MECHANISMS RESPONSIBLE FOR OPTIMUM NITROGEN ALLOCATION WITHIN HERBACEOUS PLANTS
Phenomenology
Formation of the gradient in nL within a plant would be dependent on either light environment or age. Hirose et al. (1988)
observed that the gradient in nL in the individual plant of Lyschimacia vulgaris was less steep when the plants were grown in a thin leaf stand with much light penetration than in a dense stand, although the age of plants in these stands was identical. For a blade of Carex acutiformis (a sedge) in the stand, nL was greatest at the tip and decreased towards the blade base (Hirose et al., 1989). Because the blade of grass-type monocots elongate due to activity of the intercalary meristem at the blade base, the tip of the blade is more aged than the base. These results indicate that light is more important than age in determining nL. Analysing the light environments of leaves of various ages in a vine plant, Ackerly (1992) reached the same conclusion.
Hikosaka et al. (1994) devised an experimental system to examine separately the effects of light environment and age. They grew vines of Ipomoea tricolor horizontally to minimize the effects of mutual shading of the leaves. Photon irradiances of the respective leaves were controlled with small shade boxes. When nutrients were sufficient, nL was influenced by irradiance levels and nL was greater in the leaves receiving high light. In contrast, when the nutrient was limiting, nL decreased with leaf age even when all the leaves were exposed to the same irradiance (Hikosaka et al., 1994). Thus, age also appears to be important.
In the plant in the dense stand, there are vertical gradients in chloroplast properties such as Chl a/b and Rubisco/Chl as well as that in nL (Evans and Seemann, 1989; Terashima and Hikosaka, 1995). Such differences in chloroplast properties are well characterized by comparative studies in which plants were grown under high and low light conditions (Boardman, 1977; Anderson, 1986). When the leaves of I. tricolor that had been exposed were shaded, not only their nL but the ratios of Chl a/b and Rubisco/Chl decreased. In contrast, when nL decreased with age under constant photon irradiance, changes in Chl a/b and Rubisco/Chl were small (Hikosaka, 1996). These results suggest that re-acclimation of sun leaves to the shade is accompanied by both a decrease in nL and qualitative changes in chloroplast properties. Selective breakdown of the components related to energy transduction, such as Rubisco, and maintenance or synthesis of the components for the light harvesting system occur in re-acclimation to a shadier environment. On the other hand, when the leaves senesce in constant light, all photosynthetic components generally decreased (Hikosaka, 1996; Hikosaka, 2005).
Acclimation to sun and shade: formation of sun and shade leaves
Because construction and maintenance are complex processes, the processes should be dissected into several basic processes. The first deals with acclimation to the light environment in developing leaves and then senescing leaves.
Sun and shade leaves differentiate according to the light environments in which they are grown (Björkman, 1981). As already discussed, sun leaves are generally thicker than shade leaves. Thickening of sun leaves is mainly due to elongation of palisade tissue cells or to periclinal divisions in the palisade tissue cells, or both. By such thickening, mesophyll surface area per leaf area becomes greater in sun leaves than in shade leaves. Properties of chloroplasts are also different between the sun and shade leaves (Boardman, 1977; Anderson, 1986).
Anatomical differences between sun and shade leaves. Differentiation processes of sun and shade leaves were analysed using Chenopodium album, an annual herb. The plants were shaded in various ways and the effects of these shading treatments on the properties of the developing leaves were examined. Palisade tissue, two cell layers thick, was formed when mature leaves were exposed to high light, irrespective of the light environments of the developing leaves. On the other hand, when mature leaves were shaded, one-cell-layered palisade tissue was formed. These results clearly showed that anatomy of new leaves is determined by light environment of mature leaves. There must be a signal transduction system that conveys the signal(s) from the mature leaves to developing leaves (Yano and Terashima, 2001).
Further, a comparative developmental study of sun and shade leaves for C. album was conducted. Whether the plants were grown under typical sun or shade conditions, the number of cells in the palisade tissue per leaf was almost identical. Moreover, in sun leaves, anticlinal and periclinal divisions occur almost synchronously. Thus, the signal(s) from the mature leaves regulates the direction of cell division. In the future sun leaves, the signal probably induces periclinal division in addition to anticlinal division, while the signal from the shaded mature leaves only allows the cells to divide anticlinally (Yano and Terashima, 2004).
It is hypothesized that the signal is the abundance of photosynthate. When photosynthates from mature leaves are abundant, leaves would develop into sun leaves. Stomatal frequency could be also regulated by similar mechanisms (Lake et al., 2001).
In some deciduous trees, all leaves that will unfold in flush in the next year are prepared in the bud during the winter. In some species of this group, the anatomy of the next-year leaves is determined when they are in winter buds. For example, in Fagus crenata from the Pacific side of Japan (there are some difference among ecotypes; T. Koike, pers. comm.), the number of cell layers of the leaf is already determined in the winter bud (Uemura et al., 2000). The developing leaves may sense the light environment of the bud. However, it is more likely that differentiation between sun and shade leaves occur in the developing bud in response to matter production during the year. On the other hand, chloroplast properties are largely determined by the light environment of the developing leaves (Yano and Terashima, 2001; see below).
Roles of photoreceptors. In the plant stand, the decrease in PPFD with depth is always accompanied by a decrease in the red : far-red ratio (Anten et al., 2000; Smith, 2000). Thus, the role of phytochrome in the differentiation of sun and shade leaves has been studied.
Smith et al. (1993) demonstrated that shading of aurea mutants (deficient in phytochrome A, and also possibly other phytochromes) of tomato plants caused a similar decrease in Pmax as in wild-type plants. A series of studies using Arabidopsis thaliana with mutant photoreceptors clearly showed that phytochrome mutants are able to form sun and shade leaves in response to the light environment in which they are growing. These leaves differed in Pmax and chloroplast properties including Chl a/b and Rubisco/Chl. These results indicate that phytochromes do not play a central role in the differentiation of sun and shade leaves (Walters et al., 1999). Weston et al. (2000) examined the anatomy of leaves of photoreceptor mutants and confirmed their differentiation into sun and shade leaves.
Walters and Horton (1995) showed that acclimation of leaves was not dependent on the intensity of blue light. However, in the complete absence of blue light, acclimation to irradiance did not occur (Walters et al., 1999). In ecophysiological experiments, shading is usually done with a black screen, which does not change the spectrum of the light. In such experiments as well, differentiation between sun and shade leaves has been observed (for a review, see Björkman, 1981). Thus, sun and shade leaves develop even when the light spectrum is unchanged.
However, the above studies indicate that plants appear to sense whether they are in the light or in the dark using these photoreceptors. The decrease or increase in sugar content induces or suppresses expression of different genes (Koch, 1996) and the effects differ completely depending on whether leaves are in complete darkness or in the light (Fujiki et al., 2001).
In sucrose-uncoupling mutants, in which the expression of plastocyanin is not repressed by sucrose, various phytochrome-mediated phenomena become insensitive to sucrose (Dijkwel et al., 1997). It is also noteworthy that a mutant of A. thaliana that lacks the chloroplast inner envelope triose phosphate/phosphate translocator is defective in photosynthetic acclimation to the light environment (Walters et al., 2003). These results infer a close relationship between the sugar-sensing system or sugar metabolism and the photosensing system.
Redox control. The redox state of electron transport components between photosystems I and II reflects a balance between input and processing of light energy (Fujita, 1997). The balance can be assessed fluorometrically as the redox state of plastoquinone pool and is referred to as excitation pressure (Huner, 1998). In high light or other conditions that saturate or suppress the velocity of the Calvin–Benson cycle, the electron transport system is reduced. On the other hand, when the photosynthetic capacity is much greater than the incoming light, the electron transport system tends to be oxidized. When the leaves of Triticum aestivum and Secale cereale were developed under moderate light at a low temperature or under high light at an ordinary temperature, the maximum photosynthetic rates on a leaf area basis measured under the optimal condition increased (Gray et al., 1997). Chloroplasts in the plants developed under high excitation pressure are of the sun type.
The molecular identity of excitation pressure has been studied intensively. The redox state of plastoquinone itself appears to suppress Lhcb gene expression via phosphorelay in algae; while in higher plants, it is suppressed by reduced thioredoxin or glutathione. In the regulation of other genes, the phosphorelay, thioredoxin, glutathione and reactive oxygen species are involved (Hihara and Sonoike, 2001; Rodermel, 2001; Surpin et al., 2002; Muramatsu and Hihara, 2003). Walters et al. (1999) observed that A. thaliana defect in the y gene is not capable of light acclimation and proposed a central role of the COP/DET/FUS regulatory cluster, a focus for multiple signal transduction pathways including the redox signal.
Nitrogen abundance and cytokinin. Nitrogen availability affects the number or the total volume of chloroplasts in the leaf rather than their quality (Evans and Terashima, 1988; Terashima and Evans, 1988). Therefore, when the amounts of the photosynthetic components are expressed on a Chl basis, these values are not affected by nitrogen availability very much. However, when nitrogen is extremely limited, the quality of the chloroplast is also affected. For example, as predicted by the optimization model for nitrogen allocation (Hikosaka and Terashima, 1995), the Chl a/b ratio increases when the availability of nitrogen is extremely low (Kitajima and Hogan, 2003). On the other hand, when the nitrogen is abundant, the leaves tend to accumulate more Rubisco and thereby Rubisco/Chl increases (Terashima and Evans, 1988; Makino et al., 1994, 1997; Eichelmann and Laisk, 1999).
The abundance of nitrogen in the environment may be sensed as the concentration of nitrate or ammonium near the root surface. Nitrate also acts as a signal to induce the expression of enzymes involved in nitrogen assimilation (Crawford, 1995).
Cytokinin also plays a role as a signal. When maize seedlings are grown without exogenous nutrients, the leaves are metabolically more C3-like and their main carboxylating enzyme is Rubisco (Sugiharto et al., 1990). Addition of nitrate or ammonium to the intact nitrogen-deficient maize plants leads to rapid and marked increases in the Ppc transcript and in phosophoenolpyruvate carboxylase (PEPCase) activity. When nitrogen-deficient maize shoots without roots were fed with nitrate, PEPCase synthesis was not enhanced. Thus, the induction of PEPCase is not due to nitrate signal. Recent studies showed that cytokinin synthesized in roots in response to nitrate or ammonium is responsible for enhanced PEPCase synthesis (Takei et al., 2002). The concentration of cytokinin increases on application of nitrate or ammonium to the root. Perhaps, such regulation would occur in C3 plants as well. Light-harvesting complex II that are not essential to photosynthetic energy transduction may not be synthesized under the very limited nitrogen availability, which may result in a high Chl a/b ratio.
Appreciating importance of cytokinin from the root, Pons and Bergkotte (1996) hypothesized that the gradient in PPFD within a plant can be sensed by the different rates of transpiration in the leaves. Leaves in sunny microhabitats transpire more than those in shade microhabitats. To prove this hypothesis, they conducted an interesting experiment with growing and mature primary leaves of Phaseolus vulgaris. They enclosed one of the pair leaves and regulated the vapor pressure deficit (VPD) in the chamber. When they decreased the VPD in the chamber to suppress transpiration during leaf expansion, the leaves showed more shade-type characteristics than the leaves exposed to greater VPD. They argued that the cytokinin synthesized in the root system is preferentially transported by the transpiration stream to the leaves under high VPD and affects the nitrogen metabolism of these leaves. However, leaf expansion tends to be suppressed under dry conditions. Then, it is probable that the photosynthetic rate per leaf area increases with air dryness.
In nature, leaves exposed to the sun would be sun leaves irrespective of humidity. When the hypothesis of Pons and Bergotte (1996) for the differentiation of sun and shade leaves is followed, the cytokinin concentration in the xylem sap should increase in humid air. This possibility should be tested.
Acclimation of chloroplast properties within a leaf. In the leaf, the gradient of chloroplast properties across the leaf is formed due to acclimation of chloroplasts to the local light environment (Terashima and Inoue, 1985a, b; Yano and Terashima, 2001). The acclimation itself is not usually affected by nutrient status. The effects of local light environment were shown in the experiments in which the leaves were illuminated from the abaxial side. The inversion of a spinach leaf after full expansion induces complete inversion of the Chl a/b ratio and the partial inversion of the rate of electron transport activity (Terashima and Inoue, 1985b). The illumination of expanding leaves from the abaxial side leads to complete inversion of thylakoid morphology (Terashima et al., 1986). Analyses of the photosynthetic system in pendulous leaves such as those of Eucalyptus were also made. The results clearly showed that the Chl a/b ratio was highest on both sides and lowest in the middle (James et al., 1999). It is highly likely that the redox control is responsible for the gradient formation.
Factors regulating leaf senescence
In upright herbaceous plants, new leaves gradually shade old leaves. Senescence of the leaves is thus accompanied by shading by the upper leaves. Senescence processes may be superimposed by re-acclimation of the former sun leaves to the shade. When the whole plant is shaded, all the leaves retain their photosynthetic activity longer (Hidema et al., 1991). On the other hand, when particular leaves of the plants are shaded, such parts and leaves senesce faster (Weaver and Amasino, 2001). When all the leaves of the plant are under the same light conditions (this situation is possible in vine plants), the oldest leaves senesce fastest. Therefore, senescence patterns vary considerably. Thus, there is a need to dissect these complex experimental/natural conditions into several cases.
Phytochrome. In upright plants, young leaves shade old leaves, thereby the ratio of red light to far-red light (R/FR) received by the old leaves decreases. Phytochrome appears to be involved in changes in chloroplast properties in senescing leaves. Okada et al. (1992) and Okada and Katoh (1998) found that chlorophyll degraded rapidly in the dark in detached and attached Oryza sativa leaves, respectively. Chlorophyll degradation was reversibly suppressed by short exposure of the leaves to the weak red light. The effect of red light was suppressed by far-red light. An experiment with Helianthus annuus plants demonstrated that artificial far-red enrichment of the light penetrating through the canopy enhanced senescence of the lower leaves (Rousseaux et al., 1996). The results of these studies clearly indicate that the difference in the R/FR ratio received by leaves is involved in regulating leaf senescence (for review, see Anten et al., 2000; Smith, 2000).
Plant nitrogen status and cytokinin. When nitrogen nutrition is deficient, senescence is accelerated. The leaf may sense a decrease in nitrate and/or cytokinin levels. Effects of a decreased cytokinin level in the senescing process have been studied using transgenic tobacco plants expressing the isopentenyl transferase gene, a key enzyme of cytokinin synthesis, under the control of a highly senescence-specific promoter (SAG 12). Jordi et al. (2000) showed that the chlorophyll content was maintained in old senescing leaves in transgenic plants. However, an enhanced cytokinin level was less effective in maintaining Rubisco and Pmax. Using the same transgenic tobacco plants, Wingler et al. (1998) showed that cytokinin production decelerated the decrease in Rubisco content to some extent and that accumulation of sugars blocked the effects of cytokinin. These results indicate that enhanced production of cytokinin in the senescent leaves causes some deceleration of that senescence. Various factors regulate cytokinin production in the leaf (Jordi et al., 2000). Obviously, the cytokinin level decreases in the presence of sugar.
Cytokinin in the transpiration stream is involved in gene expression for nitrogen metabolism (Sakakibara et al., 2000; Takei et al., 2002) and the decrease in cytokinin input with the decrease in transpiration rate by shading could partly explain leaf senescence as Pons and Bergkotte (1996) and Pons et al. (2001) argued.
Sugar repression. High sugar concentration represses expression of genes for photosynthetic components (Sheen, 1990; Krapp and Stitt, 1995; Koch, 1996; Koch et al., 2000; Pego et al., 2000), which leads to the acceleration of senescence and re-translocation of nitrogenous compounds (Dai et al., 1999). Inversely, the low sugar concentration may be required for maintaining the high photosynthetic rate of the source leaf or for retardation of leaf senescence (Ono and Watanabe, 1997; Ono et al., 2001). There are some studies indicating interaction between photosensory and sugar-sensing pathways as mentioned above. Also, retardation of leaf senescence by cytokinin was cancelled by high sugar concentrations (Wingler et al., 1998). However, suppression of gene expression for photosynthetic components by sugar is strictly dependent on the developmental stage of the leaf. When leaves are actively expanding, sugar does not suppress an increase in photosynthetic activity (T. Araya, K. Noguchi and I. Terashima, unpubl. res.).
If a leaf can sense its light environment or photosynthetic status relative to those of other leaves within the plant and regulate the photosynthetic capacity accordingly, the nitrogen distribution within a plant would always change towards the optimal conditions (Ono et al., 2001). It is probable that a leaf senses its photosynthetic status by monitoring its sugar concentration. When the demand for photosynthates produced by the leaf under consideration is large, sugar concentration of this leaf would be low because of vigorous translocation to sink organs. On the other hand, when the demand for photosynthates produced by the leaf is small, its sugar concentration will increase.
Nitrogen deficiency suppresses plant growth, and thereby the sink activity of developing leaves is lowered. Then, translocation of carbon from the source leaves is also suppressed, this would lead to accumulation of carbohydrate under nitrogen deficiency (Radin and Eidenback, 1986; Paul and Stitt, 1993; Ono et al., 1996). Thus, by monitoring the abundance of non-structural carbon, nitrogen deficiency could be monitored indirectly. Perhaps, in addition to more direct nitrogen sensing systems, the leaves also sense the nitrogen status of a plant indirectly by monitoring its sugar concentration (Paul and Driscoll, 1997).
Redox control. When a leaf is shaded, the excitation pressure is lowered. This can be a signal for re-acclimation to shaded conditions (Huner et al., 1998). However, this possibility has not been tested.
Effects of partial and general shading. When the whole plants were generally shaded, senescence of all leaves was decelerated (Hidema et al., 1991). This could be explained as follows (Hikosaka, 1996, 2005). The amount of nitrogen needed for the construction of young organs will decrease due to suppression of growth. Moreover, photosynthetic production of the leaves decreases. Thus, nitrogen demand would not be large and sugar would not accumulate in all the leaves.
On the other hand, when particular leaves are shaded, they senesce faster (Weaver and Amasino, 2001). Because shading with a black cloth accelerated leaf senescence, the roles of phytochrome are not essential. Effects of such selective shading have not been assessed in detail. It is not known whether such shading increases sugar concentration. Obviously, excitation pressure decreases on shading, in particular, shade enriched with far-red light. Then, this can cue the senescence or adjustment of photosynthetic capacity to the low light conditions. Transpiration is also suppressed in shaded leaves. Thus, delivery of cytokinin also decreases.
Senescence in horizontally grown vine plants. When an Ipomoea tricolor plant was grown horizontally, all the leaves received the same irradiance. When the nitrogen nutrition was limited, nL of the oldest leaves started to decrease (Hikosaka et al., 1994). This apparently indicates the importance of actual age. In this study, however, all the lateral buds were removed to simplify the system, and only the terminal bud of the main stem was allowed to produce new leaves. Because it is known that sinks attract photosynthates from nearby leaves and young sink leaves are nearer to the apex of the main shoot, the photosynthates produced by these young leaves would be preferentially used for constructing new organs, which might keep their sugar concentration low. Then, the distance, or cost of re-translocation of carbon and nitrogen could be important. Plant hormone(s) could be involved in such regulation.
Answers to all your Biology Questions
