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Gas exchange and leaf ultrastructure of tinnevelly senna, Cassia angustifolia, under drought and nitrogen stress.

DROUGHT AND LIMITED SOIL NITROGEN are among the most important environmental constraints that limit both crop choice and productivity in a wide range of agricultural ecosystems. Adoption of crops that are drought-tolerant and responsive to low levels of nitrogen application is a cost-effective and environmentally sound way of farming drought-prone soils with nitrogen deficiency. In this regard, currently under-utilized crops with industrial value may be promising candidates.

Senna, Cassia augustifolia (syn. Cassia alexandrina, Senna attgustifolia, Cassia senna, Cassia acutifolia: as per revision by Irwin and Barneby, 1982), a non-nitrogen fixing member of Caesalpiniaceae, is included in the pharmacopeias of USA, Germany, UK, India, and many other countries mainly for its cathartic properties (Hussain et al., 1984; Lemli, 1986; Folkard, 1995, p. 352). The primary active constituents of senna are two rheindianthrone-8,8' diglucosides called sennoside A and B. Despite the availability of some synthetic products, sennoside formulations are increasingly used as safe laxatives (Atzorn et al., 1981: Al-Dakan et al., 1995). Senna, however, has not received wide recognition as a crop in many countries except for India where it is grown in arid, nutrient-poor, sandy soils (Pareek et al., 1983). Preliminary on-farm observations in the dry zone of Sci Lanka indicated that senna can be successfully planted as an unirrigated crop on alfisols in the dry season, if the residual moisture from the previous wet season is used for early plant establishment. Furthermore, senna plants can be grown by seedlings, ratooning (pruning seedling plants near the base and allowing regrowth) and cuttings. We previously reported the effects of environmental conditions, propagation methods, and cultural practices on the concentrations of sennosides in dried senna leaves, the main harvest product of senna (Ratnayaka et al., 1998). The primary objective of this study was to investigate the impact of drought and foliar nitrogen application (as urea) on gas exchange and some leaf surface characteristics of senna toward strengthening our understanding of stress tolerance mechanisms of senna. These results may also help select treatments for site-specific field experiments to evaluate senna as a potential new crop particularly in droughtprone and nitrogen-deficient agricultural ecosystems.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of senna were obtained from the Ceylon Institute of Scientific and Industrial Research (CISIR, current Industrial Technology Institute, ITI), Sri Lanka. One-year-old seedlings grown on a mixture of loam top soil:sand:peat (2:1:1 by volume: top soil, organic matter 7.7 %, pH 7.1, estimated N release at 157 kg [ha.sup.-1], P at 385 kg [ha.sup.-1], K at 224 kg [ha.sup.-1]) in 25cm-diam. (10 L) pots in a greenhouse were pruned at the height of approximately 10 cm to obtain new shoot growth in the spring (air temperature, 22-28[degrees]C; 14-h photoperiod with supplemental lighting by fluorescence lamps at photosynthetic photon flux, approximately 350 [micro]mol [m.sup.-2] [s.sup.-1]). A fertilizer mixture of urea (45-0-0), 1.7 g; triple super phosphate (0-45-0), 1.7 g, and KCl (0-0-62), 1.3 g was applied to each pot 2 wk before pruning. The emerging sprouts were deflowered to ensure adequate leaf growth for measurements. Plants were watered to field capacity (average pre-dawn water potential of about -0.3 MPa was maintained) until the treatments of drought and foliar nitrogen spray were started.

Drought and Foliar Nitrogen Treatments

Two levels of drought (D0, well-watered and D1, water-stressed) and two levels of foliar nitrogen treatment (NO, no nitrogen applied and N1, nitrogen applied) were started when the shoots were 5 wk old. These four factorial treatments (D0N0, D0N1, D1N0, and D1N1) were tested in a randomized complete block with three replications. A 1% (w/v) urea:tap water solution with two drops of a detergent per liter was used as the foliar nitrogen treatment. We tested spraying 5, 3, and 1% urea using a different set of plants and observed that both 5 and 3% caused tip burn of leaflets. The plants that were treated with foliar nitrogen were sprayed until the leaflet surfaces were fully wet (about 25 mL per plant). The foliar nitrogen treatment was applied once a week for 3 wk, each at the end of the first three drought cycles described below, and shown in Fig. 1. Control plants were sprayed with the same solution without urea. For drought treatments, one half of the plants used for each nitrogen treatment was subjected to 1 wk drought by withholding water starting a week before the beginning of foliar nitrogen application (Fig. 1). The other half of the plants in each nitrogen treatment was well watered to field capacity. A total of four drought cycles, each 1 wk long, and three weekly foliar nitrogen applications were completed. Measurements were then taken, using the leaves that developed during treatments, over the fifth drought cycle lasting 9 d. Upon completion of the above measurements, all plants were well watered and placed outside the greenhouse. They were allowed to flower and set pods with no fertilizers applied during the summer.

[FIGURE 1 OMITTED]

Gas Exchange and Leaf Water Potential

An infrared gas analyzer system (LI-6200, LI-COR Inc., Lincoln, NE) was used to measure gas exchange. Measurements were taken daily between 0800 and 0900 h (daylight saving time) on the two most distal leaflet pairs of the third fully expanded leaf from the apex of each plant. Instantaneous water use efficiency (WUE) was computed as the ratio of net photosynthesis ([P.sub.net]) to transpiration (E). Leaf xylem water potential was measured using the fifth leaf of a well-developed branch with a pressure chamber every other day during the gas exchange measurements. Gas exchange was also measured twice during pod development (seed filling stage) after the plants were transferred outside the greenhouse.

Leaf Chlorophyll Concentration

Leaf chlorophyll concentration of each plant was measured three times using the fourth fully expanded leaf from the apex, according to Wintermans and De Mols (1965), during the course of gas exchange measurements. A PerkinElmer Lambda 2, UV/VIS spectrophotometer (PerkinElmer, Norwalk, CT) was used to read the absorbance at 649, 654, and 665 nm. In addition, a leaf chlorophyll meter (Minolta SPAD 501) was used to assess leaf chlorophyll content nondestructively (Marquard and Tipton, 1987). Multiple SPAD readings (n = 110) were taken daily on the leaflets of the same leaves that were used for chlorophyll extraction. SPAD readings were linearly predictive for chlorophyll concentration (Y = 0.777x + 28.719, [r.sup.2] = 0.90, P < 0.001, n = 25) in a calibration experiment.

Leaf Area, Specific Mass and Elemental Analysis

Four well-developed leaflets were removed one each from four leaves at different heights to determine leaf area and leaf specific mass (LSM). Leaflets were photocopied and the copies of the images were cut and weighed. Leaflet area was calculated based on the average weight of a circle (based on weights of five circles) with known area removed from the same paper that was used to obtain the copies of the leaflet images, as (Ac/Wc) x Wl, where Ac is average area of the circle cut from the paper, Wc is average weight of the paper circle, and Wl is weight of a cut leaflet image from the same paper. Total leaf area per plant was estimated by multiplying the total leaflet number of each plant by leaflet area. The same leaflets used for leaflet area measurements were oven-dried at 70[degrees]C to constant weight, and LSM was calculated as the ratio of leaflet dry weight to the area. At the end of all measurements, the leaflets of the third to fifth fully expanded leaves from the apex were stripped and air-dried to constant weight at room temperature. Dried leaflet samples were analyzed by A & L Eastern Agricultural Laboratories Inc. (Richmond, VA) for macro- and micronutrients. Plants were removed from pots when the pods turned brown, and leaves, pods, stems, and roots were oven-dried at 70[degrees]C to constant weight, and weighed separately, 14 wk after the drought and foliar nitrogen treatments were stopped and plants were transferred outside the greenhouse.

Scanning Electron Microscopy

Three leaflets were stripped from the leaf that was used for gas exchange measurements in each plant on the tenth day of the fifth drought cycle. These leaflets from each plant were cut cross-wise into approximately 5 mm wide pieces, and immediately fixed in 3% (v/v) gluteraldehyde in 0.1 M Na-K phosphate buffer (pH 7.2, phosphate buffer) for 1 h at room temperature. The fixed leaflet pieces were then post-fixed for 1.5 h in 1% (w/v) Os[O.sub.4] in phosphate buffer. Upon post-fixing, the tissues were rinsed in phosphate buffer and dehydrated in graded ethanol (50, 70, 95, and 100% each three times), and then in propylene oxide three times (10 min in each). The dehydrated leaflet pieces were critical-point-dried in C[O.sub.2] with a Tousimis Samdri-790 (Tousimis Research Corporation, Rockville, MD, USA). These leaflet pieces were then coated with gold (100 [Angstrom]) in a Hummer II sputter coater. Central zones of each side of the mid vein on the adaxial and abaxial surface of ten leaflet pieces per treatment were examined using a Hitachi S-2700 scanning electron microscope. Number of stomata and trichomes were counted on the computer-acquired image at the magnification of 350 and 70, respectively. These numbers were used to calculate total stomatal and trichome counts per leaflet. Length of the stomatal aperture was measured at the 1000 magnification.

Statistical Analysis

StatView (vet. 5.0: SAS Inst. Inc.) and JMP (vet. 2.0.1: SAS Inst. Inc.) were used to test the statistical significance of main effects and interactions of treatments by ANOVA. Means were compared by Fisher's protected LSD.

RESULTS

Leaf Water Potential

Water-stressed plants had significantly lower leaf xylem water potential than the well-watered plants from the third day of the fifth drought cycle through the end of the measurements (Fig. 2). Foliar nitrogen treatment did not influence water potential in water-stressed or well-watered plants. The range of water potential was approximately 1.6 MPa in the water-stressed plants but was only approximately 0.6 MPa in the well-watered plants. Net C[O.sub.2] uptake was completely suppressed on the 10th day at a water potential of approximately -2 MPa, and at a stomatal conductance ([g.sub.w]) of < 0.02 mol [m.sup.-2][s.sup.-1].

[FIGURE 2 OMITTED]

Gas Exchange

Water-stressed plants maintained the same level of gas exchange ([P.sub.net], [g.sub.w] or E) as well-watered plants until the leaf xylem water potential dropped below approximately -0.8 MPa on the fifth day of the final drought cycle. Thus, data presented are grouped into the first 4 d and the last 5 d. However, foliar nitrogen treatment increased [P.sub.net] by 18 and 22% in well-watered and water-stressed plants, respectively, during the first 4 d (P < 0.001, Fig. 3). There was no interaction effect of foliar nitrogen treatment and drought on [P.sub.net] for the whole period of measurements. During the last 5 d of the fifth drought cycle, drought decreased [P.sub.net] by 20 and 32% in nitrogen-treated and untreated plants, respectively (P < 0.001). Foliar nitrogen treatment increased [P.sub.net] by 40 and 67% in well-watered and water-stressed plants, respectively, for the same period (P < 0.001). During pod development, 12 wk after the drought and nitrogen treatments were stopped, there was no effect of drought on [P.sub.net] (Table l). During the same time, foliar nitrogen treatment increased [P.sub.net] by 24% (P < 0.05) in well-watered, and by 44% (P < 0.001) in water-stressed plants. Foliar nitrogen treatment had an interaction effect with drought on [g.sub.w] in both periods of the fifth drought cycle (Fig. 3). In water-stressed plants, increase of [g.sub.w] because of nitrogen treatment paralleled the pattern of [P.sub.net] (Fig. 3), and a high correlation between [P.sub.net] and [g.sub.w] was observed (Fig. 4). Except for the well-watered plants during the last 5 d, E followed a similar pattern to [P.sub.net]. Instantaneous water use efficiency (WUE) was similar in all four treatment combinations during the first 4 d. During the last 5 d, there was no effect of drought on WUE, but nitrogen-treated plants had 25% greater WUE regardless of drought (well watered, P < 0.05; water stressed, P < 0.01).

[FIGURES 3-4 OMITTED]

Stomatal Count, Aperture Length, and Trichome Count

The number of stomata per leaflet ([N.sub.s]) was reduced at least by 25% (P < 0.001) because of drought on both adaxial and abaxial surfaces regardless of the foliar nitrogen treatment (Table 2). Furthermore, [N.sub.s] was reduced at least by 10% (P < 0.05) by the foliar nitrogen treatment on adaxial surface, regardless of drought treatment. The [N.sub.s] on the abaxial surface, however, increased at least by 25% (P < 0.01) because of foliar nitrogen treatment regardless of drought treatment. In nitrogen-treated plants, adaxial and abaxial stomatal numbers were similar under well-watered or waterstressed conditions. In the plants not treated with nitrogen, however, abaxial [N.sub.s] was at least 26% (P < 0.01) less than adaxial [N.sub.s], regardless of drought.

Length of the stomatal aperture was similar on both adaxial and abaxial surfaces under each of the treatment combinations (Table 2). Foliar nitrogen treatment did not influence aperture length on both surfaces in well-watered plants. In water-stressed plants, however, foliar nitrogen treatment increased aperture length by 18% (P < 0.01) on adaxial and by 21% (P < 0.01) on abaxial surfaces. Interaction between foliar nitrogen treatment and drought was significant only on the abaxial surface (P < 0.05).

Senna trichomes were mostly uniseriate (globular trichomes were rare) with micropapillate sculpturing and were 56 to 160 [micro]m long and 9 to 19 [micro]m wide. They were also bent at the base with the tip oriented toward the distal end of the leaflet (Fig. 5). Trichome count was greater on abaxial than adaxial surface by a factor of >8 across all treatment combinations (Table 2). Trichome count on the adaxial surface increased because of drought by 22% (P < 0.05) and 38% (P < 0.05) without and with foliar nitrogen treatment, respectively. Furthermore, plants deprived of foliar nitrogen treatment had at least 25% (P < 0.05) greater adaxial trichome count regardless of drought. On the abaxial surface, however, with a significant interaction between drought and nitrogen treatment (P < 0.001), trichome count increased by 14% (P < 0.0.01) in response to drought in plants not treated with foliar nitrogen but decreased by 20% (P < 0.01) in nitrogen-treated plants.

[FIGURE 5 OMITTED]

Leaf Chlorophyll, Area, and Specific Mass

There was an interaction between nitrogen treatment and drought on chlorophyll a + b concentration, with foliar nitrogen treatment causing a 47% (P < 0.01) increase only in well-watered plants (Table 3). Although the 10% increase in chlorophyll concentration, because of foliar nitrogen treatment in water-stressed plants, was not significant, the SPAD readings with greater sample size (taken daily on more leaflets than that were used for destructive measurements on the same leaves) showed a significant effect. Drought also increased chlorophyll concentration by 66% (P < 0.001) in plants deprived of nitrogen treatment.

Foliar nitrogen treatment increased leaflet area by 12% in well-watered plants (P < 0.05) but had no effect in water-stressed plants (Table 3). Drought reduced the leaflet area at least by 36% (P < 0.001). Leaf specific mass (LSM) increased by 8% (P < 0.01) in well-watered and 16% (P < 0.01) in water-stressed plants in response to foliar nitrogen treatment. Furthermore, water-stressed, nitrogen-treated plants had 10% (P < 0.05) greater LSM than the well-watered, nitrogen-treated plants. In well-watered plants, foliar nitrogen treatment resulted in 2.5 times greater leaf area per plant compared with the plants without foliar nitrogen treatment. Foliar nitrogen treatment, however, did not influence leaf area per plant in the water-stressed plants. Overall, drought caused a 78% reduction of leaf area per plant. Foliar nitrogen treatment resulted in 2.4 times higher number of stomata per plant than the untreated plants in the absence of drought.

DISCUSSION

The four drought cycles and three foliar nitrogen treatments over a period of 4 wk allowed us to use leaves that developed under drought and nitrogen treatments for all the measurements. Thus, these results reflect the long-term gas exchange and leaf developmental responses of senna to cyclic drought and foliar application of nitrogen.

Gas Exchange

The close positive relationship of [P.sub.net] to [g.sub.w] in water-stressed plants, regardless of nitrogen treatment, indicates the dominant role of stomatal regulation of gas exchange during drought in senna. Although [g.sup.w] and E declined at the late stage of drought, especially when deprived of foliar nitrogen supplement, WUE remained the same in all treatment combinations suggesting that carbon gain in drought-acclimated senna was water-efficient relative to well-watered plants. In contrast, Ehleringer (1993) showed that lower carbon isotope discrimination ([DELTA], an estimate of WUE) was associated with greater survival of the desert shrub, Encelia farinosa Gray ex Torr., during long-term droughts in the field. Increased gas exchange under foliar nitrogen treatment regardless of water status, except for E of well-watered plants during the last 5 d of the fifth drought cycle, showed nitrogen responsiveness in senna. Close parallelism between the pattern of gas exchange and leaf chlorophyll concentration, a variable closely correlated to leaf nitrogen content (Chapin et al., 1987: Wood et al., 1992), further confirmed this.

Given that all plants had received one basal nitrogen supply at the start of the experiment, it is important to consider how severe a nitrogen stress was imposed on the plants not treated with foliar nitrogen. Ratnayaka et al. (2002) reported >4.5% leaf nitrogen content and approximately 35 [micro]mol [m.sup.-2] [s.sup.-1] [P.sub.net] in senna grown in garden plots with high soil nitrogen (estimated nitrogen release of 157 kg [ha.sup.-1]) and no water stress. In the current study, leaf nitrogen content was highest under water stress, 4.0% with and 3.7% without nitrogen treatment. Under well-watered conditions, leaf nitrogen content was 3.4% with and 3.2% without nitrogen treatment. Range of these leaf nitrogen contents, and the respective rates of [P.sub.net] indicate that plants not treated with foliar nitrogen experienced nitrogen stress. Furthermore, lower leaves in well-watered plants not treated with nitrogen yellowed, and leaf area per plant was reduced compared to nitrogen-treated plants. In the same study, leaf biomass of well-watered plants with no nitrogen treatment was also reduced in proportion to leaf area per plant (Ratnayaka et al., 1998).

Foliar nitrogen treatment with 1% urea offers a promising way of supplying nitrogen during senna growth, compared with soil application of nitrogen fertilizer. We also found the foliar nitrogen treatment to have long-lasting merit, as 12 wk after its use, [P.sub.net] was still higher in nitrogen-treated plants during the period of pod growth compared to untreated plants.

Abscission of lower leaves and reduced leaflet size in water-stressed senna plants caused >75% reduction in total transpirational surface area. The remaining apical leaves, whether nitrogen-treated or not, had higher chlorophyll concentration, LSM and leaf nitrogen than well-watered plants. These responses show resource reallocation from the lower shedding leaves to the apical leaves, thus enabling high carbon gain by remaining leaves during stress. Makela et al. (1996), using a model to determine the optimal control of gas exchange during drought, found that maximizing photosynthesis per unit leaf area during an expected drought is optimal. However, reports showing that concentrations of photosynthetic pigments decline in drought are common (Giardi et al., 1996: Yordanov et al., 2000), although the plastid can reorganize the remainder of photosynthetic reaction centers for more efficient photosynthesis subsequently (Giardi et al., 1996). In those studies, however, drought treatments were temporary. Our measurements were taken with defoliation and resource reallocation to the apical leaves under cyclic drought. Other authors also have supported this experimental approach as more realistic toward understanding the capacity of a species to acclimate to drought (Pennypacker et al., 1990; Antolin and Sanchez-Diaz, 1993). We also observed that senna grown in the field allocates nearly all of the root biomass into the long tap root. Collectively, these characteristics qualify senna as a promising candidate for tropical highland agro-ecosystems characterized by frequent and unpredictable droughts and low levels of soil nitrogen.

Leaf Ultrastructure

Senna is amphistomatous with a stomatal density (on a unit leaf area basis) higher than many species (for surveys on stomatal densities see Bolhar-Nordenkampf and Draxler, 1993; Kelly and Beerling, 1995). High stomatal density and amphistomal leaves are adaptations to dry habitats (Bolhar-Nordenkampf and Draxler, 1993). We measured stomatal numbers per leaflet to evaluate the leaflet size factor as affected by treatments. Interestingly, although drought reduced stomatal number on both surfaces regardless of nitrogen stress, the effect of nitrogen stress on stomatal number depended on the specific leaf surface (adaxial vs. abaxial) regardless of drought. Smaller leaflet size under drought (about 36% decrease regardless of nitrogen treatment) largely explains reduced stomatal number. However, leaflet size cannot explain the surface-dependent differential response of stomatal number to nitrogen treatment (decrease on adaxial but increase on abaxial surface). One explanation for the increased stomatal number on the abaxial surface is that the nitrogen supplement may have increased the leaf thickness. Increased LSM in nitrogen-treated plants under both drought and well-watered conditions suggests this, although leaf thickness was not measured in this study. Parkhurst (1978) concluded that presence of stomata on both surfaces is important for thicker leaves. Another possibility is that demand for carbon required for nitrogen assimilation increased under increased leaf nitrogen and chlorophyll concentration. As a result, increased gas exchange ([P.sub.net] and [g.sub.w]) could have triggered the cells to sense water as a limiting factor (even in well-watered plants between waterings). Thus, if nitrogen treatment caused the cells to sense such physiological drought then reduction in adaxial stomatal number, and increase in abaxial stomatal number in senna is not surprising. Because a given number of stomata on the abaxial surface may allow less water loss than the same stomatal count on adaxial surface because trichome number is >8 times greater (therefore, a more stable boundary layer) on the abaxial surface than the adaxial surface. Finally, some effect of urea depositing mainly on to the adaxial surface during application, rather than equally on both surfaces, on leaf ultrastructure cannot also be completely ruled out.

Similar aperture size on both surfaces suggests that epidermal cell sizes on the two leaf surfaces were the same (see Bondada et al., 1994; Bondada and Oosterhuis, 2000). Reduced aperture size on both surfaces in the plants subjected concurrently to drought and nitrogen stress, indicates a probable reduction in epidermal cell size. Our results suggest that the two leaflet surfaces of senna respond to environmental stress in a coordinated fashion to reduce total stomatal count per leaflet under drought, but maintain the same stomatal count per leaflet under nitrogen stress, compared with respective unstressed plants. Heckenberger et al. (1998) reported that drought-stressed castorbean (Ricinus communis L.) plants had higher total adaxial stomatal count per unit surface area than the well-watered plants. They proposed an altered relationship between cell division and elongation in different tissue layers as a probable reason for this.

Epicuticular wax deposits and trichomes increase light reflectivity to maintain optimum leaf temperatures for metabolism (Ehleringer and Mooney, 1978; Bolhar-Nordenkampf and Draxler, 1993). Closely deposited network of epicuticular wax on both leaflet surfaces, and bent trichomes, in senna may be important in this regard. We examined intact leaflets attached to the plant using a light microscope and confirmed that this trichome orientation is not an artifact of tissue preparation for scanning electron microscopy. Micropapillate sculpturing on senna trichomes also may increase light scattering compared to a smooth surface. The greater trichome number on abaxial surface compared with adaxial surface is likely to be important during the paraheliotropic leaflet movement characteristic to senna in which abaxial surfaces are exposed to sun, past midday under high temperatures.

Senna leaflets have tightly packed palisade mesophyll cells under both adaxial and abaxial epidermal layers. This isobilateral leaf anatomy restricts the interconnected network of free, wet cell surfaces of spongy mesophyll to a thin central strip. Having spongy mesophyll away from the stomatal connection to the dry atmosphere may help minimize transpiration (Bolhar-Nordenkampf and Draxler, 1993). Occurrence of palisade cells with tightly packed chloroplasts (as seen in transmission electron micrographs not shown) on both sides of the leaflet may also contribute to the high rate of [P.sub.net], and efficient utilization of incident or diffused light when leaflets are either horizontal or paraheliotropically angled. Furthermore, large bundle sheath extensions could provide support against wilting during stress.

Thus, our results indicate that a multitude of physiological, developmental, and morphological characteristics collectively confer tolerance to drought and nitrogen stress in senna. Maintenance of high carbon gain and water use efficiency over a wide range of stress levels, drought-deciduousness with re-allocation of resources to apical leaves, plasticity of stomatal and trichome numbers on a given leaflet surface, densely deposited epicuticular wax, isobilateral leaf anatomy with large bundle sheath extensions, and paraheliotropic leaflet movement are important in this regard. Further research should be conducted to understand the mechanisms underlying the increased abaxial stomatal count under foliar nitrogen treatment, and its possible practical implications. Foliar treatment of senna with 1% urea is a promising alternative to soil-application of nitrogen fertilizer during plant growth and should be tested in sitespecific field experiments particularly in tropical and subtropical dry farming systems where senna is a potential new crop.

Abbreviations: Ac, average area of a cut paper circle: ANOVA, analysis of variance; E, transpiration; [g.sub.w], stomatal conductance; LSD, least significant difference: LSM, leaf specific mass; [N.sub.s], number of stomata per leaflet; [P.sub.net], net photosynthesis; SPAD, Minolta SPAD 501 chlorophyll meter; We, average weight of a cut paper circle; Wl, weight of the leaflet image cut from the paper; WUE, instantaneous water use efficiency.
Table 1. Net photosynthesis ([micro]mol [m.sup.-2] [s.sup.-1]) of senna
during pod development 12 wk after the treatments, drought and foliar
nitrogen application, were stopped.

Foliar nitrogen treatment    Well-watered    Water-stressed

No foliar nitrogen             19.34            17.17
Foliar nitrogen                24.14 *          24.77 ***
Mean                           21.74            20.97

* Significantly greater than no foliar nitrogen treatment in a given
water status, at P < 0.05.

** Significantly greater than no foliar nitrogen treatment in a given
water status, at P < 0.001.

Table 2. Stomatal and trichome numbers per leaflet, and aperture length
of senna as affected by drought and foliar nitrogen treatment.

                                   Adaxial surface

Treatment               Stomata       Trichomes    Aperture

                                 x1000             [micro]m

Well-watered
  No foliar nitrogen    64 463 *        629 *      15.30
  Foliar nitrogen       57 504          445        15.66
  Mean                  60 983 ***      537        15.48
Water-stressed
  No foliar nitrogen    48 547 *        772 *      13.02
  Foliar nitrogen       37 980          616        15.36 **
  Mean                  43 263          694 *      14.19

                                   Abaxial surface

Treatment               Stomata       Trichomes    Aperture

                                x1000              [micro]m

Well-watered
  No foliar nitrogen    47 229        6451         16.11
  Foliar nitrogen       59 120 **     6230         16.05
 Mean                   53 174 ***    6340         16.08
Water-stressed
  No foliar nitrogen    24 562        7397 ***     12.87
  Foliar nitrogen       39 841 **     4976         15.54 **
  Mean                  32 201        6186         14.20

* Significantly greater than other foliar nitrogen treatment in a given
water status, and greater than other water status mean, at P < 0.05.

** Significantly greater than other foliar nitrogen treatment in a
given water status, and greater than other water status mean, at
P < 0.01.

*** Significantly greater than other foliar nitrogen treatment in a
given water status, and greater than other water status mean, at
P < 0.001.

Table 3. Leaf characteristics of senna as affected by drought and
foliar nitrogen treatment. LSM = leaf specific mass. Values per plant
were estimated by multiplying the value per leaflet by the number of
leaflets per plant.

Treatment               Chlorophyll a + b    SPAD units    Leaflet area

                            [micro]g
                           [cm.sup.-2]                     [cm.sup.-2]

Well-watered
  No foliar nitrogen        21.52            46.98           1.71
  Foliar nitrogen           31.62 **         57.00 ***       1.97 *
  Mean                      26.57            51.99           1.85 ***
Water-stressed
  No foliar nitrogen        35.06            58.69           1.09
  Foliar nitrogen           38.55            60.50 ***       1.21
  Mean                      36.80 ***        59.59 ***       1.15

Treatment                   LSM                Leaf area

                         [micro]g
                        [mm.sup.-2]    [cm.sup.-2] [plant.sup.-1]

Well-watered
  No foliar nitrogen     59.32                 474.39
  Foliar nitrogen        64.49 **             1213.41 ***
  Mean                   61.91                 843.90 ***
Water-stressed
  No foliar nitrogen     61.85                 195.95
  Foliar nitrogen        71.06 **              175.55
  Mean                   66.45 *               185.75

Treatment                   Stomata                  Stomata

                          x[10.sup.4]
                        [leaflet.sup.-1]    x[10.sup.4] [plant.sup.-1]

Well-watered
  No foliar nitrogen        11.08                   30.03
  Foliar nitrogen           11.73                   72.25 **
  Mean                      11.40 **                51.11 **
Water-stressed
  No foliar nitrogen         7.31                   13.14
  Foliar nitrogen            7.77                   11.29
  Mean                       7.54                   12.22

* Significantly greater than other foliar nitrogen treatment in a given
water status, and greater than other water status mean, at P < 0.05.

** Significantly greater than other foliar nitrogen treatment in a
given water status, and greater than other water status mean, at
P < 0.01.

*** Significantly greater than other foliar nitrogen treatment in a
given water status, and greater than other water status mean, at
P < 0.001.


ACKNOWLEDGMENTS

The authors are grateful to Dr. Tracy Sterling of New Mexico State University for the encouragement during writing, and for editing the draft of this manuscript. They thank Mr. David Cain for maintaining the greenhouse facilities, Dr. Thomas Jensen, and Mr. Michael Baxter and Mr. Rajendra Gharbaran for assistance during electron microscopy. Lehman College and Graduate School of City University of New York provided financial assistance for this research.

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H. H. Ratnayaka * and D. Kinaid

H.H. Ratnayaka, Dep. of Biology, Xavier Univ. of Louisiana. 1 Drexel Drive, New Orleans, LA 70125: D. Kincaid, Dep. of Biological Sciences, Lehman College and the Graduate School of the City Univ. of New York, 250 Bedford Park Boulevard West. Bronx, NY 10468. Received 23 July 2003. Crop Ecology, Management & Quality. * Corresponding author (hratnaya@xula.edu).

doi: 10.2135/cropsci2003.737
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Title Annotation:CROP ECOLOGY, MANAGEMENT & QUALITY
Author:Ratnayaka, H.H.; Kincaid, D.
Publication:Crop Science
Date:May 1, 2005
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