• Stomatal formation is affected by a plant's external environment, with long-distance signaling from mature to young leaves seemingly involved. However, it is still unclear what is responsible for this signal. To address this question, the relationship between carbon isotope discrimination (Δ) and stomatal density was examined in cowpea (Vigna sinensis).
• Plants were grown under various environments that combined different amounts of soil phosphorus (P), soil water, and atmospheric CO2. At harvest, stomatal density was measured in the youngest fully expanded leaf. The 13C : 12C ratio was measured in a young leaf to determine the Δ in mature leaves.
• Results indicated that stomatal density is affected by P as well as by amounts of water and CO2. However, stomatal responses to water and CO2 were complex because of strong interactions with P. This suggests that the responses are relative, depending on some internal factor being affected by each external variable. Despite such complicated responses, a linear correlation was found between stomatal density and Δ across all environments examined.
• It is proposed that the Δ value is a good surrogate for the long-term mean of the intercellular (Ci) to the atmospheric (Ca) CO2 concentration ratio (Ci : Ca) and may be useful in understanding stomatal formation beyond complicated interactions.
Stomatal density is known to be affected by environmental variables such as light and atmospheric CO2 concentration (Bergmann, 2004). The majority of stomata are formed well before a leaf is fully expanded (Lake et al., 2002). The laminae of developing leaves often curl around the apical meristem because their abaxial surfaces differentiate earlier and grow faster than their adaxial ones (McConnell & Barton, 1998). Thus, at the time of stomatal formation, the young leaves are not fully exposed to the external atmosphere because of their curling nature, making it difficult to detect their actual environments (Lake et al., 2002). Therefore, it is reasonable that fully developed mature leaves should be responsible for detecting the aerial environment and regulating stomatal density in new leaves.
Currently, a long-distance signal is suggested as being involved in the process, with environmental changes being detected by mature leaves and their signals transported acropetally, to regulate stomatal density in the new leaves (Schoch et al., 1980; Lake et al., 2001; Lake et al., 2002; Thomas et al., 2003). The involvement of long-distance signaling in stomatal formation was first discovered when the response of stomatal formation to shading was investigated. Schoch et al. (1980) revealed that shading of mature leaves of cowpea plants reduced stomatal index in new leaves. Thereafter, similar phenomena have been observed in Arabidopsis (Lake et al., 2001) and tobacco (Thomas et al., 2003). Thomas et al. (2003) further reported that the application of high irradiance to mature leaves of tobacco plants increased stomatal density in the new leaves. Although little attention was paid to stomata, Yano & Terashima (2001) also reported that mature leaves function as light sensory sites when the anatomy of new leaves changes in response to shading. Such a systemic control has also been revealed for CO2. Lake et al. (2001) found that CO2 enrichment only around mature leaves reduced stomatal density in new leaves of Arabidopsis.
Both light and CO2 can influence photosynthetic rate. Hence, sugar as the photosynthetic product has been assumed to play a role in signal mediation between mature and new leaves (Coupe et al., 2006). However, shading and CO2 enrichment have opposite effects on sugar production but similarly reduce stomatal density in new leaves, effectively ruling out the model that considers sugar production to be the signal for stomatal formation (Bergmann, 2006). Miyazawa et al. (2006) recently found a positive correlation between stomatal conductance in mature leaves and stomatal index in new leaves. Since shading and CO2 enrichment generally reduce stomatal conductance, the authors proposed that the information of reduced stomatal conductance in mature leaves is transmitted to new leaves, lowering their stomatal index.
While Miyazawa et al. (2006) appears to resolve the paradox in sugar signal hypothesis, it is still unclear how new leaves perceive the information of changes in stomatal conductance in mature leaves. It would seem that there must be a signal (or signals) that corresponds to changes in stomatal status. To address this question we have examined the carbon isotopic composition (the 13C : 12C ratio) in leaf tissues because isotopic composition in leaf tissues is strongly affected by stomatal status (Farquhar et al., 1989). In general, most of the carbon (C) in young, developing apical leaf tissues is derived from that fixed by mature, basal leaves (Joy, 1964). Thus, the 13C content in the more apical leaf tissues reflects the stomatal status of the mature leaves. Therefore, the present study attempts to investigate the relationship between 13C content and stomatal density in young leaves.
Schoch et al. (1980) demonstrated that, in cowpea plants, stomatal formation of excised young, unfolding leaves responds to an environmental condition, but that of intact leaves is fully controlled by a signal(s) from mature leaves. This suggests that although young, expanding leaves are potentially capable of local response to environmental conditions, the signal(s) from mature leaves overwhelms the capacity of young leaves. In the present study, stomatal density was altered in cowpea plants by subjecting them to various amounts of phosphorus (P), soil water and atmospheric CO2 concentration. Although soil water (Quarrie & Jones, 1977; Bañon et al., 2004) and atmospheric CO2 (Bergmann, 2004) are known to affect stomatal density, as far as we know, any relation between P and stomatal density is still unresolved. Furthermore, interactive effects of P with the other environmental variables have rarely been investigated. Thus, this study attempts to elucidate whether or not soil P concentration, including interactive effects with the other variables studied, can affect stomatal density; and whether the 13C content of young leaf tissues correlates with their stomatal density under the influence of various P, soil water, and CO2 environments.
Materials and Methods
Effects of soil P and soil water
Plastic pots (5 cm diameter and 15 cm height) were each filled with 350 g of Andisol (0.1 mg Truog-P kg−1 dry soil) amended with 100 mg ammonium sulphate, 40 mg potassium chloride, and either 0 (0P, 0-P), 50 (LP, low P), 150 (MP, mid-P) or 450 mg (HP, high P) superphosphate (a mixture of Ca(H2PO4)2·H2O and CaSO4). Three pre-germinated seeds of cowpea (Vigna sinensis L., cv HAF-43) were sown in each pot and the pots were placed in a growth chamber (conditions during the 12 h photoperiod were 30 ± 2°C and 60 ± 5% relative humidity (RH) with 400 ± 50 µmol m−2 s−1 photon flux density; conditions during the 12 h dark period were 25 ± 2°C and 60 ± 5% RH). Seedlings were thinned to leave just one per pot on the third day after sowing. At the same time, three amounts of soil water content were induced: 0.6 g g−1 (HSW, high soil water, 80.0% of field capacity), 0.4 g g−1 (MSW, mid-soil water, 53.3% of field capacity) and 0.2 g g−1 (LSW, low soil water, 26.7% of field capacity). The amounts of water required to maintain these soil water contents were determined gravimetrically by weighing each pot every day. The combinations of four different P contents and three different water contents gave 12 treatments in all (HP-HSW, HP-MSW, HP-LSW, MP-HSW, MP-MSW, MP-LSW, LP-HSW, LP-MSW, LP-LSW, 0P-HSW, 0P-MSW and 0P-LSW). Each treatment was replicated five times. All 60 plants were harvested 40 d after sowing by cutting at the base.
Effects of soil P and atmospheric CO2
Similar (5 × 15 cm) plastic pots were filled with 350 g Andisol amended with 100 mg ammonium sulphate, 40 mg potassium chloride, and either 50 mg (LP) or 450 mg (HP) superphosphate. Three pre-germinated seeds of cowpea were sown in each pot and these were placed in one of two different growth chambers. The growth chambers were maintained under the same conditions with respect to temperature, humidity and light (during the 12 h photoperiod 30 ± 2°C and 60 ± 5% RH with 200 ± 50 µmol m−2 s−1 photon flux density; during the 12 h dark period 25 ± 2°C and 60 ± 5% RH) but under different CO2 concentrations. In one chamber, atmospheric CO2 was maintained at 1000 ± 150 µmol mol−1 (HC, high CO2,) and, in the other, CO2 concentration was maintained at 400 ± 100 µmol mol−1 (LC, low CO2) by a CO2 controller (CO2 concentration control unit, Koito Industries Ltd, Yokohama, Japan). The seedlings were thinned to leave just one per pot on the third day after sowing. Then, the pots were placed in trays and water was applied from the bottom so as to maintain a water depth of approx. 8 mm throughout the experiment. In each chamber (HC and LC), P concentration (HP and LP) was replicated five times (split-plot design). All 20 plants were harvested 32 d after sowing by cutting at the base.
Stomatal density, stomatal index, leaf area and shoot dry matter weight
Fingernail polish imprints were taken from the middle portions of both the adaxial and abaxial surfaces of the youngest fully expanded leaf of each plant (see Fig. 1). The imprints were observed under an inverted microscope (IX 70, Olympus, Japan) equipped with a digital camera, and the images were presented using image-editing software (Cool SNAP, Roper Scientific, Tucson, AZ, USA) on a computer screen. Ten images were randomly selected from each imprint, and the number of stomata and epidermal cells in each image was counted. Then, the area of each image was determined using NIH Image version 1.60 image-analysis software, and stomatal density was calculated by dividing the number of stomata counted by the area of each image. Epidermal cell density was also calculated by dividing the number of epidermal cells counted by the area of each image. Stomatal index was then calculated as the value of stomatal density divided by the sum of stomatal density and epidermal cell density. The leaves were removed from the stems and their areas were measured using a leaf-area meter (LI-3100C, Li-Cor, Lincoln, NE, USA). The leaves and stems were then dried at 80°C for 48 h and shoot dry matter weights were measured.
Carbon isotopes composition
The dried leaves were ground to powder for analysis. Approximately 80 µg subsamples were encapsulated in 0.15 ml tin foil and combusted in an automatic NCS analyzer (NA2500, CE Instruments, Milan, Italy). The carbon dioxide gas from combustion was introduced through an elemental analyzer-IRMS interface (ConFlo II, Thermo Fisher Scientific Inc., Worcester, MA, USA) into a stable isotope ratio mass spectrometer (Delta plus, Thermo Fisher Scientific Inc. Worcester, MA, USA) and analyzed for its carbon isotope compositions (δ). The δ values were expressed in delta notation relative to standard (PDB, PeeDee belemnite limestone) as:
δ (‰) = (Rsample/Rstandard − 1) × 1000(Eqn 1)
where R refers to the carbon isotopes’ composition (13C : 12C ratio) of sample and standard, respectively.
The young leaves, including the youngest fully expanded leaf, were used to determine isotopic composition (δp). Following a method employed by others (Marino & McElroy, 1991; Polley et al., 1993; Hanba et al., 1997), carbon isotope discrimination (Δp) was determined as follows. Plastic pots (5 × 15 cm) were filled with 400 g loamy sand amended with 160 mg of a compound fertilizer (N : P : K = 12 : 16 : 14). The pots were placed in trays and water was applied from the bottom so as to maintain a water level of c. 1 cm depth. Five seeds of maize (Zea mays L. cv. Royal-dent) were sown in each pot and were placed adjacent to the cowpea plants in the experiment monitoring the effects of soil P and atmospheric CO2 in the two growth chambers maintained with differing concentrations of CO2. The maize seedlings were thinned to leave just one plant on the fifth day after sowing. On the day of the cowpea harvest, maize plants were also harvested to determine δ values (δz). The δ value of atmospheric CO2 (δa) was calculated by the following equation using δz:
δa = Δz (1 + δz/1000) + δz(Eqn 2)
where Δz is carbon isotope discrimination in maize and assumed to be 3.3‰ (Marino & McElroy, 1991). Thereafter, Δp was calculated using δp and δa by the following equation:
Δp= (δa − δp)/(1 + δp/1000) (Eqn 3)
In the experiment looking at the effects of soil P and soil water, the data were analyzed by a fixed model, two-way analysis of variance (ANOVA) in which sources of variation were P concentration (HP, MP, LP or 0P), soil water content (HSW, MSW or LSW) and their interactions. Since the experiment monitoring the effects of soil P and atmospheric CO2 was conducted in a split-plot design, the data were analyzed by a mixed-model ANOVA with concentration of CO2 (HC or LC) as a main-plot factor and P concentration as a subplot factor (Gomez & Gomez, 1983). The data were checked for deviations from normality and for homogeneity of variance and log-transformed if necessary. Correlations were analyzed between Δp and stomatal density using the Pearson correlation coefficient test.
Leaf area and shoot dry matter weight
Effects of soil P and soil water ANOVA demonstrates that each of the two growth parameters was significantly increased by increases in P (Fig. 2). The increase in soil water also significantly increased the two growth parameters. Significant interactions between P and soil water on both growth parameters indicate that growth promotion as a result of P was greater under MSW and HSW conditions than under LSW.
Effects of soil P and atmospheric CO2 ANOVA demonstrates that each of the two growth parameters was significantly increased by increases in P (Fig. 2). The increase in CO2 significantly increased shoot dry matter weight but had no significant effect on leaf area. Significant interaction between P and CO2 was found on leaf area but not on shoot dry matter weight.
Stomatal density and stomatal index
Figure 3 shows stomatal images of cowpea plants observed in both experiments (for the effects of soil P and soil water experiment, only the images under HSW are shown). Stomatal densities and indices are shown in Figs 4 and 5, respectively.
Effects of soil P and soil water ANOVA demonstrates that the two main effects (P and water) on stomatal density were significant for both adaxial and abaxial leaf surfaces (Fig. 4). Looking at the mean values for each main effect, either higher P (HP, 133 ± 6; MP, 121 ± 6; LP, 97 ± 3; and 0P, 88 ± 7 for the adaxial surface; and HP, 339 ± 12; MP, 286 ± 12; LP, 261 ± 14 and 0P, 233 ± 14 for the abaxial surface), or lower soil water (HSW, 108 ± 8; MSW, 102 ± 5; and LSW, 119 ± 6 for the adaxial surface; and HSW, 206 ± 17; MSW, 264 ± 13; and LSW, 309 ± 11 for the abaxial surface) increased stomatal density. However, such a generalization is dangerous because the interaction between these was also significant on both surfaces. Indeed, the increase of stomatal density under the LSW condition was distinct under 0P, LP and MP conditions, but was reversed under HP conditions on both leaf surfaces.
The effect of P on stomatal index was significant, particularly for the abaxial surface (P < 0.001), but also (very weakly) for the adaxial surface (P = 0.083) (Fig. 5). Higher P resulted in greater stomatal index. However, the effect of soil water on stomatal index was not significant on either leaf surface. An interaction between P and soil water was significant on the abaxial surface. Thus, the increase of stomatal index as a result of P was greater under the HSW condition than under MSW and LSW on the abaxial surface. The significant increases in both stomatal density and index as a result of higher P indicate that P promotes stomatal formation. Contrasting with the effect of P, the significant increase of stomatal density as a result of lower soil water should be caused by reduction of epidermal cell expansion, as soil water has no significant effect on stomatal index.
Effects of soil P and atmospheric CO2 ANOVA shows that the two main effects (P and CO2) were significant on both leaf surfaces (Fig. 4). For the adaxial surface, either higher P or higher CO2 resulted in greater stomatal density. An interaction between P and CO2 was significant on the abaxial surface. As a result, stomatal density increased 1.5 times under elevated CO2 with HP, but only 1.2 times with LP.
Carbon isotope discrimination Δp
Effects of soil P and soil water ANOVA demonstrates that increased P significantly reduced Δp (Table 1). Contrasting with the effect of P, increases in soil water significantly increased Δp. No interaction was found between P and soil water.
Table 1. Effects of soil phosphorus (P), soil water and atmospheric CO2 on carbon isotope discrimination (‰) in mature leaves of cowpea (Vigna sinensis cv. HAF-43)
Effects of soil P and soil water
Effects of soil P and atmospheric CO2
Soil water (W)
Treatments are HP, MP, LP and 0P, representing 450, 150, 50 and 0 mg of superphosphate per pot; HSW, MSW and LSW, representing 0.6, 0.4 and 0.2 g g−1 of soil water; and HC and LC, representing 1000 and 450 µmol mol−1 of atmospheric CO2.
Each value is a mean ± SE (n = 5). Effects were analyzed by a fixed-model, two-way ANOVA for the experiment considering the effects of soil P and soil water; and by a mixed-model ANOVA for the experiment considering the effects of soil P and atmospheric CO2. Probabilities are for phosphorus (P), soil water (W) and CO2 (C).
16.7 ± 0.1
16.1 ± 0.3
16.1 ± 0.1
16.2 ± 0.1
16.9 ± 0.2
19.0 ± 0.1
18.1 ± 0.4
17.7 ± 0.2
17.4 ± 0.3
16.5 ± 0.3
17.2 ± 0.2
17.4 ± 0.1
17.3 ± 0.1
16.4 ± 0.1
17.0 ± 0.1
17.8 ± 0.2
19.4 ± 0.3
18.6 ± 0.3
17.3 ± 0.1
17.4 ± 0.2
16.6 ± 0.2
17.1 ± 0.1
17.3 ± 0.1
17.0 ± 0.2
16.4 ± 0.1
16.9 ± 0.1
17.4 ± 0.2
19.2 ± 0.2
18.3 ± 0.3
P < 0.001
P = 0.039
P < 0.001
P = 0.001
P × W
P = 0.110
P × C
P = 0.255
Effects of soil P and atmospheric CO2 ANOVA demonstrates that increased P significantly reduced Δp (Table 1). The increase in CO2 also reduced Δp. No interaction was found between P and soil water.
Relation between stomatal density and Δp
Figure 6 shows the relationship between stomatal density and Δp. Significant correlations were found between Δp and stomatal densities on adaxial (r = − 0.685), abaxial (r = −0.737) and adaxial + abaxial (r = −0.748) leaf surfaces. These correlations suggest that mature leaves send some signal, which is strongly associated with carbon isotope discrimination, affecting stomatal density in young leaves.
Our results have further revealed that soil P has interactive effects with soil water and with atmospheric CO2 on stomatal density, particularly on the leaf's abaxial surface (Fig. 4). While CO2 enrichment increased stomatal density under both HP and LP, the increment was greater in HP than in LP. In the case of the interaction with soil water, we have found opposite responses of stomatal density, depending on the P concentration; increases in soil water increased stomatal density under HP but decreased it under the lower P concentrations. Such strong interactions suggest that the response of stomatal density to any particular environmental variable will be relative rather than constant. We assume that each of the external environmental variables can affect some internal factor in either a positive or a negative way. It seems to be the integrated status of that factor that determines stomatal density in the young leaves.
Beyond such complicated interactions, we have found a fairly simple, linear relationship between stomatal density and Δ in young leaves across a range of external environments (Fig. 6). The Δ value represents a long-term mean of the intercellular (Ci) to the atmospheric (Ca) CO2 concentration ratio (Ci : Ca) (reviewed in Farquhar et al., 1989). Since most carbohydrates in young, expanding leaves originate from mature leaves (Joy, 1964), the Δ values measured in this study would seem to reflect the Δ values within mature leaves. Thus, the results indicate that there is a correlation between Δ values, and hence the Ci : Ca fluctuations, in mature leaves and stomatal density in young leaves.
In fact, significant differences in Δ caused by P, by water and by CO2 suggest that these external variables have changed the Ci : Ca ratio (Table 1). An increase in P nutrition increases photosynthetic rate (Jacob & Lawlor, 1991), while soil dryness reduces stomatal aperture (Kramer & Boyer, 1995). CO2 enrichment is known to reduce stomatal aperture but to increase photosynthetic rate (Assmann, 1999). Both the increased photosynthetic rate and the reduced stomatal aperture similarly lower the Ci : Ca ratio, leading to a reduction in Δ. We therefore suggest that external environment, soil P, soil water and atmospheric CO2 primarily affects the Ci : Ca ratio in mature leaves, and stomatal density in young leaves responds to the integrated status of the Ci : Ca ratio.
The Ci : Ca ratio is determined by the balance between mesophyll demand for CO2 (photosynthetic rate) and the CO2 supply through the stomata (stomatal conductance) (reviewed in Farquhar et al., 1989). This suggests that these two parameters could be involved in the long-distance signaling. According to Miyazawa et al. (2006), in mature leaves in steady-state environments, only stomatal conductance (and not photosynthetic rate) is responsible for stomatal formation in young leaves. However, under nonsteady-state conditions, as in this study, plants are likely to open and close their stomata to maintain the Ci : Ca ratio (Wong et al., 1979; Field et al., 1983; Yoshie, 1986; Roelfsema et al., 2002; Hashimoto et al., 2006). Moreover, stomatal conductance is affected not only by environmental variables but also by its own daily fluctuation (Bates & Hall, 1982; Collatz et al., 1991), by leaf age (Raschke & Zeevaari, 1976; Vos & Oyarzún, 1987) and by patchy stomatal closure (Terashima et al., 1988; Mott & Buckley, 2000). Recognizing that stomatal formation is a process that takes place over a timescale of days, rather than of minutes or seconds, a long-term parameter such as Δ , rather than an instantaneous one such as stomatal conductance, can be a more suitable criterion to understand stomatal formation in response to various external variables.
In conclusion, we have found that P nutritional status can affect stomatal density by promoting production rate of stomata per epidermal cell. Moreover, it has been revealed that strong interaction effects of P with water or CO2 make it difficult to predict stomatal responses to changing multiple factors only by investigating each factor individually. Despite this complexity of responses, a strong correlation has been found between stomatal density and Δ. The inconsistent results in the literature (e.g. in response to CO2 enrichment) may also be explained by investigating Δ. Hence, we propose that the Δ value in mature leaves should be considered a good surrogate for the long-term mean of the Ci : Ca ratio, and that it may also be useful to help us understand stomatal formation in young leaves better, because it integrates the fluctuations in both stomatal conductance and photosynthetic rate that occur under natural, nonsteady-state conditions.
We thank Yasuko Kato and Chiemi Sakuma for their technical assistance. We are also grateful to Akira Yamauchi for providing laboratory facilities. This study was supported financially by the Research Fellowships for Young Scientists from the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Industrial Technology Research Grant Program in 2005 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.