Micro-vesicles, pyriform vesicles and macro-vesicles associated with the plasma membrane in the root hairs of Vicia hirsuta after freeze-substitution.
ROBERT W. RIDGE.
Department of Biology, Division of Natural Sciences, International Christian University, 3-10-2 Osawa, Mitaka-shi, Tokyo, 181 Japan.
Figures (4 figs at 72dpi total 800k - could take 4 to 5 minutes to download - but worth it!!)
Abstract
After freeze-substitution, micro-vesicles were found only in close proximity to the plasma membrane. Macro and pyriform vesicles were found throughout the cytosol, but also 'packaged' close to the plasma membrane, the package delineated by electron transparent outlines similar to the endoplasmic reticulum. These outlines appeared to be continuous with nearby endoplasmic reticulum and were always associated with Golgi bodies and microtubules. Micro-vesicles were found only in grazing sections of the plasma membrane made between the apical dome and the region of the nucleus, where the cell is the most cytoplasmic, and only in close proximity to the plasma membrane. Micro-vesicles were also found in close proximity to microtubules as well as other vesicle types. From the results it is suggested that pyriform and micro-vesicles may have specialised roles in root hair tip growth.
Keywords
Freeze-substitution - Macro-vesicles - Micro-vesicles - Nod-factors - Plasma membrane -Pyriform vesicles
Introduction
Root hairs are tip-growing cells that grow by the addition of cell wall material delivered to the tip inside vesicles. Vesicles in root hairs are mainly found close to the tip region, where there is a distinct ÔfrothÕ of vesicles that are presumably emanating from the endomembrane system. In the cytoplasm-rich region between the nucleus and the tip, there is a large amount of endoplasmic reticulum and Golgi bodies that form the production and modification centre of cell wall precursors. The nucleus follows the growing tip at a set distance, presumably because continuous transcription of the genes for proteins involved in cell wall production is needed. Indeed, the cytoplasm in this area is dense with ribosomes, suggesting that translation is a continuous process (see e.g. Ridge 1988, 1990a, and figures in this paper).
The flow of vesicles and cell wall precursors to the tip is apparently controlled by the cytoskeleton (Lloyd et al. 1987), and the flow of vesicles within the apical dome may be controlled by a calcium gradient (Jaffe 1982). The cytoskeleton is also involved in maintaining the integrity of the hair shape (Lloyd 1984) and in positioning of the nucleus, which follows the tip as it grows. Excess membrane from the tip is likely recycled by a clathrin-based system at the base of the dome (Ridge 1993, see also Emons and Traas 1986).
Recent advances in the knowledge of root hair biology have resulted from the use of dry cleaving and freeze fracturing (e.g. Emons and Traas 1986), by rapid-freeze, freeze-substitution for electron microscopy (e.g. Emons 1987, 1988, Ridge 1988, 1990a) and by drug studies aimed at determining the role of the cytoskeleton (Lloyd et al. 1987, Ridge 1990a). The root hairs of legumes are particularly important to study because they are the entry point for Rhizobium in its symbiosis with many of the world's major legume food crops. Ideas on root hair growth and Rhizobium infection are summarized and modeled in Ridge (1993).
After freeze-substitution treatment, several kinds of vesicle have been found in root hairs, viz: smooth vesicles, coated vesicles, clathrin-coated vesicles and the unusual pyriform vesicle (Ridge 1988). It is possible that the smooth and coated vesicles are involved in the transport and deposition of cell wall substances to the tip, and can be categorised as secretory vesicles. However, clathrin-coated vesicles are clearly involved in membrane recycling (see discussion in Emons 1987, and in Ridge 1988, 1993). A role for pyriform vesicles has yet to be deduced by experimentation.
In this paper I report the occurrence of another kind of vesicle, defined as a micro-vesicle, that is found only at the plasma membrane, and the occurrence of macro-vesicles and pyriform vesicles in discrete compartment-like zones or ÔpackagesÕ close to the plasma membrane.
Material and Methods
Seed germination
Vicia hirsuta seed was treated with concentrated sulphuric acid for 45 min (they have a very hard seed coat), rinsed ten times, and then left in water for several hours. Swollen seeds were left to germinate on damp absorbent paper in a sealed dish, in the dark, for 3 days at room temperature. Seedlings with well-developed hairs were used for rapid freezing.
Electron microscopy
The methods of Ridge (1988, 1990b) were used, which were essentially as follows:
Roots were cut at about 1 cm from the root tip, held by very fine tweezers at the cut end, and frozen by rapidly plunging into liquid propane from a height of about 50 cm. The liquid propane was prepared by condensing propane gas into an aluminium container cooled with liquid nitrogen to about -196 C. After freezing, specimens were transferred to vials containing 0.3% OsO4 in dry acetone over molecular sieve (Bio-Rad type 3a) and left for 3 days in a -80 C freezer. The containers were then transferred to -20 C for 8 hr, given 2 changes of clean dry acetone over 4 hr, and then warmed to room temperature over 4 hr. Samples were infiltrated by gradually increasing the concentration of Spurr's resin over 2 days plus another day at 100%, and embedded at 60 C. Sections (ca 70nm) were stained with 2% uranyl acetate in 50% ethanol for 15 minutes followed by lead citrate for 5 min and viewed in a JEOL 1200DX electron microscope at 80kv.
Results
Grazing sections of root hairs taken between the nucleus and the apical dome revealed distinct compartment-like zones or 'packages' of vesicles at the cytoplasm/plasma membrane interface (Figs. 1-3). The packages were found to be delineated by electron transparent outlines similar to the endoplasmic reticulum and always associated with Golgi bodies (dictyosomes) (Fig. 1). From consecutive serial sections the packages were found to be of irregular shape. The vesicle population in these packages consisted mostly of secretory vesicles (which are electron transparent) but with associated pyriform vesicles (electron dense); coated vesicles were never found in the packages. Secretory vesicles, which are always spherical, were found to have the greatest diameter of approximately 120 nm, while pyriform vesicles, which are spherical but have distinct 'tails', have a maximum diameter of approximately 95 nm. Macro-vesicles and pyriform vesicles were also found within the cytoplasm of the cell, mostly within the apical dome of the root hair tip.
Microtubules were found inside the packages in close association with the vesicles, either adjacent to the plasma membrane and inside the package, or deeper in the cytoplasm next to the package (Figs. 2 and 3). Microfilaments were not found within or close to the packages, although they were found in other parts of the cell and often within contact distance of the plasma membrane. The packages were not found at the extreme tip of the hair, where there is a high density of secretory vesicles that subtend the plasma membrane and where organelles are usually absent. The exact distribution of the packages was not studied, except that they were only found in the cytoplasm-rich region between nucleus and tip.
A population of micro-vesicles was found in grazing sections made at a high angle (close to parallel), and only close to the plasma membrane in the region between the nucleus and apical dome (Fig. 4). They were not found endoplasmically (i.e. within the cytoplasm), and they could not be found on sections made at right angles to the plasma membrane. The micro-vesicles were not uniform in shape or perfectly spherical (Fig. 4) but of largest dimensions of approximately 50 nm (compare to the spherical secretory vesicles in Fig. 4). Because electron microscopy sections are generally about 70 nm in thickness, it is likely that many complete micro-vesicles were seen in one section. If this were to be the case, then the micro-vesicles seen in Fig. 4 range in size of from 15 to 50 nm, and are thus, in their smallest dimensions, close to the size of ribosomes after the same rapid-freeze treatment.
Micro-vesicles were found in close association with macro-vesicles, as well as with microtubules (Fig. 4).
Discussion
Vesicle 'packages'
Macro-vesicles and pyriform vesicles have already been described, but they have not previously been shown to be 'packaged' close to the plasma membrane. Pyriform vesicles were first described in root hairs (Ridge 1988) and as far as I know have never been described for any other organism. Observations of the packages close to the membrane, associated with both Golgi and microtubules, leads me to suggest that the packages possibly move to the tip in close association with the plasma membrane, guided by the microtubules and supplied with vesicles by the Golgi, perhaps moving as a unit towards the tip. The plasma membrane of many types of cell is a dynamic ÔorganelleÕ in its own right, in which many kinds of proteins and other membrane-bound molecules are able to move. It is possible therefore that the plasma membrane is responsible for movement of these packages to the tip, which is the inevitable direction of all secretory vesicles in plant tip-growing cells. Clearly also, pyriform vesicles may have a specialised role to play in the organisation and fate of other kinds of vesicle, although it is equally valid to say that they may have a role in other aspects of the cell, such as the delivery of special molecules such as those required for signal transduction.
Micro-vesicles
This is the first description of micro-vesicles in root hairs. Micro-vesicles have been described for fungal mycelium, where they are also called chitosomes (Ruiz-Herrera et al. 1977). Bartnicki-Garcia (1990) gives a value of 40-70 nm in diameter for conventionally-fixed material, and states that freeze-substituted fungus gives significantly smaller values. The results presented here show that Vicia hirsuta root hair micro-vesicles range in size from 15 to 50 nm, which is in the range of the dimensions suggested by Bartnicki-Garcia.
As in fungal hyphae, micro-vesicles in root hairs may have a different function to the larger macro-vesicles. Fungal micro-vesicles are known to be involved in the delivery of chitin synthetase, which is synthesised at the plasma membrane-cell wall interface (Bartnicki-Garcia 1990). If root hair micro-vesicles have a parallel role, then it could be possible that these micro-vesicles carry substances with special tasks to the root hair tip. For example it is known that plants produce chitinases (Boller et al. 1983, Schlumbaum et al. 1986). Indeed, Krause et al. (1994) have made a cDNA bank from mRNA isolated from Vigna unguiculata root hairs after stimulation by Rhizobium Nod-factor, and have found, amongst others, cDNAs coding for chitinase I, III and IV.
It is well established in the literature that Nod-factors are the signal molecules that initiate the Rhizobium/legume symbiosis (for review see Fisher and Long 1992) , and it is therefore clear that the Nod-factors must interact with a signal receptor protein that is most likely to be located on the plasma membrane. Yet at the growing tip of root hairs, where Rhizobium attaches and eventually invades, membrane is constantly being deposited and flows away back down the apical dome for recycling by a clathrin-based system. The supply of any molecules involved in recognition and transmission of a signal molecule must be constantly renewed, and I suggest that the various vesicles found in root hair tips may have such different and specialised roles. That is, spherical macro-vesicles deposit cell wall precursors for growth of the tip, but pyriform and/or micro-vesicles may be involved in different roles, such as depositing special domains of membrane containing proteinaceous molecules that transduce signal messages by interaction with external signal molecules, such as Nod-factors.
The close association of micro-vesicles with microtubules, close to the plasma membrane, also indicates that microtubules are probably guiding the flow of micro-vesicles, presumably to the hair tip.
Further work on the biological role of specialised vesicles in the legume root hair is needed.
References
Bartnicki-Garcia, S. 1990. Role of vesicles in apical growth and a new mathematical model of hyphal morphogenesis. In I.B. Heath ed., Tip Growth in Plant and Fungal Cells. Academic Press, California. pp. 211-232.
Boller, T., Gehri. A., Mauch, F. and Vogeli, U. 1983. Chitinase in bean leaves: induction by ethylene, purification, properties and possible function. Planta 157: 22-31.
Emons, A.M.C. 1987. The cytoskeleton and secretory vesicles in root hairs of Equisetum and Limnobium and cytoplasmic streaming in root hairs of Equisetum. Ann. Bot. 60: 625-632.
Emons, A.M.C. 1988. Methods for visualising cell wall texture. Acta Bot. Neerl. 37: 31-38.
Emons, A.M.C., Traas, J.A. 1986. Coated pits and coated vesicles on the plasma membrane of plant cells. Eur. J. Cell Biol. 41: 57-64.
Fisher, R.F. and Long, S.R. 1992. Rhizobium-plant signal exchange. Nature 357: 655-660.
Jaffe, L.F. 1982. Developmental currents, voltages and gradients. In S. Subtelny and P.B. Green, eds., Developmental Order: its Origin and Regulation. Alan R. Liss, New York pp. 183-215.
Krause, A., Sigrist, C.J.A., Dehning, I., Sommer, H. and Broughton W.J. 1994. Accumulation of transcripts encoding a lipid transfer-like protein during deformation of nodulation competent Vigna unguiculata root hairs. Mol. Plant-Microbe Interact. 7: 411-418.
Lloyd, C.W. 1984. Helical microtubular arrays in onion root hairs. Nature 305: 311-313.
Lloyd, C.W., Pearce, K.J., Rawlins, D.J., Ridge, R.W. and Shaw, P.J. 1987. Endoplasmic microtubules connect the advancing nucleus to the tip of legume root hairs, but F-actin is involved in basipetal migration. Cell Motil. Cytoskel. 8: 27-36.
Ridge, R.W. 1988. Freeze-substitution improves the ultrastructural preservation of legume root hairs. Bot. Mag. Tokyo 101:427-441.
Ridge, R.W. 1990a. Cytochalasin-D causes organelle-crowding and abnormal ingrowths in legume root hairs. Bot. Mag. Tokyo 103: 93-102.
Ridge, R.W. 1990b. A simple apparatus and technique for the rapid freezing and freeze-substitution of single-cell algae. Jap. J. of Electron Microsc. 39: 121-125.
Ridge, R.W. 1993. A model of legume root hair growth and Rhizobium infection. Symbiosis 14: 359-373.
Ruiz-Herrera, J., Lopez-Romero, E. and Bartnicki-Garcia, S. 1977. Properties of chitin synthetase in isolated chitosomes from Mucor rouxii. J. Biol. Chem 252: 3338-3343.
Schlumbaum, A., Mauch, A., Vogeli, U. and Boller, T. 1986. Plant chitinases are potential inhibitors of fungal growth. Nature 324: 365-367.