Abstract: The development of chloroplasts in the mesophyll cells of control and urea-applied satsuma mandarin leaves were studied. At 20 days after budding (DAB) active differentiation of chloroplasts in urea-applied trees was evident. However, on a length times width basis, chloroplasts were small in both palisade (1.99 × 0.70 μm2) and spongy (2.78 × 1.50 μm2) layers that contained few small (0.53 - 0.72 × 0.39 - 0.57 μm2) starch grains. The thylakoid layers per chloroplast were lesser (2.80) in this treatment compared to control (3.72). At 40 DAB, both control and urea-applied trees reached chloroplast maturity, but control trees had bigger chloroplast (4.87 × 2.39 μm2) in the palisade layer cells which contained extremely large and numerous starch grains with few dilated thylakoid layers. Degenerating chloroplasts in the spongy layer cells of control trees were characterized by the disappearance of the outer membrane. While urea-applied trees had smaller chloroplast (4.56 × 2.39 μm2) in the palisade layer. Further, few small starch grains, thylakoid-filled stroma and many lipid droplets were found. Moreover, thylakoid layers were about to dilate but outer membrane of the chloroplasts remained intact in both palisade and spongy layer cells. Compared to the palisade layer cells, chloroplasts in the spongy were bigger (5.32 x 1.95 μm2).
Introduction
Plastids are probably the most metamorphic and ubiquitous DNA-containing organelles present in plant cells. These organelles carry out numerous metabolic functions including photosynthesis, starch synthesis and steps in lipid, terpenoid, amino acid, tetrapyrrole and plant hormone biosynthesis (Mullet, 1993; Harrak et al., 1995). The various important functions of plastids complimented the need for meticulous investigations of these organelles.
Generally, the development of leaves is derived from meristematic cells of shoots that contain proplastids. Subsequently, as these meristematic cells develop into leaf cells, proplastids differentiate into chloroplasts. As a result, the number of chloroplasts increases dramatically during mesophyll cell expansion producing large population of chloroplasts in each mature mesophyll cells (Pyke and Leech, 1992) or simply, chloroplasts develop from proplastids through a process that involves an increase in volume and membrane expansion (Reiter et al., 1994).
Although chloroplast differentiation appears to start very early during plant development (Leon et al., 1998), the stages of chloroplast differentiation at various ages of plants or leaf tissues in a variety of species are not yet well established. It can also be asserted that the chloroplast development could be dependent on plant species, environmental factors and different growing conditions.
In some monocot species, a more or less thorough investigation on chloroplast development has been done particularly in C4 plants where further chloroplast differentiation concurrent with mesophyll differentiation in the bundle sheath has been reported (Nelson and Langdale, 1989). But in citrus, information regarding chloroplast development is very limited particularly with nitrogen foliar- applied trees.
The verification of chloroplast development in a particular growth stage of citrus will provide us a more comprehensive information on the reaction mechanisms between nitrogen and chloroplast and the subsequent development of other organelles in the leaf tissues.
The present study was conducted in an effort to describe the developmental stages of chloroplast in the mesophyll cells of satsuma mandarin leaves foliar sprayed with urea at 20 and 40 days after budding.
Materials and Methods
The experiment was conducted in the Citriculture Laboratory, Faculty of Agriculture, Ehime University during spring 1998. Three-year-old satsuma mandarin (Citrus unshiu Marc. cv. Okitsu Wase) trees of similar vigor were planted in potted sandy soil mixed with granite and maintained in plastic house conditions. Light intensity of approximately 60,000 lux and temperature range of 23-26°C were maintained in the plastic house. Heavy pruning was done by removing the old shoots and leaves on April 14, 1998. Spring flushes were allowed to grow for about 3-5 cm before spraying. Three sprayings of urea (Wako Pure Chemicals Industries Ltd., Osaka, Japan) at 2,000 ppm were applied to two sets of 3 potted trees 11 set for 20 DAB and another set for 40 DAB at 33 ml/tree in the morning of April 28, 30 and May 2. The same number of trees were sprayed with tapwater which served as the control. Pots were covered with polyethylene bags to prevent entry of spray solution into the soil. Leaf sampling for 20 and 40 days after budding were done on May 3 and 23, 1 and 20 days after the last spray application, respectively.
Ecological data such as leaf length, width, mesophyll thickness and leaf color were recorded. Moreover, chlorophyll content determined by MINOLTA-SPAD 501 reading values and nitrogen content by Kjeldahl analysis were also included. Prior to nitrogen analysis, samples for electron microscopy were prepared by cutting the central portion of the leaf blades into small pieces (1 mm x 2 mm) and were fixed in paraformaldehyde-glutaraldehyde for 24 hrs at 10°C, postfixed in 1 percent osmiun tetroxide for 2 hrs at room temperature, dehydrated in a graded alcohol series and embedded in epoxy resin. Ultrathin sections were cut with diamond knife using the ultramicrotome (UT-1000) and double stained with uranyl acetate and lead citrate. Sections were examined and photographed under HITACHI H-7100 transmission electron microscope at 100 kV.
A total of 130 photomicrographs (x10,000) were considered for investigations on the different chloroplast features. Chloroplast and starch grain areas were traced from approximately 25 photomicrographs and were measured using leaf area meter MK2. Cell, chloroplast and starch grain sizes were measured on a length (L) and width (W) basis and the number of starch grain and thylakoid layer per chloroplast were directly counted from the photomicrographs. Means of these parameters were analyzed using the Duncan's Multiple Range Test (DMRT).
Results
Comparatively, urea-applied trees had bigger leaves and thicker mesophyll, higher chlorophyll and nitrogen contents than the control (Table 1).
| Table 1: | Ecological data of control and urea-applied satsuma mandarin leaves 20 and 40 days after budding. |
In undifferentiated mesophyll cells 20 days after budding (20 DAB), the first 3 layers of palisade and spongy cells in both urea and control trees had remarkable differences in cell size, chloroplast area and size, starch grain area and size, number of starch grains and thylakoid layers per chloroplast (Tables 2a and 2b). Apparently, urea applied trees had bigger cell size than the control (Table 2b). The chloroplast area as well as the size, the starch grain area and the number of starch grains per chloroplast were greater in palisade layer 3 and spongy layers 1-2 but smallest in palisade layer 1 of control trees (Table 2a). The starch grain size was bigger in palisade layer 3 and spongy layers 1-3 than in the palisade layers 1 and 2, while the number of thylakoid layer per chloroplast was more in the spongy layers 1-3. At this particular phase, 20 DAB, different stages of chloroplast were observed, however, each mesophyll layer was dominated by a certain chloroplast stage. The chloroplast developmental stages in palisade layers 1-3 were generally featured with the formation of lamellar system (Figs. 1 A, C and E). Spongy layers 1-3 had similar plastid developmental stages where formation of stroma and grana lamellae were at an advanced phases and were almost at the full grown stage of chloroplast (Figs. 1 G, I and K).
Table 2b represents the urea-applied trees where chloroplast area and size, starch grain area, size and number per chloroplast and number of thylakoid layer per chloroplast were bigger and numerous in spongy layers 1-3, and least in palisade layers 1 and 2. Bigger cell size and more thylakoid layer per chloroplast in the mesophyll cells were observed in this treatment compared to the control trees. Majority of the chloroplasts in palisade layers 1 and 2 were at the developing stage of forming internal lamellar system (Figs. 1 B and D). The palisade layer 3 cells had chloroplasts mostly in the stage of forming stroma lamellae (Fig. 1 F). Spongy layer 1 had chloroplasts generally forming grana (Fig. 1 H) and the spongy layers 2 and 3 mostly contained chloroplasts forming both grana and stroma lamellae (Figs. 1 J and L).
At 40 DAB, mesophyll cells of both urea and control plants have a clearly differentiated palisade and spongy layer cells which were bigger than those of 20 DAB. Tables 3a and 3b illustrate that the cell size in the mesophyll of urea-applied and control trees at 40 DAB did not greatly differ but, within the 2 palisade and 3 spongy layer cells, differences were observed. In terms of length, the palisade layer 1 cells were the longest followed by palisade layer 2 and in terms of width, spongy layer 3 cells were the widest. The chloroplast area in the spongy layer 3 cells and the chloroplast size in palisade layers 1 and 2 were bigger in control trees (Table 3a) than in urea-applied trees (Table 3b). The biggest starch grain area and the largest starch grain were observed in the chloroplasts located in the palisade layers 1 and 2 respectively, in control trees (Table 3a). Numerous starch grains were also found in the chloroplasts of palisade layer 1 cells. The number of thylakoid layer per chloroplast was many in the spongy layer 3 cells where the cell size, chloroplast area, chloroplast size and the number of starch grain per chloroplast were least.
| Table 2a: | Cell and chloroplast structures in satsuma mandarin leaves 20 days after budding. |
| *Means in columns are separated by Duncan's Multiple Range Test at 5% level. 1 : length; 2: width |
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| Table 2b: | Cell and chloroplast structures in urea-applied satsuma mandarin leaves 20 days after budding. |
| *Means in columns are separated by Duncan's Multiple Range Test at 5% level 1 : length; 2: width; -: no starch grain |
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| Table 3a: | Cell and chloroplast structures in satsuma mandarin leaves 40 days after budding. |
| *Means in columns are separated by Duncan's Multiple Range Test at 5% levell 1 : length; 2: width |
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| Table 3b: | Cell and chloroplast structures in urea-applied satsuma mandarin leaves 40 days after budding. |
| *Means in columns are separated by Duncan's Multiple Range Test at 5% levell 1 : length; 2: width |
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| Fig. 1: | Electron micrographs of chloroplast in the mesophyll cells of control and urea-applied satsuma mandarin leaves 20 days after budding, A, C, E: control - palisade layers 1, 2 and 3; B, D, F: urea-applied-palisade layers 1, 2 and 3; G, l, K: control-spongy layers 1, 2 and 3; H, J, L: urea-applied - spongy layers 1, 2 and 3. Chloroplasts of urea-applied trees were smaller and at early stages of development compared to the control. Bar 0.5 μm. |
| Fig. 2: | Electron micrographs of chloroplast development in the mesophyll cells of control and urea-applied satsuma mandarin leaves 40 days after budding. A: Control - palisade layer; B: urea-applied - palisade layer; C: control spongy layer; D urea-applied - spongy layer. The outer membrane of chloroplasts in control start to disappear. Bar = 1 μm. |
Chloroplasts at this stage (40 DAB) have already reached advanced maturity phase for both control and urea-applied trees. Chloroplasts found in palisade layers 1 and 2 were similar in structure and so with the chloroplasts in the spongy layer cells. In control trees, palisade layers 1 and 2 were indicated with chloroplasts that were irregularly globular with extremely large and numerous starch grains and with few dilated thylakoid layers (Fig. 2 A). Spongy layers 1-3 were identified with smaller chloroplasts that were ellipsoidal, containing less starch grains but more thylakoid layers than the choloroplasts in the palisade layer accompanied with a sign of degenerating chloroplast characterized by the disappearance of the outer membrane (Fig. 2 C).
Table 3b shows the starch grain area and chloroplast size of urea-applied trees that were almost similar in any mesophyll layer cells. Fewer starch grains were contained in the chloroplasts of spongy layer cells than in the palisade. The number of thylakoid layer was numerous chloroplasts located in the spongy layer 3 cells where the chloroplast area was biggest and starch grain was srnalle Chloroplasts in this group were ellipsoidal, have more li droplets and thylakoid-filled strorna. Although thylakoid observed were about to dilate, the outer membrane was intact in both palisade and spongy layer cells. Chloroplast in the spongy cells were bigger compared to those palisade (Figs. 2 B and D).
Discussion
Present study indicated that the nitrogen content in the leaves of urea-applied trees was higher than in control However, the accumulation of nitrogen in the leaves decreased to about 16 percent at 40 days after budding. This phenomenon has been revealed in the studies Embleton et al. (1973) and Feigenbaum et al. (1987), indicative. that the nitrogen concentration of citrus leaves tends to decline with age. Although both control and urea-applied trees had similar mesophyll differentiation at 20 and 40 DAB, differences in terms of nitrogen and chlorophyll contents and subsequently, the chloroplast ultrastructures were observed in both treatments.
The bigger cell size exhibited by urea-applied trees could be considered a positive indication of nitrogen effect on the growth of leaves at an early stage. The different developmental stages of chloroplast observed in the mesophyll cells of both control and urea-applied trees at 20 DAB is considered to be a consequential event related to the early stage of leaf development where differentiation of plastids occur. An interesting feature of smaller chloroplasts observed in the urea-applied trees is not to be regarded as being due to slow or non-growing chloroplasts but rather can be viewed as developing plastids which have resulted from an active division of preexisting chloroplasts triggered by the application of nitrogen (Possingham, 1980). Whereas, the control trees, which had bigger chloroplasts, can be considered as the same chloroplasts that preexisted in the cell and continued to develop but were not actively dividing unlike those of urea-applied trees. Thomson and Whatley (1980) however, reported that during the early stages of cellular differentiation, differences in plastid size apparently become established as a result of differential expansion of the plastids prior to division and as some cells become fully differentiated, plastids undergo dedifferentiation to an eoplast state similar to some of the chloroplasts found in urea-applied trees, which were usually small (Figs. 1 B, D and F). Some of these small chloroplasts were dumbbell-shaped suggestive that these were in the early stage of plastid division (Pyke and Page, 1998).
Based from the dominating chloroplast ultrastructure per mesophyll layer cells observed, palisade and spongy layer cells in control trees had chloroplasts which were at advanced stages of development compared to the urea-applied trees. This phenomenon could have been the result of an active division and redifferentiation of plastids in urea-applied trees. Consequently, the formation of these early developmental stages of chloroplast could have been the result from prolonged juvenile stages of chloroplast that occurred in urea-applied trees.
The cell sizes of both control and urea-applied plants at 40 DAB were almost similar. This similarity could be attributed to their capacity to have reached the full size or maturity stage (Figs. 2 C and D). However, the chloroplasts with smaller and lesser starch grain and more thylakoid layers in urea-applied trees need to be considered in elaborating its differences with the control trees.
The accumulation of extremely large and numerous starch grains in the chloroplasts of control trees in both palisade and spongy layers is indicative of non-active chloroplasts. Negative starch degradation process due to the absence of necessary degradative enzymes caused by nitrogen deficiency is assumed in these non-active chloroplasts. Similar studies in different plant species have been observed in this regard (Thomson and Weier, 1962; Whatley, 1971; Chonan et al., 1977), reflecting large starch grains in the chloroplasts of nitrogen deficient plants. In the present study, it seems that the size of chloroplast is directly affected by the continuous accumulation of starch grains. As these starch grains increase in size and become enlarged, the walls of the chloroplasts are being pushed in order to accommodate the starch grains without breaking the seemingly elastic chloroplast membrane. Degradative enzymes for starch grains in these chloroplasts are suspected to be lacking due to the low nitrogen content. Moreover, starch accumulation decreases photosynthetic activities in plants (Makino and Osmond, 1991; Nii et al., 1993, Wykoff et al., 1998) and may disrupt chloroplast organizations (Carmi and Shomer, 1979). Thus, as reflected in the present results, the accumulation of starch was accompanied by the destruction and disorientation of grana and thylakoids (Figs. 2 A and C).
An appealing observation of outer membrane disappearance in the spongy chloroplast of control trees implies a degenerating chloroplast (Fig. 2C). This could be attributed to nitrogen deficiency resulting in a premature senescence of leaves (Possingham, 1980). This chloroplast stage has not been observed in urea-applied trees. Although chloroplasts in this group were smaller compared to palisade chloroplast of control trees, a well-developed chloroplast ultrastructures were evident, which contained less and smaller starch grains, lamellae-filled stroma and more lipid droplets (Figs. 2 B and D). A similar observation was noted by Kirchanski (1975) on nutrient-applied, Zea mays, where mature chloroplast ultrastructure possessed numerous lipid droplets.
Collectively, present results indicated that the developmental stages of chloroplasts in control and urea-applied satsuma mandarin leaves collected at 20 and 40 days after budding (DAB) differed. A more active differentiation of chloroplasts in urea-applied trees were evident at 20 DAB thus, chloroplasts in this treatment were smaller with few and small starch grains and with lesser thylakoid layers per chloroplast as compared to the control. Chloroplast maturity was attained in both control and urea-applied trees at 40 DAB. Although similar developmental stages of chloroplast were observed in the mesophyll tissues for both treatments, control had extremely large and numerous starch grains with few dilated thylakoids. Chloroplasts found in the spongy layer cells were degenerating characterized by the disappearance of outer membrane which could be considered a sign of senescence. Urea-applied trees had smaller chloroplasts in palisade layer cells with few and small starch grains, thylakoid-filled stroma and contained many lipid droplets. Chloroplasts in this treatment had thylakoid layers that were almost to dilate but the outer membrane remained intact. Therefore, urea application prevented an early degeneration of chloroplasts at 40 DAB, where clear differentiation and fully developed mesophyll cells were reached.