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Intracisternal granules in the endosperm cells of the barley grain
258
J. ULTRASTRUCTURERESEARCH4, 258--263 (1960)
Intracisternal Granules in the Endosperm Cells of the Barley Grain M. S. BUTTROSE,1 A. FREY-WYSSLINGand K. MUHLETHALER
Laboratory for Electron Microscopy, Department of General Botany, Federal Institute of Technology, Ziirich, Switzerland Received May 25, 1960 Endosperm cells of barley grown under conditions of constant, low intensity light have dense homogeneous granules of 0.5-0.8/z diameter occurring inside the vesicular elements of the endoplasmic reticulum. Similar granules have not been observed in material obtained from plants grown under natural conditions. The granules resemble on the one hand intracisternal granules of pancreas exocrine cells identified to be of zymogen nature, and on the other hand fat droplets. It is noteworthy that these bodies appear in the vesicles of the endoplasmic reticulum and not in the cytoplasm. This observation may help in understanding the functions of the endoplasmic reticulum. While studying the submicroscopic development of starch in the endosperm cells of barley (2), it was found that in material taken from plants grown under low intensity light the elements of the endoplasmic reticulum contained spherical bodies. In the case of animal tissues granular inclusions of the elements of the endoplasmic reticulum were first described by Palade (4) and studied by Siekevitz and Palade (5) working with pancreas exocrine cells. Similar granular inclusions have since been observed in such tissues as cancerous human mammary gland (3) and in the vitelline body of the spider oocyte (1). In the case of higher plants, however, no report has been found of particulate inclusions in the elements of the endoplasmic reticulum, and the present paper describes the findings made with barley endosperm material.
MATERIALS AND METHODS Plants of barley (Hordeum distichum var. Prior) were grown in compost accommodated in a constant environment cabinet. Lighting was supplied by HPL mercury lamps (ultraviolet light absent), and light intensity in the vicinity of the plants was 400 foot-candles. Illumination was continuous from planting to maturity. Temperature was constant within the range 20°-22°C. Humidity was not controlled. Kernels were removed from ears at various stages of development from flowering to maturity, small blocks of endosperm tissue removed from the center of the "cheeks," approximately 1 Permanent address: Waite Agricultural Research Institute, Adelaide, South Australia.
INTRAC1STERNALGRANULESIN BARLEYGRAIN ENDOSPERM
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midway between base and apex of the kernel. They were fixed for 2 hours in 1% OsO4 in acetate-veronal buffer (pH 7.2) at 2°C, washed, dehydrated, embedded in methacrylate or Araldite, and sectioned for electron microscopy according to the usual procedures. As a control, material was taken from plants grown in the open garden under natural conditions. OBSERVATIONS In cells from endosperms harvested up to 2 weeks after flowering, the endoplasmic reticulum appeared as cisternal, tubular or vesicular elements with a homogeneous low-density content. The single membranes had typically associated with them, towards the cytoplasm, dense RNA granules or ribosomes. Such granules were not found to be associated with any other membrane system in the cell. In material taken between two and three weeks after flowering the elements of the endoplasmic reticulum appeared mainly as circular or irregularly elongated vesicles, and many of them contained relatively large discrete bodies embedded in the normal low density matter. In Fig. 1 is presented a low magnification micrograph to show the disposition of these elements. The cytoplasm of such older cells contains predominantly starch granules, vesicular elements of the endoplasmic reticulum, occasional mitochondria, and otherwise is relatively "empty." The membranes originally surrounding the starch granules have been disorganized during preparation, a common occurrence with such older tissue. The walls of the vesicles present commonly appear irregular or amoeboid in outline, which is in contrast to their usual appearance in other tissues. Few ribosomes are present free in the cytoplasm, but regularly appear in association with the membranes of the vesicles. As this is also so in the case of vesicles containing dense bodies, it is felt that these bodies may be denoted as intracisternal granules, the name used by Palade (4) for similar bodies in pancreas exocrine cells. The intracisternal granules appearing in Fig. 1 are round to oval in outline, and this constancy of outline in section indicates that they have an approximately spherical shape with a diameter of 0.5-0.8 ,~z. They have a relatively high density, and their content is homogeneous. Many have a darker layer of about 100 A thickness at the periphery, but no evidence of a bounding membrane has been found. Siekevitz and Palade (5) observed a darker peripheral shell in intracisternal granules, but likewise concluded that no membrane was present. Variations in sharpness of the boundary can be accounted for on the basis of obliqueness of sectioning. Granules usually lie free in the low density matter of the vesicles, and only in some cases is one in contact with the vesicle membrane. Normally one granule occurs in a vesicle profile, but up to five have been observed, and as shown in Fig. 1, two are quite commonly present together. Very often where two or more granules are included in one vesicle they are in close contact. In Fig. 2 they have the appearance of droplets that have flowed together.
260
M. S. BUTTROSE, A. FREY-WYSSLING AND K. MUHLETHALER
FIG. 1. Portion of barley endosperm cell showing dense bodies in the endoplasmic reticulum (ER). Plant from growth cabinet. S - starch, x 20,000. FIG. 2. Occasionally two or more intracisternal granules may aplzear in union, x 27,000. FIG. 3. The cytoplasm is reduced to a layer of ribosomes covering the E R membranes, x 37,000.
INTRACISTERNAL GRANULES IN BARLEY GRAIN ENDOSPERM
261
FIG. 4. C o n t r o l o u t d o o r material. E R vesicles f r o m barley e n d o s p e r m . × 50,000. FIG. 5. C o n t r o l o u t d o o r material. Starch granules enclosed by a s m o o t h plastid m e m b r a n e , x 45,000.
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M. S. BUTTROSE, A. FREY-WYSSLING AND K. MUHLETHALER
It should be noted that by no means all the elements of the endoplasmic reticulum contain granules (Fig. 1), and the vesicles which do are larger than normal. They attain the relatively large size of 1 # diameter, while granule-free vesicles lie mostly in the range 0.2-0.4/z, which is normal for such vesicles in this tissue. Vesicles containing granules may therefore be distinguished from others, apart from granule content, by their larger size and amoeboid outline. That the two forms occur freely intermixed suggests that originally they were identical, and that differences have resulted following the formation of granules. In Fig. 3 is illustrated the extent to which cytoplasm may be reduced in various areas of the cell. The cytoplasm is represented in section by narrow canals completely surrounding each endoplasmic reticulum vesicle, many of the latter containing intracisternal granules. Similar formations were found from time to time in various parts of the cell. Ribosomes present appear both in association with the membranes and free in the cytoplasm. As their density in respect of numbers is similar to that found in other parts of the cell (Figs. 1 and 2), it would appear unlikely that the narrow cisternae of cytoplasm arose by contraction of a much more extensive volume during tissue preparation, but that the observed formation reflects the condition in the living cell. The condition in control material, taken from outdoor plants, is shown in Figs. 4 and 5. Numerous vesicles of the endoplasmic reticulum may be found, with associated ribosomes and a low density homogeneous content. No such vesicles have been observed to contain granules. In Fig. 5 are presented examples of the extremely numerous small starch granules present in this normal tissue. As is usual, the starch granules appear in the electron microscope to have little electron scattering power, and are readily distinguished from the intracisternal granules. It is seen that they are surrounded by a double membrane without associated ribosomes. A comparison of control and growth cabinet material showed that the number of small starch granules was enormously reduced in the latter.
DISCUSSION The question arises as to the nature of the substance which can be accumulated as dense bodies in the endoplasmic reticulum. According to the osmic acid staining properties of the intracisternal granules they could be either proteinaceous or lipoid in content. In the light microscope it is easy to distinguish cellular protein inclusions from oil droplets by microchemical reactions. In the present case such an identification was impossible as the granules are submicroscopic or just at the limit of the light microscope resolving power, so that staining does not yield any recognizable coloration.
INTRACISTERNAL GRANULES IN BARLEY GRAIN ENDOSPERM
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Siekevitz and Palade identified intracisternal granules of similar appearance in pancreas exocrine cells to be of zymogen nature (5). They further investigated with carbon-14 how this probable protein material is synthesized by the ribosomes on the outer face of the cisternal membranes, penetrates these membranes and is amassed inside the endoplasmic reticulum (6). The pancreas exocrine has a specialized function of producing and secreting enzymes, while the endosperm has quite a different function. Nevertheless, owing to the similarity of the granules under discussion with those of pancreas exocrine cells in respect of location, order of size, and osmic acid staining properties, it becomes necessary to consider the possibility that they too have a zymogen nature. As endosperm intracisternal granules were not found in tissues of control plants, it would appear that they were abnormalities induced by artificial environment conditions. The most important factor involved was probably lowness of light intensity, as plants showed a pronounced tendency to etiolation. Furthermore, the reduction in number of starch granules could be due to a reduced photosynthesis associated with low light intensity. If nitrogen supply and enzyme synthesis in the endosperm were normal, then it can be understood that with depletion in carbohydrate substrate supply, zymogen material could be in excess of metabolic requirements. In this case such material might be amassed near the site of synthesis in elements of the endoplasmic reticulum, and thus be stored in isolation from the cytoplasm. Alternatively, one must consider the possibility that the material is of lipoid nature. As has been mentioned, the appearance of the intracisternal granules is equally indicative of lipid as it is of protein. It could be suggested that with low intensity, but continuous, light the starch-synthesizing mechanism was to some extent disturbed, resulting in a partial diversion of materials to lipid synthesis. The end result of such a process would be the accumulation of droplets in the endoplasmic reticulum. No evidence in support of either postulate can be presented at this stage. However, it seems worthwhile to emphasize that the endoplasmic reticulum is capable of transforming and storing considerable quantities of material. This gives a further insight into its nature and function. REFERENCES 1. 2. 3. 4. 5.
ANDRe, J. and ROUILLER,C., or. Biophys. Biochem. Cytol. 3, 977 (1957). BUTTROS~,M. S., in preparation. HOLLMANN,K. H., J. Ultrastructure Research 2, 423 (1959). PALADE,G. E., or. Biophys. Biochem. Cytol. 2, 417 (1956). SIEKEVITZ,P. and PALADE,G. E., or. Biophys. Biochem. Cytol. 4, 309 (1958). 6. - ibid. 4, 557 (1958). 18 -- 60173313 J . Ultrastructure t~esearch
J. ULTRASTRUCTURERESEARCH4, 258--263 (1960)
Intracisternal Granules in the Endosperm Cells of the Barley Grain M. S. BUTTROSE,1 A. FREY-WYSSLINGand K. MUHLETHALER
Laboratory for Electron Microscopy, Department of General Botany, Federal Institute of Technology, Ziirich, Switzerland Received May 25, 1960 Endosperm cells of barley grown under conditions of constant, low intensity light have dense homogeneous granules of 0.5-0.8/z diameter occurring inside the vesicular elements of the endoplasmic reticulum. Similar granules have not been observed in material obtained from plants grown under natural conditions. The granules resemble on the one hand intracisternal granules of pancreas exocrine cells identified to be of zymogen nature, and on the other hand fat droplets. It is noteworthy that these bodies appear in the vesicles of the endoplasmic reticulum and not in the cytoplasm. This observation may help in understanding the functions of the endoplasmic reticulum. While studying the submicroscopic development of starch in the endosperm cells of barley (2), it was found that in material taken from plants grown under low intensity light the elements of the endoplasmic reticulum contained spherical bodies. In the case of animal tissues granular inclusions of the elements of the endoplasmic reticulum were first described by Palade (4) and studied by Siekevitz and Palade (5) working with pancreas exocrine cells. Similar granular inclusions have since been observed in such tissues as cancerous human mammary gland (3) and in the vitelline body of the spider oocyte (1). In the case of higher plants, however, no report has been found of particulate inclusions in the elements of the endoplasmic reticulum, and the present paper describes the findings made with barley endosperm material.
MATERIALS AND METHODS Plants of barley (Hordeum distichum var. Prior) were grown in compost accommodated in a constant environment cabinet. Lighting was supplied by HPL mercury lamps (ultraviolet light absent), and light intensity in the vicinity of the plants was 400 foot-candles. Illumination was continuous from planting to maturity. Temperature was constant within the range 20°-22°C. Humidity was not controlled. Kernels were removed from ears at various stages of development from flowering to maturity, small blocks of endosperm tissue removed from the center of the "cheeks," approximately 1 Permanent address: Waite Agricultural Research Institute, Adelaide, South Australia.
INTRAC1STERNALGRANULESIN BARLEYGRAIN ENDOSPERM
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midway between base and apex of the kernel. They were fixed for 2 hours in 1% OsO4 in acetate-veronal buffer (pH 7.2) at 2°C, washed, dehydrated, embedded in methacrylate or Araldite, and sectioned for electron microscopy according to the usual procedures. As a control, material was taken from plants grown in the open garden under natural conditions. OBSERVATIONS In cells from endosperms harvested up to 2 weeks after flowering, the endoplasmic reticulum appeared as cisternal, tubular or vesicular elements with a homogeneous low-density content. The single membranes had typically associated with them, towards the cytoplasm, dense RNA granules or ribosomes. Such granules were not found to be associated with any other membrane system in the cell. In material taken between two and three weeks after flowering the elements of the endoplasmic reticulum appeared mainly as circular or irregularly elongated vesicles, and many of them contained relatively large discrete bodies embedded in the normal low density matter. In Fig. 1 is presented a low magnification micrograph to show the disposition of these elements. The cytoplasm of such older cells contains predominantly starch granules, vesicular elements of the endoplasmic reticulum, occasional mitochondria, and otherwise is relatively "empty." The membranes originally surrounding the starch granules have been disorganized during preparation, a common occurrence with such older tissue. The walls of the vesicles present commonly appear irregular or amoeboid in outline, which is in contrast to their usual appearance in other tissues. Few ribosomes are present free in the cytoplasm, but regularly appear in association with the membranes of the vesicles. As this is also so in the case of vesicles containing dense bodies, it is felt that these bodies may be denoted as intracisternal granules, the name used by Palade (4) for similar bodies in pancreas exocrine cells. The intracisternal granules appearing in Fig. 1 are round to oval in outline, and this constancy of outline in section indicates that they have an approximately spherical shape with a diameter of 0.5-0.8 ,~z. They have a relatively high density, and their content is homogeneous. Many have a darker layer of about 100 A thickness at the periphery, but no evidence of a bounding membrane has been found. Siekevitz and Palade (5) observed a darker peripheral shell in intracisternal granules, but likewise concluded that no membrane was present. Variations in sharpness of the boundary can be accounted for on the basis of obliqueness of sectioning. Granules usually lie free in the low density matter of the vesicles, and only in some cases is one in contact with the vesicle membrane. Normally one granule occurs in a vesicle profile, but up to five have been observed, and as shown in Fig. 1, two are quite commonly present together. Very often where two or more granules are included in one vesicle they are in close contact. In Fig. 2 they have the appearance of droplets that have flowed together.
260
M. S. BUTTROSE, A. FREY-WYSSLING AND K. MUHLETHALER
FIG. 1. Portion of barley endosperm cell showing dense bodies in the endoplasmic reticulum (ER). Plant from growth cabinet. S - starch, x 20,000. FIG. 2. Occasionally two or more intracisternal granules may aplzear in union, x 27,000. FIG. 3. The cytoplasm is reduced to a layer of ribosomes covering the E R membranes, x 37,000.
INTRACISTERNAL GRANULES IN BARLEY GRAIN ENDOSPERM
261
FIG. 4. C o n t r o l o u t d o o r material. E R vesicles f r o m barley e n d o s p e r m . × 50,000. FIG. 5. C o n t r o l o u t d o o r material. Starch granules enclosed by a s m o o t h plastid m e m b r a n e , x 45,000.
262
M. S. BUTTROSE, A. FREY-WYSSLING AND K. MUHLETHALER
It should be noted that by no means all the elements of the endoplasmic reticulum contain granules (Fig. 1), and the vesicles which do are larger than normal. They attain the relatively large size of 1 # diameter, while granule-free vesicles lie mostly in the range 0.2-0.4/z, which is normal for such vesicles in this tissue. Vesicles containing granules may therefore be distinguished from others, apart from granule content, by their larger size and amoeboid outline. That the two forms occur freely intermixed suggests that originally they were identical, and that differences have resulted following the formation of granules. In Fig. 3 is illustrated the extent to which cytoplasm may be reduced in various areas of the cell. The cytoplasm is represented in section by narrow canals completely surrounding each endoplasmic reticulum vesicle, many of the latter containing intracisternal granules. Similar formations were found from time to time in various parts of the cell. Ribosomes present appear both in association with the membranes and free in the cytoplasm. As their density in respect of numbers is similar to that found in other parts of the cell (Figs. 1 and 2), it would appear unlikely that the narrow cisternae of cytoplasm arose by contraction of a much more extensive volume during tissue preparation, but that the observed formation reflects the condition in the living cell. The condition in control material, taken from outdoor plants, is shown in Figs. 4 and 5. Numerous vesicles of the endoplasmic reticulum may be found, with associated ribosomes and a low density homogeneous content. No such vesicles have been observed to contain granules. In Fig. 5 are presented examples of the extremely numerous small starch granules present in this normal tissue. As is usual, the starch granules appear in the electron microscope to have little electron scattering power, and are readily distinguished from the intracisternal granules. It is seen that they are surrounded by a double membrane without associated ribosomes. A comparison of control and growth cabinet material showed that the number of small starch granules was enormously reduced in the latter.
DISCUSSION The question arises as to the nature of the substance which can be accumulated as dense bodies in the endoplasmic reticulum. According to the osmic acid staining properties of the intracisternal granules they could be either proteinaceous or lipoid in content. In the light microscope it is easy to distinguish cellular protein inclusions from oil droplets by microchemical reactions. In the present case such an identification was impossible as the granules are submicroscopic or just at the limit of the light microscope resolving power, so that staining does not yield any recognizable coloration.
INTRACISTERNAL GRANULES IN BARLEY GRAIN ENDOSPERM
263
Siekevitz and Palade identified intracisternal granules of similar appearance in pancreas exocrine cells to be of zymogen nature (5). They further investigated with carbon-14 how this probable protein material is synthesized by the ribosomes on the outer face of the cisternal membranes, penetrates these membranes and is amassed inside the endoplasmic reticulum (6). The pancreas exocrine has a specialized function of producing and secreting enzymes, while the endosperm has quite a different function. Nevertheless, owing to the similarity of the granules under discussion with those of pancreas exocrine cells in respect of location, order of size, and osmic acid staining properties, it becomes necessary to consider the possibility that they too have a zymogen nature. As endosperm intracisternal granules were not found in tissues of control plants, it would appear that they were abnormalities induced by artificial environment conditions. The most important factor involved was probably lowness of light intensity, as plants showed a pronounced tendency to etiolation. Furthermore, the reduction in number of starch granules could be due to a reduced photosynthesis associated with low light intensity. If nitrogen supply and enzyme synthesis in the endosperm were normal, then it can be understood that with depletion in carbohydrate substrate supply, zymogen material could be in excess of metabolic requirements. In this case such material might be amassed near the site of synthesis in elements of the endoplasmic reticulum, and thus be stored in isolation from the cytoplasm. Alternatively, one must consider the possibility that the material is of lipoid nature. As has been mentioned, the appearance of the intracisternal granules is equally indicative of lipid as it is of protein. It could be suggested that with low intensity, but continuous, light the starch-synthesizing mechanism was to some extent disturbed, resulting in a partial diversion of materials to lipid synthesis. The end result of such a process would be the accumulation of droplets in the endoplasmic reticulum. No evidence in support of either postulate can be presented at this stage. However, it seems worthwhile to emphasize that the endoplasmic reticulum is capable of transforming and storing considerable quantities of material. This gives a further insight into its nature and function. REFERENCES 1. 2. 3. 4. 5.
ANDRe, J. and ROUILLER,C., or. Biophys. Biochem. Cytol. 3, 977 (1957). BUTTROS~,M. S., in preparation. HOLLMANN,K. H., J. Ultrastructure Research 2, 423 (1959). PALADE,G. E., or. Biophys. Biochem. Cytol. 2, 417 (1956). SIEKEVITZ,P. and PALADE,G. E., or. Biophys. Biochem. Cytol. 4, 309 (1958). 6. - ibid. 4, 557 (1958). 18 -- 60173313 J . Ultrastructure t~esearch