responses to treatments of changeover in N availability at mean effective fractional leaf areas of 7 5% (, leaf 11) and 15% (, leaf 1), 34% (C, leaf 1) and 58% (D, leaf 1). See Table 1 for fractional leaf expansion stages of target leaves in expt 3. Values for controls CL and CH are shown for comparison in all panels. ars represent least significant differences (P 5, n 4) among treatments for each leaf section, and in the interest of clarity they are only shown in and C. TLE 3. Leaf area of completely expanded lea es (L max ), mesophyll cell number in the ascular tissue plane per leaf (N cell ), leaf area per cell (L cell ) of lea es of sunflower plants grown hydroponically under high (315 ppm N, CH) or low (21 ppm N, CL) nitrogen le els L max (cm ) N cell (millions) L cell (microns ) Expt 1 Expt 2 Expt 3 Expt 1 Expt 2 Expt 3 Expt 1 Expt 2 Expt 3 Treatments L8 L1 L1 L11 L8 L1 L1 L11 L8 L1 L1 H11 CH CL LSD (P 5) Cellular le el responses to N measured in fully expanded lea es The responses of L max to N are due to effects on the components of leaf area at the cellular level, i.e. area per cell and number of cells. Mesophyll cell area, as measured at L max in the plane of minor veins ( cell ), did not differ significantly among the various leaf sections within each treatment (Fig. 3), being significantly higher in CH than in CL leaves in most cases (Fig. 3). Changes in N availability during leaf expansion always caused a reduction (HL treatments) or increase (LH treatments) in cell to values similar to CL and CH treatments, respectively, regardless of the developmental stage at which the transfer took place. No significant differences among leaf sections were found either for mesophyll cell number per field or for leaf area per mesophyll cell (L cell ) (data not shown). Consequently, cell and L cell data for each leaf section were pooled to analyse the response of the entire leaf. oth L cell and cell number per leaf in the plane of the vascular tissue (N cell ) of completely expanded leaves differed significantly (P 5) between CH and CL leaves (Table 3). In the transfer treatments, L cell either increased significantly (P 5) in response to increased N supply

6 64 Tra pani et al. Sunflower Leaf Cell Number and Size L cell Relative increase (%) 1 5 L cell (µm 2 ) 5 25 space 5 25 space (µm 2 ) cell (µm 2 ) 35 4 FIG. 5. Leaf area per cell (L cell, ) and intercellular area ( space, ) of leaf 1 mesophyll cells as a function of area per cell ( cell ). Points are means per treatment (n 4). Intercellular area was calculated as: L cell cell (expt 3). Relative reduction (%) Percent of L max N cell (millions) FIG. 4. Percent increase () and reduction () of maximum leaf area (L max ; ), leaf area per cell (L cell ; ) and number of cells observed in the vascular tissue plane (N cell ; ) relative to the corresponding control (, low N supply;, high N supply) when plants of expts 1 to 3 were transferred from a low to a high N supply () and ice ersa () during different expansion stages of the target leaf expressed as percentage of L max (see Table 1 for details of target leaves and fractional leaf expansion stages). Horizontal lines indicate the range of fractional L max at changeover for which final treatment values of L max, L cell (upper line) and N cell (lower line) differed significantly (P 5) from the respective control value (NOV were applied to the absolute values of the variables for each target leaf in each experiment). Note that the range of L max at changeover which produced significant responses was the same for both L max and L cell. N cell (millions) (Fig. 4) or decreased when N supply decreased (Fig. 4) in all of the leaf expansion stages studied. In contrast, significant increases in N cell (P 5) were only found when N level was augmented early during leaf development and did not respond to increased N supply at later stages. Similarly, restricting N supply only caused significant (P 5) reductions in N cell when leaf area reached approx. 1% of L max (Fig. 4). These results are consistent with cell division becoming unimportant once the early phase of leaf expansion is completed. The association between L cell and cell of L1 (Fig. 5) and L11 (data not shown) of expt 3, was significant [L cell ( ) ( 71 11) cell, R 57, n 32, P 5; L1] while the association between cell and intercellular space ( space, Fig. 5) was not significant (slope of the relationship did not differ from zeo). These results Days before emergence FIG. 6. Dynamics of cell number (N cell ) in unemerged leaves 9 () and 1 () of hydroponically grown sunflower plants under high ( ) and low ( ) nitrogen supply. ars indicate one s.e. (n 3) and are shown when larger than symbols (expt 4). indicate that N affected the expansion of cells but not the separation between them, at least at the mesophyll level. The relationship between mesophyll cell number and epidermal cell number, both measured per unit area, in L1 of expt 3 (mean s.e ) did not differ significantly among treatments: neither N availability nor transfer

7 Tra pani et al. Sunflower Leaf Cell Number and Size 65 between N levels affected this relationship. Limited data from expt 2 showed that the relationship (mean s.e ) did not differ between CH and CL treatments. These results indicate similar responses to N availability of both mesophyll and epidermal cells, supporting the notion that N affects cell expansion of both tissues to the same degree. Cell production in unemerged lea es The intercepts on the x-axis of the linear regression fitted to N cell s. time relationships for unemerged L9 and L1 did not differ significantly between CH and CL plants (Fig. 6), and there were no differences in timing of leaf emergence between N treatments for either L9 or L1. In contrast, N stress significantly (P 5) affected the rate of cell production. In L1, rate of cell production was reduced from millions of cells per day (CH plants) to millions of cells per day (CL plants) while in L9 the corresponding rates were (CH plants) and 24 2 (CL plants). These results suggest that N supply did not affect the duration of cell production during the unemerged phase of leaf development and that N supply had little, if any, effect on cell number in the leaf primordium. Thus the rate of cell production in the unemerged leaf was the main determinant of the number of cells per leaf at the time of leaf appearance. In spite of differences in methodology, the results obtained for unemerged leaves in response to N supply are consistent with those obtained in emerged leaves for number of cells per leaf (Table 3). DISCUSSION Responses of leaf area to N levels and to N transfer treatments were very marked and, in the case of transfers, rapid (Fig. 1). Transfer between N levels modified L max to different extents depending on the direction (LH or HL) of the change in N supply and the timing of transfer. In general the effects of transferring plants between N levels were greater when applied early during leaf expansion (i.e. HL1 and LH1, Figs 1 and 4) and when N supply was increased rather than reduced (Fig. 4). In fact, maximum increases of 1% in L max were registered in response to increases in N availability, while the maximum reductions in L max were in the order of 4% when N supply was reduced (Fig. 4). Treatment effects were strongly linked to changes in LER rather than duration of leaf expansion; a single L max LER relationship could describe all the data (Fig. 2). Clearly, the rate of leaf expansion remains the main determinant of the final area a leaf will achieve under conditions of both constant and varying N supply during the expansion phase. Nitrogen availability did not affect SL (Table 2) in spite of the large (up to 1%) responses in leaf area (Fig. 1, Table 3) suggesting that this attribute was modulated by factors other than N. In contrast, SLN at L max showed a strong response to changes in N availability, even when they took place at stages close to 5% of L max (Table 2), indicating that actual N availability influenced the partitioning of N to the leaf even in advanced phases of leaf development. This is consistent with the notion that a continuing flux of N to the leaf is necessary for its growth during the last two thirds of the leaf expansion phase (Tra pani and Hall, 1996). The effects of N supply and transfer between treatments on cell cross-sectional area ( cell ) did not differ significantly among leaf sections (Fig. 3). These findings are consistent with those of Granier and Tardieu (1998) who found no differences in epidermal cell area among leaf sections of completely expanded leaf 8 of unstressed sunflower. Maximum leaf area was strongly affected by N stress through effects on both number and size of mesophyll cells, though with a greater impact on cell number (Table 3). Leaf area per cell (L cell ) and cell were clearly associated (Fig. 5); L cell showed significant responses to N increments or reductions in all transfers investigated (Fig. 4) indicating a plastic response of cell expansion in the face of variation in N availability, while N cell only increased (or decreased) significantly when N availability was enhanced (or restricted) when leaf area was lower than 1 15% of L max (Fig. 4). Our data are consistent with results of Lecoeur et al. (1995) on pea, which suggest that cell division finished before leaf area was around 2% of L max, but contrast with data of Yegappan et al. (198) on sunflower. These authors considered that palisade cell division ceased when the leaf reached about 35% of L max. The discrepancy between results on sunflower can be considered minor given that in Fig. 2 of Yegappan et al. (198) no standard errors are indicated, precluding an exact definition of fractional L max at which cell division ceases. The effects of N availability on the number of mesophyll cells per unit area were proportional to those registered in epidermal cells. These results contrast with those obtained for Festuca arundinacea (Macdam et al., 1989) where the number of mesophyll cells associated with each epidermal cell decreased with N stress. Moreover, epidermal cell size did not change with N stress in Festuca arundinacea (Macdam et al., 1989). These discrepancies are likely to arise from the intrinsic differences between mono- and dicotyledon-leaf types of development and morphology (e.g. Lecoeur et al., 1995). The rate of cell production in unemerged leaves was significantly reduced by N stress (Fig. 6), while effects of N on initial primordium size were minor, if any. This result is consistent with the limited effects of N stress (except for extreme conditions) on plant leaf number and on dynamics of sunflower leaf emergence (Steer and Hocking, 1983; Tra pani and Hall, 1996). The effect of N stress on leaf cell number at the stage of leaf appearance (Fig. 6) compares well, in relative terms, with that for the fully expanded leaf (i.e. 4% reduction under N stress; Table 3). nalogous to the lack of effects of N availability on the duration of expansion of emerged leaves, N stress did not affect the duration of the unemerged leaf phase as evaluated by the date of target leaf appearance in both CH and CL plants (Fig. 6). Taken together, our results identify the rate of cell production in unemerged leaves and the rate of expansion of emerged leaves as the main determinants of the final area that leaves would achieve in response to N levels and to changeovers in N availability. Responses to N changeovers are similarly rapid but smaller in extent when

8 66 Tra pani et al. Sunflower Leaf Cell Number and Size restricting N (Fig. 4). The effects of N stress are linked to limited cell production in the unemerged leaf and during the early phase of expansion of emerged leaves, cell expansion being restricted by N stress during later phases. Clearly, cell area appears as the plastic leaf character in the face of late changes in N supply, as it can respond significantly (increase, decrease) until the advanced phases of leaf development. lthough increments in cell expansion can compensate leaf area when N stress is relieved shortly after leaf emergence, the number of cells established in the cell production phase will set a limit to the response. Responses to changes in N supply on the final phases of leaf development (i.e. 6 to 1% of L max ) remain to be investigated. Our results suggest that a framework for modelling N effects on individual sunflower leaf area, along the lines of that proposed by Lecoeur, Wiry and Sinclair (1996) for water-stressed pea, need to incorporate effects on cell number of unemerged leaves and L cell of emerged leaves, with small additive effects of N cell in the first phase of expansion ( 1% of L max ) of emerged leaves. The duration of the expansion phases of unemerged and emerged leaves could be taken as N-insensitive. This should be sufficient, at least for the range of N concentrations explored here, which covers most of the range seen in Tra pani and Hall (1996) applicable to N levels which do not affect leaf emergence rates. Outside these levels other approximations would be needed. CKNOWLEDGEMENTS The work reported here was supported by Universidad de uenos ires (U G 122) and ID 82 OC R, PID PVT No We gratefully acknowledge P. Gundel and R. Ramondo for technical assistance. LITERTURE CITED urrus M, Chanabe CH, libert G, idney D Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.). Plant Cell Reports 1: Connor DJ, Hall J, Sadras VO Effect of nitrogen content on the photosynthetic characteristics of sunflower leaves. ustralian Journal of Plant Physiology 2: Dizeo de Strittmatter C Nueva te cnica de diafanizacio n. oletin Sociedad rgentina de ota nica 15: Garnier C, Tardieu F Spatial and temporal analyses of expansion and cell cycle in sunflower leaves. Plant Physiology 116: Hunt R Plant growth analysis. London: Edward rnold. Lecoeur J, Wery J, Sinclair TR Model of leaf area expansion in field pea subjected to soil water deficits. gronomy Journal 88: Lecoeur J, Wery J, Turc O, Tardieu F Expansion of pea leaves subjected to short water deficit: cell number and cell size are sensitive to stress at different periods of leaf development. Journal of Experimental otany 46: Macdam JW, Volenec JJ, Nelson CJ Effects of nitrogen on mesophyll cell division and epidermal cell elongation in tall fescue leaf blades. Plant Physiology 89: Ma Q, Longnecker N, Dracup M Nitrogen deficiency slows leaf development and delays flowering in narrow-leafed Lupin. nnals of otany 79: Moore TC Research experience in plant physiology. laboratory manual. 2nd Edn. New York: Springer Verlag. Morton G, Watson DJ physiological study of leaf growth. nnals of otany NS 12: Palmer SJ, erridge DM, McDonald JS, Davies WJ Control of leaf expansion in sunflower (Helianthus annuus L) by nitrogen nutrition. Journal of Experimental otany 47: Sadras VO, Tra pani N Leaf expansion and phenological development: key determinants of sunflower plasticity, growth and yield. In: Smith DL, Hamel C, eds. Crop yield, physiology and processes. erlin Heidelberg: Springer-Verlag, Steer T, Hocking PJ Leaf and floret production in sunflower (Helianthus annuus L) as affected by nitrogen supply. nnals of otany 52: Steer T, Coaldrake PD, Pearson, CJ, Canty PJ Effects of nitrogen supply and population density on plant development and yield components of irrigated sunflower (Helianthus annuus L.). Field Crops Research 13: Sunderland N Cell division and expansion in the growth of the leaf. Journal of Experimental otany 11: Takami S, Turner NC, Rawson H Leaf expansion of four sunflower (Helianthus annuus L.) cultivars in relation to water deficits. I. Pattern during plant development. Plant, Cell and En ironment 4: Tra pani N, Hall J Effects of level of insertion and nitrogen supply on the expansion of leaves of filed grown sunflower (Helianthus annuus L.). Plant and Soil 184: Trewavas pivotal role for nitrate and leaf growth in plant development. In: aker NR, Davies WJ, Ong CK, eds. Control of leaf growth. Cambridge: Cambridge University Press, Yegappan TM, Paton DM, Gates CT, Muller WJ Water stress in sunflower (Helianthus annuus L.): I Effect on plant development. nnals of otany 46: 61 7.