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- Materials and Methods
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.
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- Materials and Methods
The present study has confirmed that stomatal density in cowpea varies according to the several external environments: with soil P, with soil water and with atmospheric CO2. It has previously been reported that stomatal formation is affected by light (Schoch et al., 1980; Thomas et al., 2003), CO2 (Woodward, 1987; Woodward & Bazzaz, 1988; Knapp et al., 1994; Woodward & Kelly, 1995; Woodward et al., 2002), O2 (Ramonell et al., 2001), O3 (Frey et al., 1996), NO2 (Siegwolf et al., 2001), UV-B (Gitz et al., 2004) and soil water (Quarrie & Jones, 1977; Bañon et al., 2004). In addition to these variables, it is a novel finding that P concentration in soil can also affect stomatal density (Fig. 4).
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.
In this study, CO2 enrichment has increased stomatal density in cowpea plants (Fig. 4). However, observations in the literature of the effect of CO2 enrichment on stomatal density vary, and include little effect (Radoglou & Jarvis, 1990; Ryle & Stanley, 1992), a decreasing effect (Woodward, 1987; Woodward & Bazzaz, 1988; Knapp et al., 1994; Woodward & Kelly, 1995; Woodward et al., 2002), and an increasing effect (Rowland-Bamford et al., 1990; Royer, 2001; Marchi et al., 2004). Such inconsistency may result from interspecific differences (Gray et al., 2000), but at the same time we cannot deny the involvement of strong interactions between several environmental variables, as revealed in this study.
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.