Jerry R. Barrow 2. Mary E. Lucero 2.
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Jerry R. Barrow 2. Mary E. Lucero 2. Ronald E. Aaltonen 2. Henry Dunant Lipid bodies are universal components of plant cells and provide a mobilized carbon source for essential biological processes. Oil plants harvested for food and fuel depend on these lipid bodies. Plants also host diverse populations of endophytic fungi, which easily escape microscopic detection. This study reviews data from previous surveys of endophyte distribution in native plants to specifically examine the physical association between endophytic fungi and plant lipid bodies.
Plant tissues stained with trypan blue and sudan IV prior to differential interference contrast microscopy exhibited lipid bodies tightly associated with fungal hyphae and with trypan blue stained fungal networks. The abundance of endophyte-associated lipids in healthy plant tissues suggests endophyte involvement in carbon oil metabolism and transport.
More research, particularly at the molecular level, is merited to assess the significance of this plant feature which is conserved in grasses and shrubs. Exploring the interfaces where plant cells and endophyte cells exchange organic carbon by using modern imaging, molecular and genomic analysis could transform current understanding of plant carbon metabolism.
Oils serve as a carbon and energy reserves and are precursors for membrane lipid and steroid biosynthesis in plants Hanisch et al. Oil or lipid bodies called oleosomes are spherical storage organelles embedded within the cytoplasm of eukaryotes Waltermann and Steinbuchel; ; Martin and Parton, Oleosomes occur in cells of most plant tissues and are prominent in seeds, pollen grains, embryos and endosperm.
They are also in stems, fruits and leaves, where they are frequently associated with plastids Lersten et al. The most common forms of lipids stored in plants are triacylglycols TAG , triesters of fatty acids that are attached to glycerol Moellering and Benning, ; Goncalves et al. Oil bodies consist of a TAG matrix covered by a stabilizing layer of phospholipids and unique proteins, which are thought to originate from the endoplasmic reticulum Goldberg et al.
These universal components of eukaryotic cells provide a rapidly mobilized lipid source for many important biological processes Martin and Parton, ; Rezanka and Sigler, To function normally, embryos, seeds and pollen are physiologically programmed to cope with severe desiccation during maturation and storage Liu et al. Regulation of fatty acid composition may be one mechanism by which plants regulate tolerance to temperature and moisture stress Volk et al.
TAGs in Cuphea sp. After storage, if seeds were imbibed with water before TAGs liquefy, cells suffer irreversible membrane damage and fail to germinate. Oleosins, proteinaceous matrices associated with the surfaces of lipid bodies, are thought to maintain stability of lipid bodies in seeds of desiccation-tolerant plants to prevent them from coalescing during seed dehydration and germination Murphy, , Purkrtova et al. Oleosins that coat oil bodies in soybeans protect them from environmental stresses Iwanaga et al.
Pollen grains characteristically contain large numbers of oil bodies that serve as energy reserves for subsequent germination Piffanelli et al. Surface proteins contribute to oil body stability in lily Lillium longiflorum Thumb.
This stability may enhance pollen viability as it is subjected to environmental extremes in transit by insects or wind.
Adequate lipid storage compounds are essential for successful development during final stages of somatic embryogenesis in Picea abies Grigova et al. Lersten et al. They found that oil bodies were common in eudicots and less frequent in monocots. They observed that in recent years oil bodies have been neglected because of a shift in plant tissue preparation.
Early microscopic studies involved analysis of freehand sections that allowed vivid staining of lipids. Later methods involved killing fixatives and alcoholic dehydration that dissolved lipids and obscured their microscopic detection.
In addition to physiological roles, plant oils have wide economic importance. Two major plant oil classes are recognized: 1 volatile, low-molecular mass oils that are used commercially in perfumes and food extracts and 2 larger molecular mass, non-volatile oils that are commercially extracted from seeds of canola, castor, corn, soybean and other important crop plants Lersten et al. Lipid bodies structurally similar to those found in vascular plants are also common in microbes. Microbial oils have potential commercial value as food supplements, pharmaceuticals, and biofuels Peng and Chen, These may be produced more economically than those extracted from agricultural crops.
Peng and Chen isolated fungal endophytes with large and copious quantities of lipid bodies within their hyphae from oleagenous plants. Those with enzymatic capability to decompose wheat straw are candidates for low cost microbial oil production. Plants host diverse populations of cryptic, symbiotic fungi Arnold et al. It is well documented that fungal symbionts contribute multiple benefits that enhance tolerance to abiotic and biotic stress, but the mechanisms of inducing stress tolerance are not well understood.
In our research, we have heavily utilized a dual staining approach to reveal trypan blue staining fungal chitin and Sudan IV staining lipid bodies which are associated with fungal symbionts within plant tissues Barrow et al. We have also used molecular techniques to demonstrate the presence of complex fungal consortia that associate with plants even under aseptic conditions Lucero et al. Although Sudan IV stain would also reveal lipid bodies associated strictly with the plant, our collective experience suggests this is rare.
Our objective in this study was to review dual stained images of plant tissues taken from studies examining endophyte distribution and other plant-fungal associations to specifically consider the association between plant lipid bodies and endophytic fungi. Micrographs of plant tissues retrieved from data archives of Jerry Barrow were prepared in association with several studies that occurred between and Root and leaf samples were collected from actively growing grasses and shrubs.
Staining methods are described in Barrow and Aaltonen and Barrow Briefly, hand sectioned tissues were cleared with potassium hydroxide followed by staining with a solution containing trypan blue, which stains fungal cell walls blue, and Sudan IV, which stains lipid bodies red.
Slides containing small secondary roots and leaves were mounted and examined with a Zeiss Axiophot microscope with conventional and differential interference contrast DIC optics at X and X magnification. Digital images were captured and processed using Auto-Montage 3D software by Syncroscopy. Analysis of root and leaf tissues frequently revealed amorphous trypan blue stained fungal structures associated with lipid bodies on leaf surfaces Figure 1a , root meristems Figure 1b , lateral roots Figure 1c and hydrated seeds Figure 1d of Bouteoula eriopoda 1a - c , and Sporobolus airoides 1d , respectively.
Although hyphae were sometimes visible Figures 1a - c , fruiting bodies and other clear details useful for identification were typically absent. Sometimes only blue stain, suggestive of fungal chitin, could be detected Figure 1d. This amorphous fungal morphology observed among endophytes has been previously described Barrow, Similar amorphous fungal structures, thought to be derived from yeasts, have been reported in surface sterilized seeds of Atriplex canescens and in micropropagated shoots of Bouteloua eriopoda Osuna and Barrow, and of Atriplex canescens , both of which were demonstrated to contain DNA representing diverse fungal taxa Lucero et al.
Trypan blue stain reveals a hyaline branched fungal network fh , associated with red, sudan IV stained lipid bodies lb. Figure 1b. A lateral root initial of Bouteloua eriopoda. Sudan IV stains intercellular lipid bearing fungal protoplasts ich and lipid bodies lb within meristematic cells.
Finely branched fungal hyphae fh and amorphous fungal structures appear blue. Figure 1c. Sudan IV stained lipid bodies in vascular cells of developing lateral roots of Bouteloua eriopoda.
Hyaline fungal hyphae remain unstained and clear fh. These hyphae surround many of the large lipid bodies near the center of the page. Lipid body inclusions are clear in the hyaline hypha indicated with a black arrow on the lower right corner.
Figure 1d. Endosperm cells of hydrated Sporobolus airoides seeds stained with trypan blue and sudan IV. A trypan blue stained fungal network fn with attached sudan IV stained lipid bodies lb. In our studies of plants native to the Chihuahuan Desert we found extensive colonization by dark septate endophytes DSE Figure 2e Barrow, DSE fungi are generally characterized by stained or melanized hyphae and microsclerotia Jumpponen and Trappe, , which were most prevalent in dormant plants Figure 2a.
Melanin, a natural dark pigment that makes DSE structures microscopically visible in plant tissues, was most prevalent in dormant plant tissues mh.
Lipid bodies associated with melanized hyphae in dormant plants were rare, but the abundance in physiologically active plants was remarkable Figure 2b - d. Fungal endophytes were also detected as non-staining hyaline, clear, hyphae Figure 1a , c , 2a , d or with amorphous, trypan blue staining networks Figure 1a , b , d. Lipid bodies were typically associated with all of these fungal structures.
Brown, melanized mh and fine hyaline hyphae fh are visible. A hyaline hypha near the center of the image contains weakly staining lipid bodies lb that are less developed than the lipid bodies seen during active growth stages. Figure 2b. A melanized hypha on the surface of B. Figure 2c. Figure 2d. Dual stained, physiologically active roots of B. Figure 2e. Mycelia my emerging from aseptic roots of B. Analysis of leaf tissues revealed lipid bodies integrated with fungal structures in photosynthetic mesophyll and bundle sheath and cells of the stomatal complex of grasses and shrubs Barrow, ; Barrow and Aaltonen, In both shrub and grass species, lipid bodies were most prevalent in the root meristems, cortex and phloem.
In leaves, they were most conspicuously present in photosynthetic mesophyll, bundle sheath and cells of the stomatal complex. Color images in the above cited references vividly illustrate lipid bodies associated with hyphae and other structures of endophytic fungi in tissues of native plants. Like Lersten et al. However, while Lersten et al. Lipid bodies were rarely observed in dormant plants Figure 2a. Unlike Lersten et al. We presume that the differences in observations are due to different research objectives our group was primarily interested in distribution of endophytic fungi , differences in imaging Lersten et al.
Nonetheless, we feel that highlighting this difference is important, particularly because recent interest in plant oils for biofuels, in addition to agricultural value, is climbing.
Epichloë endophytes in pastures of the Iberian Peninsula.
Presencia de hongos endofitos Neotyphodium spp. Actas de la E1 pasto: El recurso mas barato. Although Sudan IV stain would also reveal lipid bodies associated strictly with the plant, our collective experience suggests this is rare. Two distinct steroleosins are present in seed oil bodies. Stable oil bodies sheltered by fndofitos unique oleosin in lily pollen. Eukaryotic lipid body proteins in oleogenous actinomycetes and their targeting to intracellular triacylglycerol inclulsions: Avalaibility of Phosforus in a calcareous soil treated with rock-phosphat dissolving fungi. Symbiotic bacteria as a determinant of plant community structure and plant productivity in dune grassland.