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Marginal and laminar hydathode-like structures in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius Welw
Abstract
The fan-shaped leaves of the resurrection plant Myrothamnus flabellifolius Welw. fold during episodes of drought and consequent desiccation of the tissue. The leaf teeth of M. flabellifolius have several features characteristic of hydathodes. Tracheary elements from the three vein endings that converge in each leaf tooth subtend and extend into a cluster of cells significantly smaller than those of the adjacent mesophyll. The stomata overlying this putative epithem are larger than the other stomata on the leaf surface. Crystal violet is absorbed via these stomata in non-transpiring leaves, suggesting that they are water pores. Two to four such water pores occur per hydathode and are readily distinguished in desiccated leaves. Laminar hydathodes apparently also occur in the leaves of M. flabellifolius. Branched vein endings that terminate in short, wide tracheary elements subtend the outer edges of the abaxial leaf ridge, which otherwise lack stomata, and coincide with regions of crystal violet uptake. Guttation could not be induced in M. flabellifolius. However, desiccated leaves readily absorb liquid water through the leaf surface. The use of Calcafluor White to trace the pathway of apoplastic water movement suggests a role for both types of hydathode in foliar water uptake during rehydration while the accumulation of Sulphorhodamine G (indicating solute retrieval from the apoplast) in the epithem of transpiring plants suggests the hydathodes may be a pathway of water loss in the desiccating leaf.
... Finally, laminar hydathodes, also called paginal hydathodes by Belin-Depoux (12), are observed over the entire leaf surface (Supplemental Figure 1c) and are found in three dicot families (Crassulaceae, Moraceae, Urticaceae) (Supplemental Table 1). Myrothamnus flabellifolius, a plant from the Myrothamnaceae family shows both laminar and marginal hydathodes (33). Yet wherever their location on the leaf surface, these hydathodes share a conserved pattern of tissular organization with a highly vascularized epithem connected to the outside by water pores (see sidebar titled Epithemal Hydathodes). ...
... For instance, Crassula species collected from the Namib desert can absorb water through their hydathodes (87). In the resurrection plant M. flabellifolius, leaves rehydrated in the presence of calcofluor showed an intense staining of the epithem compared to surrounding mesophyll cells and a bright fluorescence in the vasculature, indicating a water uptake through both marginal and laminar hydathodes suggestive of hydathode importance in maximizing the use of available rainwater for rapid rehydration (33). Similarly, colored aqueous dyes can easily penetrate via hydathodes into the vascular system of maize and squash leaves (31). ...
... Guttation is dependent on the presence of water pores in the epidermis of the hydathode. In few plants such as M. flabellifolius (33,134), F. diversifolia (83), and Physocarpus opulifolius (80) guttation was not observed and could not be induced. This inability to show guttation even under favorable conditions in P. opulifolius was correlated with the presence of immature water pores (80). ...
Hydathodes are organs found on aerial parts of a wide range of plant species that provide almost direct access for several pathogenic microbes to the plant vascular system. Hydathodes are better known as the site of guttation, which is the release of droplets of plant apoplastic fluid to the outer leaf surface. Because these organs are only described through sporadic allusions in the literature, this review aims to provide a comprehensive view of hydathode development, physiology, and immunity by compiling a historic and contemporary bibliography. In particular, we refine the definition of hydathodes. We illustrate their important roles in the maintenance of plant osmotic balance, nutrient retrieval, and exclusion of deleterious chemicals from the xylem sap. Finally, we present our current understanding of the infection of hydathodes by adapted vascular pathogens and the associated plant immune responses.
... Hydathodes are often overlooked foliar structures that are relatively common among vascular plants (for review see Cerutti et al. 2019), being responsible for the process of guttation (i.e. the exudation of apoplastic fluid; Bellenot et al. 2022). Marginal and apical hydathodes are the most prevalent (Cerutti et al. 2019;Jauneau et al. 2020;Rios et al. 2020), while laminar hydathodes, which are found over the entire leaf surface, are restricted to Crassulaceae and three other eudicot families, which have very few or no succulent representatives: Moraceae, Urticaceae and Myrothamnaceae (Lersten and Peterson 1974;Lersten and Curtis 1991;Chen and Chen 2005;Drennan et al. 2009). The noteworthy anatomy of hydathodes in Crassula and their contrasting foliar distribution among different species have led to several exquisitely illustrated publications through the years (de Bary 1884;Sporer 1915;Rost 1969;Smirnova 1973;Voronin et al. 1976). ...
... This may be even more crucial for species occurring within the hyper-arid Gariep centre (sensu van Wyk and Smith 2001), the northernmost region of the Succulent Karoo biome and habitat to C. ausensis , C. deceptor , C. plegmatoides and C. sericea (Fig. 1 ). Besides Crassula , other plants that occur sympatrically in western southern Africa are also believed to benefit from frequent fog and dew through fog drip, selfirrigation and maybe even FWU (Snow 1985;Andrews et al. 2011;Vogel and Müller-Doblies 2011;Roth-Nebelsick et al. 2012), including leaf succulents in the Aizoaceae (Niesler 1997;Matimati et al. 2013) and the desiccation-tolerant resurrection plant Myrothamnus flabellifolius (Myrothamnaceae; Drennan et al. 2009). Interestingly,Myrothamnus is also one of the rare cases in which laminar hydathodes occur, suggesting the possibility of FWU. ...
Hydathodes are usually associated with water exudation in plants. However, foliar water uptake (FWU) through the hydathodes has long been suspected in the leaf-succulent genus Crassula (Crassulaceae), a highly diverse group in southern Africa, and, to our knowledge, no empirical observations exist in the literature that unequivocally link FWU to hydathodes in this genus. FWU is expected to be particularly beneficial on the arid western side of southern Africa, where up to 50% of Crassula species occur and where periodically high air humidity leads to fog and/or dew formation. To investigate if FWU is operational in different Crassula species we used the apoplastic fluorescent tracer Lucifer Yellow in combination with different imaging techniques. Our images of dye-treated leaves confirm that hydathode-mediated FWU does indeed occur in Crassula and that it is probably widespread across the genus. Hydathodes in Crassula have been repurposed as moisture-harvesting structures, besides their more common purpose of guttation, an adaptation that has likely played an important role in the evolutionary history of the genus. Our observations suggest that FWU ability is independent of geographical distribution and its associated environmental conditions, as FWU is possible in species occurring within the fog belt of western southern Africa but also in those from the rather humid eastern side. We did not find a strong apparent link between FWU ability and leaf surface wettability. Instead, the hierarchically sculptured leaf surfaces of several Crassula species may facilitate FWU due to hydrophilic leaf surface microdomains, even in seemingly hydrophobic species. Overall, these results confirm the ecophysiological relevance of FWU in Crassula and reassert the importance of atmospheric humidity for some arid-adapted plant groups.
... Marginal and apical hydathodes are the most prevalent (Cerutti et al., 2019;Jauneau et al., 2020;Rios et al., 2020), while laminar hydathodes, which are found over the entire leaf surface, seem to be restricted to Crassulaceae and three other eudicot families that have very few or no succulent representatives: Moraceae, Urticaceae and Myrothamnaceae (Chen & Chen, 2005;Drennan et al., 2009;Lersten & Curtis, 1991;Lersten & Peterson, 1974). The noteworthy anatomy of hydathodes in Crassula and their contrasting foliar distribution among different species have led to several exquisitely illustrated publications through the years (de Bary, 1884;Rost, 1969;Smirnova, 1973;Sporer, 1915;Voronin et al., 1976). ...
Hydathodes are usually associated with water exudation in plants. However, foliar water uptake (FWU) through the hydathodes has long been suspected in the leaf-succulent genus Crassula (Crassulaceae), a highly diverse group in southern Africa, and, to our knowledge, no empirical observations exist in the literature that unequivocally link FWU to hydathodes in this genus. FWU is expected to be particularly beneficial on the arid western side of southern Africa, where up to 50% of Crassula species occur and where periodically high air humidity leads to fog and/or dew formation. To investigate if hydathode-mediated FWU is operational in different Crassula species, we used the apoplastic fluorescent tracer Lucifer Yellow in combination with different imaging techniques. Our images of dye-treated leaves confirm that hydathode-mediated FWU does indeed occur in Crassula and that it might be widespread across the genus. Hydathodes in Crassula serve as moisture-harvesting structures, besides their more common purpose of guttation, an adaptation that has likely played an important role in the evolutionary history of the genus. Our observations suggest that ability for FWU is independent of geographical distribution and not restricted to arid environments under fog influence, as FWU is also operational in Crassula species from the rather humid eastern side of southern Africa. Our observations point towards no apparent link between FWU ability and overall leaf surface wettability in Crassula. Instead, the hierarchically sculptured leaf surfaces of several Crassula species may facilitate FWU due to hydrophilic leaf surface microdomains, even in seemingly hydrophobic species. Overall, these results confirm the ecophysiological relevance of hydathode-mediated FWU in Crassula and reassert the importance of atmospheric humidity for some arid-adapted plant groups.
... It was known in isiZulu as Uvukakwabafile, which means 'awake from the dead'. This might originate from its adaptability to extreme drought surroundings, which benefits from evolved powerful survival strategies including a well-developed root system and the capability to recover from dehydration [22][23][24]. However, few genes related to the drought tolerance of M. flabellifolia have been characterized, and the underlying molecular mechanisms are largely unknown. ...
Drought and salinity have become major environmental problems that affect the production of agriculture, forestry and horticulture. The identification of stress-tolerant genes from plants adaptive to harsh environments might be a feasible strategy for plant genetic improvement to address the challenges brought by global climate changes. In this study, a dehydration-upregulated gene MfWRKY7 of resurrection Plant Myrothamnus flabellifolia, encoding a group IId WRKY transcription factor, was cloned and characterized. The overexpression of MfWRKY7 in Arabidopsis increased root length and tolerance to drought and NaCl at both seedling and adult stages. Further investigation indicated that MfWRKY7 transgenic plants had higher contents of chlorophyll, proline, soluble protein, and soluble sugar but a lower water loss rate and malondialdehyde content compared with wild-type plants under both drought and salinity stresses. Moreover, the higher activities of antioxidant enzymes and lower accumulation of O2− and H2O2 in MfWRKY7 transgenic plants were also found, indicating enhanced antioxidation capacity by MfWRKY7. These findings showed that MfWRKY7 may function in positive regulation of responses to drought and salinity stresses, and therefore, it has potential application value in genetic improvement of plant tolerance to abiotic stress.
... Welw. is a unique dwarf shrub worldwide and grows in poor rock conditions [24,25]. A long period of evolution, as well as mighty adaptability to extreme drought surroundings, make M. flabellifolia develop a powerful survival strategy including a well-developed root system and the capability to recover from dehydration [26][27][28]. Based on the previous study, there are a variety of TFs that play a part in the transcriptional regulatory networks during the dehydration process in M. flabellifolia, in which MfbHLH38 was obviously up-regulated in initial period of dehydrating treatment [29]. ...
Background
The basic helix-loop-helix (bHLH) proteins, a large transcription factors family, are involved in plant growth and development, and defensive response to various environmental stresses. The resurrection plant Myrothamnus flabellifolia is known for its extremely strong drought tolerance, but few bHLHs taking part in abiotic stress response have been unveiled in M. flabellifolia.
Results
In the present research, we cloned and characterized a dehydration-inducible gene, MfbHLH38, from M. flabellifolia. The MfbHLH38 protein is localized in the nucleus, where it may act as a transcription factor. Heterologous expression of MfbHLH38 in Arabidopsis improved the tolerance to drought and salinity stresses, as determined by the studies on physiological indexes, such as contents of chlorophyll, malondialdehyde (MDA), proline (Pro), soluble protein, and soluble sugar, water loss rate of detached leaves, reactive oxygen species (ROS) accumulation, as well as antioxidant enzyme activities. Besides, MfbHLH38 overexpression increased the sensitivity of stomatal closure to mannitol and abscisic acid (ABA), improved ABA level under drought stress, and elevated the expression of genes associated with ABA biosynthesis and ABA responding, sucha as NCED3, P5CS, and RD29A.
Conclusions
Our results presented evidence that MfbHLH38 enhanced tolerance to drought and salinity stresses in Arabidopsis through increasing water retention ability, regulating osmotic balance, decreasing stress-induced oxidation damage, and possibly participated in ABA-dependent stress-responding pathway.
... Colleter secretion plays an important protective role in meristems and young leaves against dehydration and pathogens (Klein et al., 2004;Tresmondi et al., 2015;Fernandes et al., 2016). The secretory activity of those glands provides adequate conditions for leaf primordia and young leaves to develop and perform their metabolic processes under more appropriate conditions in terms of hydration and protection against herbivory and parasitism (Fahn, 1988;Morris et al., 2005;Drennan, Goldsworthy & Buswell, 2009;Singh, 2014;Tresmondi et al., 2015;Silva et al., 2017). In the case of hydathodes and EFNs, their secretory activity usually persists throughout the entire life cycle of the leaf, with the glands being associated with water flow and with mediating interactions between the plant and other organisms, respectively (Burgess & Dawson, 2004;Koulman et al., 2007;Shepherd & Wagner, 2007;Gish, Mescher & Moraes, 2015;Nahas, Gonzaga & Del-Claro, 2017). ...
Leaf teeth are projections on the leaf blade margin. They are structurally variable, with characters that are important for taxonomy and phylogeny, but there is a paucity of information on the anatomy of these structures and little understanding of the features and their functions. Here we describe and compare the leaf tooth anatomy of 47 eudicot species. Toothed margin samples from leaves at different developmental stages were collected, fixed and studied under light and scanning electron microscopy. We identified eight leaf tooth morphotypes, six of which occurred with glands. Hydathodes were the most common glands, being found in 11 species; colleters were found in ten species and extrafloral nectaries were found in two species. Cunonioid teeth either devoid of glands or associated with hydathodes were found in Lamiales, Asterales and Apiales. Dillenioid teeth associated with hydathodes were found in Dilleniales. Spinose teeth associated with colleters were found in Aquifoliales. In rosids, we found begonioid, malvoid, theoid, urticoid and violoid teeth, which may be associated with either colleters or nectaries or lack an associated gland. For each family studied, there was only one type of association between gland and tooth, demonstrating the systematic potential of these glands in eudicots.
... (Myrothamnaceae), a short shrub from southern Africa, is known as the only wooden resurrection plant for its DT features [38]. To survive in the extremely dry mountain environment, the unique fan leaves of M. flabellifolius can fold and roll up tightly when plant tissue is dehydrated, which makes the plants turn quickly into a long-term desiccant state and rehydrate rapidly after contact with water [39,40]. In the meantime, the primary problem faced by photosynthesis under drought stress is that photosynthesis agencies can provide the possibility of generating toxic reactive oxygen species (ROS) [41]. ...
Phytochrome-interacting factors (PIFs), a subfamily of basic helix-loop-helix (bHLH) transcription factors (TFs), play critical roles in regulating plant growth and development. The resurrection plant Myrothamnus flabellifolia possesses a noteworthy tolerance to desiccation, but no PIFs related to the response to abiotic stress have been functionally studied. In this study, a dehydration-inducible PIF gene, MfPIF1, was cloned and characterized. Subcellular localization assay revealed that MfPIF1 is localized predominantly in the nucleus. Overexpression of MfPIF1 in Arabidopsis thaliana led to enhanced drought and salinity tolerance, which was attributed to higher contents of chlorophyll, proline (Pro), soluble protein, and soluble sugar, activities of antioxidant enzymes as well as lower water loss rate, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation in transgenic lines compared with control plants. Moreover, MfPIF1 decreased stomatal aperture after drought and abscisic acid (ABA) treatment, and increased expression of both ABA biosynthesis and ABA-responsive genes including NCED3, P5CS, and RD29A. Overall, these results indicated that MfPIF1 may act as a positive regulator to drought and salinity responses, and therefore could be considered as a potential gene for plant genetic improvement of drought and salinity tolerance.
... Secretory products of cavity pore may serve to minimize loss of water during desiccation (Naidoo et al., 2009). Special stoma-like structures on the leaf surface of Myrothamnus flabellifolius which can absorb water also help to survive in the extreme arid environment (Drennan et al., 2009). Ayensu (1969Ayensu ( , 1974 described elongated mesophyll cells, which connected the epidermis to the bundle sheaths on the adaxial side of the leaf in some Velloziaceae taxa. ...
Water uptake and water use efficiency of poikilochlorophyllous resurrection plants considerably differ from other vascular plants due to their special ecological adaptation strategies. Histological traits of the living and dead leaves of desiccation-tolerant Xerophyta scabrida (Pax) Th., Dur. et Schinz. were investigated after safranin staining to examine the dynamics of rehydration and the process of water transport by safranin impregnation differences. Staining that appeared on the leaf's surface epidermal glands after 30 min of remoistening in living X. scabrida leaves suggested that glandular complexes could take part in the water uptake from the beginning of the rehydration process, when the xylem might not have been filled up, yet. The leaves of living X. scabrida became fully impregnated faster than the dead leaves: Sclerenchyma staining started from the xylem while dead X. scabrida leaves were not able to rehydrate fully. The dynamics of rehydration by the orientation of immersing leaves was also studied. Leaves immersed into the solution with their base downward became fully impregnated earlier than the leaves sank into the dye with their apex downward. Faster water supply in the former case might have been related to the earlier recovery of xylem integrity and the resultant spatially continuous water supply. The results indicate that the living leaves of desiccation-tolerant X. scabrida were able to uptake significant water amount not only (and not primarily) through their vascular tissues but by external water conduction. Due to the specific binding of safranin to lignifying cell walls and its fast spread from the xylem to the sclerenchymatous bundle sheaths also confirm the potential role of sclerenchyma in the water movement of leaf tissues.
... 18 Hydathodes are usually connected to the vascular tissue, 21 and it has been suggested that hydathodes may be able to take up moisture deposited on leaves by dew or fog. 22,23 Flooding is another serious plant stress and is a large contributor to plant mortality as it lowers oxygen levels. 9,24 However, some plants have adapted to live in aquatic habitats such as lakes and ponds, including one species of Crassula. ...
The genus Crassula contains a number of highly adaptable species, which can inhabit a wide range of environments. This investigation aimed to
examine whether there are any differences in the anatomical adaptations in relation to water availability of four species
of Crassula: the New Zealand pygmy weed, Crassula helmsii (T Kirk) Cockayne (from an aquatic habitat); the fairy crassula: Crassula multicava Lemaire ssp. multicava (from a subtropical habitat); the jade plant, Crassula ovata (Miller) Druce; and the anteelplakkie, Crassula socialis Schönland (both from semi-arid habitats). Plants were grown in a greenhouse and the anatomical features of stems and leaves
were examined using light microscopy. Plant material was sectioned by hand and sections were stained with Toluidine blue O.
Cuticle thicknesses were measured by treating sections with Sudan black B. Stomatal and hydathode densities on leaves and
stems were measured using epidermal peels. Two measures of leaf succulence were used: degree of succulence and succulence
quotient. The aquatic species C. helmsii had significantly fewer features associated with conserving water, including the thinnest cuticles on the adaxial leaf (P < 0.001) and abaxial leaf (P < 0.001). In contrast, the semi-arid species C. ovata had significantly the highest hydathodes on adaxial leaf surfaces (P < 0.001). Crassula ovata also had significantly the highest degree of succulence (P < 0.001), while C. socialis had the highest succulence quotient. The subtropical species, C. multicava, had significantly the thickest cuticles on adaxial leaf (P < 0.001) and stem (P < 0.001). Crassula species from arid environments had significantly more water conserving anatomical features, such as reduced stomatal densities,
than those from less arid environments. However, all species studied possessed varying degrees of similar anatomical features.
These features make Crassula a highly adaptable genus able to inhabit a wide range of environments.
... To determine whether local cell proliferation is linked to tooth formation, we examined cuc2-3, which is known to form smooth leaf margins (Nikovics et al., 2006). Hydathodes, identified as open vein endings connected to small parenchyma cells (Drennan et al., 2009), are typically formed at the tips of teeth (Candela et al., 1999), and thus we used the hydathodes as a marker of differentiation of the tooth tip (Tsukaya and Uchimiya, 1997). Hydathodes were seen as a cluster of small cells, and their cell wall was difficult to visualize under differential interference contrast microscope (DIC). ...
Serration found along leaf margins shows species-specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simple-leaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP-SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2-3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis-express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development.
Hydathodes are typically found at leaf teeth in vascular plants and involved in water release to the outside. Although morphological and physiological analysis of hydathodes has been performed in various plants, little is known about the genes involved in hydathode function. In this study, we performed fluorescent protein-based imaging and tissue-specific RNA-seq analysis in Arabidopsis hydathodes. We used the enhancer trap line E325, which has been reported to express green fluorescent protein (GFP) at its hydathodes. We found that E325-GFP was expressed in small cells found inside its hydathodes (named E cells) that were distributed between the water pores and xylem ends. No fluorescence of the phloem markers pSUC2:GFP and pSEOR1:SEOR1-YFP was observed in the hydathodes. These observations indicate that Arabidopsis hydathodes are composed of three major components: water pores, xylem ends, and E cells. In addition, we performed transcriptome analysis of the hydathode using the E325-GFP line. Microsamples were collected from GFP-positive or negative regions of E325 leaf margins with a needle-based device (~130 µm in diameter). RNA-seq was performed with each single microsample using a high-throughput library preparation method called Lasy-Seq. We identified 72 differentially expressed genes. Among them, 68 genes showed significantly higher expression in the hydathode, while four genes showed significantly lower expression in the hydathode. Our results provide new insights into the molecular basis for hydathode physiology and development.
Atrazine is one of the most used herbicides and has been associated with persistent surface and groundwater contamination, and novel formulations derived from nanotechnology can be a potential solution. We used poly-epsilon caprolactone nano-encapsulation of atrazine (NC+ATZ) to develop a highly effective herbicidal formulation. Detailed structural study of interaction between the formulation and Brassica juncea plants was carried out with evaluation of the foliar uptake of nanoatrazine and structural alterations induced in the leaves. Following post-emergent treatment, NC+ATZ adhered to the leaf and penetrated mesophyll tissue mainly through the hydathode regions. NC+ATZ was transported directly through the vascular tissue of the leaves and into the cells where it degraded the chloroplasts resulting in herbicidal activity. Nanocarrier systems, such as the one used in this study, have a great potential for agricultural applications in terms of maintenance of herbicidal activity at low concentrations and a substantial increase in the herbicidal efficacy.
El conocimiento de la biología de las plantas, especialmente de la tolerancia que estas pueden tener frente a las actuales alteraciones ambientales en lo que hace referencia al cambio climático, es de gran importancia para identificar aquellas especies capaces de resistir situaciones de estrés hídrico que se producen en las plantas debido al aumento de su tasa de evapotranspiración y deshidratación. Por ello, mediante el presente estudio, se comparó la tasa de deshidratación foliar en once de las principales especies de plantas presentes en dos ecosistemas estratégicos de Cartagena, Colombia (caño Juan Angola y cerro de La Popa), los cuales pertenecen en su mayoría (36%) a la familia Fabaceae, seguida de las Malvaceae, Rubiaceae, Ancaradiaceae, Sapindaceae, Combrataceae, Apocynaceae y la Rizophoraceae, con 9% cada una. Los resultados ubican a la especie Laguncularia racemosa como la de mayor tasa de deshidratación foliar (23,35 ± mgH2O/hr); mientras que Guazima ulmifolia fue la especie con menor tasa de deshidratación (3,51 ± 1,63 mgH2O/hr). Se discuten los resultados encontrados en el estudio, con respecto a la importancia de la información obtenida para la gestión ecológica y ambiental de los ecosistemas locales de la ciudad ante un futuro escenario de cambio climático.
Guttation is a process of natural secretion of fluid from leaves via specialised structures called 'hydathodes', which are located at the tips, margins, and adaxial and abaxial surfaces of leaves. Hydathodes form natural openings but, unlike stomata, are open permanently and offer little resistance to the flow of fluid out of leaves. Each hydathode is formed of colourless cells, and appears as stomata-like pores in the epidermis or epithem, also known as 'transfer tissue'. The cells of epithem are soft and made of loosely arranged thin-walled parenchyma cells and without chloroplast, and are involved in absorption and secretion. Internally, they are connected by tracheary endings to a large chamber with masses of thin-walled parenchymatous tissue surrounded by a sheath layer. Ultrastructurally, the epithem cells have a dense cytoplasm, numerous mitochondria, an extensive endoplasmic reticulum system, many small Golgi-derived vesicles, numerous peroxisomes, and are interconnected by abundant plasmodesmata. Functionally, there are two types of hydathodes, namely, epidermal ones that actively exude fluid, and epithemal hydathodes that passively exude fluid. Natural guttation is often observed during early morning or late hours of the day. However, it can also be induced as desired in intact or excised plants under pneumatic pressure. Earlier notions regarding harmful effects on plants of guttation have now been addressed by botanical and physiological research discoveries regarding the basic and practical utility of guttation. This knowledge could lead to new health care applications on the one hand and ease global food-security concerns on the other.
Guttation is a physiological phenomenon resulting in oozing of drops of xylem sap on the tips or edges of leaves of some vascular plants. Of 11 species belonging to different botanical families, only rice (Oryza sativa L.) was found to guttate heavily. Therefore, the process of guttation in rice was studied in depth. Guttation was found to occur from margins, tips, adaxial and abaxial surfaces of leaves. It was the leaf margins that exuded most (151 μl), followed by adaxial surface (135 μl), and tips (98 μl), in that order. Further, the “ridged” portions exuded more fluid than “smooth” portions of the same leaf blade. The rate of guttation was 132 μl at anthesis, 120 μl at tillering, 112 μl at heading and milky stages, and 42 μl at dough stage. The wind velocity and soil moisture stress, as external factors, played dominant roles in the regulation of guttation. Genotypic variability existed for guttation among six cultivars, with cv. NDRH-2 registering the highest value of 110 μl and Mahsuri the lowest (62 μl). The guttational rates exhibited by various cultivars were positively correlated with their panicle weights, i.e., the yield sink capacity, which may be used as a selection criterion for breeding for enhanced yield. These findings on various aspects of guttation in rice are novel. The information should be useful to breeders for creating a new efficient rice plant having altered genetic makeup for increased guttation for yield improvement.
Morpho-anatomical traits of the rarely studied dicotyledonous desiccation-tolerant shrub Myrothamnus moschatus were examined and compared for the first time to Myrothamnus flabellifolius under the aspect of desiccation tolerance. Both species almost exclusively occur on rock outcrops and differ mainly in their geographic range and leaf morphology (fan-shaped in M. flabellifolius, lanceolate in M. moschatus) but have a very similar leaf and wood anatomy, except for the lack of hydathodes in M. moschatus. Both species adopt the parallel leaf venation of monocots, although this is more pronounced in M. moschatus. This provides a mechanical and protective advantage over the net venation pattern of most dicots and facilitates the reversible, drought-induced, accordion-like leaf contraction. The sclerenchyma, as a stabilising tissue, is mainly confined to vascular bundles in leaves of both species. Here, mechanical support seems to be less crucial for survival in long periods of drought than other morpho-anatomical traits (e.g. parallel leaf venation).
The mechanisms of protection against mechanical and oxidative stress were identified and compared in the angiosperm resurrection plants Craterostigma wilmsii, Myrothamnus flabellifolius and Xerophyta humilis. Drying-induced ultrastructural changes within mesophyll cells were followed to gain an understanding of the mechanisms of mechanical stabilisation. In all three species, water filled vacuoles present in hydrated cells were replaced by several smaller vacuoles filled with non-aqueous substances. In X. humilis, these occupied a large proportion of the cytoplasm, preventing plasmalemma withdrawal and cell wall collapse. In C. wilmsii, vacuoles were small but extensive cell wall folding occurred to prevent plasmalemma withdrawal. In M. flabellifolius, some degree of vacuolation and wall folding occurred, but neither were sufficient to prevent plasmalemma withdrawal. This membrane was not ruptured, possibly due to membrane repair at plasmodesmata junctions where tearing might have occurred. In addition, the extra-cytoplasmic compartment appeared to contain material (possibly similar to that in vacuoles) which could facilitate stabilisation of dry cells.
Photosynthesis and respiration are particularly susceptible to oxidative stress during drying. Photosynthesis ceased at high water contents and it is proposed that a controlled shut down of this metabolism occurred in order to minimise the potential for photo-oxidation. The mechanisms whereby this was achieved varied among the species. In X. humilis, chlorophyll was degraded and thylakoid membranes dismantled during drying. In both C. wilmsii and M. flabellifolius, chlorophyll was retained, but photosynthesis was stopped due to chlorophyll shading from leaf folding and anthocyanin accumulation. Furthermore, in M. flabellifolius thylakoid membranes became unstacked during drying. All species continued respiration during drying to 10% relative water content, which is proposed to be necessary for energy to establish protection mechanisms. Activity of antioxidant enzymes increased during drying and remained high at low water contents in all species, ameliorating free radical damage from both photosynthesis and respiration. The nature and extent of antioxidant upregulation varied among the species. In C. wilmsii, only ascorbate peroxidise activity increased, but in M. flabellifolius and X. humilis ascorbate peroxidise, glutathione reductase and superoxide dismutase activity increased, to various extents, during drying. Anthocyanins accumulated in all species but this was more extensive in the homoiochlorophyllous types, possibly for protection against photo-oxidation.
The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.
O. L. LANGE, P. S. NOBEL, C. B. OSMOND, and H. ZIEGLER In the original series of the Encyclopedia of Plant Physiology, plant water relations and photosynthesis were treated separately, and the connection between phenomena was only considered in special chapters. O. STOCKER edited Vol ume III, Pjlanze und Wasser/Water Relations of Plants in 1956, and 4 years later, Volume V, Parts I and 2, Die COrAssimilation/The Assimilation of Carbon Dioxide appeared, edited by A. PIRSON. Until recently, there has also been a tendency to cover these aspects of plant physiology separately in most text books. Without doubt, this separation is justifiable. If one is specifically inter ested, for example in photosynthetic electron transport, in details of photophos phorylation, or in carbon metabolism in the Calvin cycle, it is not necessary to ask how these processes relate to the water relations of the plant. Accordingly, this separate coverage has been maintained in the New Series of the Encyclopedia of Plant Physiology. The two volumes devoted exclusively to photosynthesis are Volume 5, Photosynthesis I, edited by A. TREBST and M. AVRON, and Volume 6, Photosynthesis II, edited by M. GIBBS and E. LATZKO. When consider ing carbon assimilation and plant water relations from an ecological point of view, however, we have to recognize that this separation is arbitrary.
The vacuoles of three "resurrection" plants, Myrothamnus flabellifolia, Anastatica hierochuntica and Selaginella dregei were found to contain large quantities of osmiophilic material which may be part of the "resurrection" mechanism. Myrothamnus differed from the others by having mitochondria, and possibly plastids, which are separated from the remainder of the cytoplasm by sheaths or membranes during desiccation. Upon "resurrection" these barriers appear to be perforated and explain in part the faster rate of "resurrection" in Myrothamnus than in other "resurrection" plants. The chloroplasts of Myrothamnus are remarkable in that they possess "staircase" granum stacks of a type not previously described in any other plant tissue.
The extensive early literature on foliar uptake of moisture, spanning nearly three centuries, has been reviewed by Stone (1957a, 1970) and Gessner (1956a). Early field studies by a number of workers demonstrated water uptake by leaves or stems of intact plants (Lloyd 1905; Wetzel 1924; Krause 1935). These and other laboratory experiments (see Stone 1957 a, 1970) led to a unanimity of opinion on the ability of leaves to absorb water, but a strong divergence of views on the ecological and physiological significance of this uptake. Remarkably, however, the flow of papers on the subject over the past 50 years has done little to resolve the questions of significance, and foliar water uptake remains as controversial a subject as ever.
New studies on the anatomy of the leaf, stem, stolen and root of 17 taxa of Gunnera have revealed several interesting anatomical features. Scalariform perforation plates occur in most vessel elements of stolons and roots, although simple perforation plates are more frequent in the stems of large leafed-species. The leaf surface and internal anatomy have not been studied since the late 19th and early 20th centuries. In this paper, detailed studies of the leaf include features such as marginal and laminar hydathodes, 'warts', hair types, and groups of 2-5 palisade-like cells connected to each other by short communicating tubes. The anatomy of Gunnera herteri is described in detail for the first lime. Nostoc colonies in the leaf are illustrated. (C) 2000 Thr Linnean Society of London.
Studies of heat and mass exchange between leaves and their local environment are central to our understanding of plant-atmosphere interactions. The transfer across aerodynamic leaf boundary layers is generally described by non-dimensional expressions which reflect largely empirical adaptations of engineering models derived for flat plates. This paper reviews studies on leaves, and leaf models with varying degrees of abstraction, in free and forced convection. It discusses implecations of finding for leaf morphology as it affects – and is affected by – the local microclimate. Predictions of transfer from many leaves in plant communities are complicated by physical and physiological feedback mechanisms between leaves and their environment. Some common approaches, and the current challenge of integrating leaf-atmosphere interactions into models of global relevance, are also briefly addressed.
Bruniaceae have peculiar leaf tips, composed of thin walled cells containing reddish-brown accumulations; these cells often break open at maturity. Files of cells (which also contain the deposits) are produced by a meristem subadjacent to the tip cells; no hydathode occurs at the leaf tip, and no lateral teeth are present on leaves. All of these features occur in Geissoloma, and nearly all in Myrothamnus, in which the meristem is lacking. These similarities are thus considered indicative of relationship among these families. SEM studies of epicuticular waxes and longitudinally-oriented cuticular relief on leaves of the three families reveal similarities. The presence of ultrastructual grooving on trichomes, the uniseriate nonglandular nature of the trichomes, and the nonlignified nature of walls in the hairs are features by which Geissolomataceae resemble Bruniaceae. Multiple epidermis is newly reported for Geissoloma. The possible significance of intracellular pectates in the epidermal cells and the multiple epidermis of Geissoloma is discussed.
Single-membrane-bounded organelles containing crystalline inclusions surrounded by a granular matrix are found abundantly in the cells of epithem. Special attention is devoted to changes which occured in the fine structure of these crystalline inclusions and variations in their morphology are described.
The crystalline structure consists of highly organized units of macromolecular dimensions and, in thin section, the crystal is digested by pepsin.
Myrothamnus flabellifoliusWelw. is a desiccation-tolerant (‘resurrection’) plant with a woody stem. Xylem vessels are narrow (14 μm mean diameter) and
perforation plates are reticulate. This leads to specific and leaf specific hydraulic conductivities that are amongst the
lowest recorded for angiosperms (ks0.87 kg m−1MPa−1s−1; kl3.28×10−5kg m−1MPa−1s−1, stem diameter 3 mm). Hydraulic conductivities decrease with increasing pressure gradient. Transpiration rates in well watered
plants were moderate to low, generating xylem water potentials of -1 to -2 MPa. Acoustic emissions indicated extensive cavitation
events that were initiated at xylem water potentials of -2 to -3 MPa. The desiccation-tolerant nature of the tissue permits
this species to survive this interruption of the water supply. On rewatering the roots pressures that were developed were
low (2.4 kPa). However capillary forces were demonstrated to be adequate to account for the refilling of xylem vessels and
re-establishment of hydraulic continuity even when water was under a tension of -8 kPa. During dehydration and rehydration
cycles stems showed considerable shrinking and swelling. Unusual knob-like structures of unknown chemical composition were
observed on the outer surface of xylem vessels. These may be related to the ability of the stem to withstand the mechanical
stresses associated with this shrinkage and swelling.Copyright 1998 Annals of Botany Company
Caffeine at concentrations between 0.1% and 1% caused the condensation of phenolics inside the vacuoles of phenolic-storing
cells prepared for electron microscopy. Caffeine added to the glutaraldehyde and washing buffer during fixation prevented
the leaching of the phenolics into the cytoplasm. Phenolic-storing cells so treated had a cytoplasm similar in appearance
to that of cells not storing phenolics. In contrast, mature phenolic-storing cells fixed without caffeine had a dense, osmiophilic
cytoplasm. The appearance of the cytoplasm in these cells thus appears to represent an artefact of fixation.
Laminar hydathodes are known from only three dicot families. InUrticaceae they are associated with minor vein junctions in all five tribes, as surveyed from cleared leaves of 43 species in 30 genera. Only one species lacked hydathodes. Exclusively adaxial hydathodes were found in 28 genera. In tribeElatostemeae, laminar hydathodes inPilea andPellionia species are abaxial, adaxial, or on both surfaces. Guttation was observed in four species.Urtica dioica (adaxial) andPilea pumila (abaxial) were studied anatomically in detail. Hydathodes in the former have normal bundle structure but xylem gaps sometimes occur. In the latter, phloem is displaced in three previously undescribed ways: 1) ends abruptly near hydathode, 2) curves into connecting vein at adjacent junction, or 3) departs xylem, skirts hydathode independently, and rejoins adjacent xylem strand. Laminar hydathodes are a unifying character of theUrticaceae, and they also strengthen its close relationship to theMoraceae.
The single leaf trace branches to form two additional stem cortical traces; these enter the leaf and form a three-dimensional pattern. Most veins terminate in epithem hydathodes, which are distributed over the entire adaxial surface and as an irregular submarginal row on the abaxial surface. Each hydathode consists of the flared end of a vein in contact with epithem parenchyma which lacks obvious intercellular spaces. The epithem abuts on the epidermis and is in contact with the outside environment through a cluster of eight to ten hydathode stomata. Tanniniferous cells form a bundle sheath around the hydathode. In addition, they are scattered throughout the mesophyll and form a continuous subepidermal layer. The close association of each vein ending with a hydathode is considered to be important for mineral nutrition in the peculiar natural environment of this plant.
A technique for clearing and staining gymnosperm leaves is described that employs safranin with a fast green counterstain, resulting in the differential staining of cell types in whole leaves. It is also applicable to leaves and other organs of angiosperms and lower vascular plants. The procedure is simple and is recommended for teaching as well as research.
: Epidermal hydathodes were found on leaves of 46 of 48 species of Crassula collected from the Namib Desert in southern Africa. The possibility that these structures might allow the absorption of surface water was investigated in 27 species (including subspecies). The presence of hydathodes on leaf epidermi correlated, in most cases, with increases in leaf thickness and enhanced rates of nocturnal, and sometimes diurnal, CO2 uptake following wetting of the leaves during the night. The precise nature of these responses varied depending on the species. In addition, wetting only the older leaves on the lower portion of the shoot of C. tetragona ssp. acutifolia not only resulted in increased thickness of these leaves, but also effected an increase in leaf thickness and stimulation of CO2 uptake rates in the distal, younger portion of the shoot that was not wetted. Overall, foliar hydathodes were implicated in the absorption of surface water in many species of Crassula such that the ecophysiology of these desert succulents was positively affected. Although rainfall in the Namib Desert is infrequent, surface wetting of the leaves is a more common occurrence as a result of nighttime dew or fog deposition. Presumably, species with hydathodes benefit directly from this source of moisture. These findings have important implications in understanding a relatively unexplored adaptation of some xerophytes to an extremely arid environment.
Hydathodes have been reported as rare for the Asteraceae, but we collated published reports of guttation, water pores, and hydrathodes from ca. 80 genera of 10 tribes. Only a few incomplete anatomical descriptions were found. Leaf teeth with guttation droplets were removed for anatomical study from Ambrosia trifida, Arctium minus, Erigeron annuus, Eupatorium rugosum, Lactuca scariola, Rudbeckia laciniata, and Silphium perfoliatum. Conspicuous xylem, but very little phloem, enters each tooth. Phloem ends well before xylem termination. Xylem diverges distally into individual files of tracheary elements separated by parenchyma. Progressively toward the tooth apex, xylem parenchyma proliferates, and successive cells are at first more elongate with increasingly sinuous walls, then shorter, broader, and lobed. The epithem beyond xylem termination resembles ordinary mesophyll chlorenchyma. The bundle sheath is variably developed but always incomplete. Water pores resemble ordinary stomata but are mostly sunken, ...
This is the first anatomical study of hydathodes from subfamily Spiraeoideae. Fresh leaves of Physocarpus opulifolius were cleared, or processed for paraffin and plastic sections and scanning electron microscopy. Each marginal tooth apex bears an achlorophyllous hydathode, which is visible adaxially as a smooth epidermal pad studded with 15–25 small, sunken "water pores" usually covered by an unbroken cuticle. Ordinary stomata are larger, raised, and abaxial. Internally, an epithem of small, loosely arranged cells extends from the adaxial epidermis to the laterally broadened, single vein ending. Bundle and epithem are bounded by the bundle sheath, which extends to the epidermis at the periphery of the pad. Guttation neither was seen naturally nor could it be induced. Cleared leaves of herbarium specimens of the six Physocarpus species showed all degrees of hydathode reduction to complete absence.
Marginal epithem hydathodes may, in some conditions, absorb water. A toothed leaf margin can be regarded as a water-holding structure. It is suggested that the lack of hydathodes in north-temperate evergreens and in the leaves of rain forest trees is related to the need for such species to reduce foliar water absorption. The high proportion of deciduous species with hydathodes in woodland vegetation is thought to be related to the summer potential soil water deficit in Britain, leaf-absorbed water supplementing water from the soil. The term ‘potomorphic’ is suggested for leaves with water absorbing structures.
In Ficus diversifolia, “mistletoe fig”, hydathodes are scattered over the leaf lamina. They open to the adaxial surface via a circular, slightly depressed epidermal pad studded with 20–30 water pores. The epithem is a cylinder of small cells, irregular in shape, with conspicuous intercellular spaces, bounded by a close-fitting sheath of unpigmented cells. Hydathodes occur above characteristic junctions of three or more minor veins, from which short tracheary strands extend upward into the epithem and end blindly. Phloem does not seem to be involved. Unsuccessful attempts were made to induce guttation. Red pigment spots occur abaxially in the axils of major veins. They are compact internal disks of parenchyma cells containing granules of various sizes. A vascular bundle arches downward, passes over the disk, then curves upward to rejoin other bundles. The central large disk has a palisade epidermis of slender elongate cells separated from each other by cuticle; the other disks have a normal epidermis.
New studies on the anatomy of the leal’, stem, stolon and root of 1 7 taxa of Gumma have revealed several interesting anatomical features. Scalariform perforation plates occur in most vessel elements of stolons and roots, although simple perforation plates are more frequent in the stems of large leafed-species. The leaf surface and internal anatomy have not been studied since the late 19th and early 20th centuries. In this paper, detailed studies of the leaf include features such as marginal and laminar hydathodes. ‘warts’, hair types, and groups of 2 5 palisade-like tells connected to each other by short communicating tubes. The anatomy of Gumma lierleri is described in detail for the first time. .Xostoc colonies in the leaf are illustrated.
The rapid flow of the transpiration stream through major veins to leaf teeth was followed in leaves of Populus balsamifera L., using the tracer sulphorhodamine G (SR), which probes for cells with H+-extrusion pumps. The tracer accumulated quickly in the hydathodes of the teeth. It was shown by freeze-substitution and anhydrous processing that SR was taken up by phloem parenchyma and epithem cells of the hydathode. When 14C-labelled aspartate was fed to the leaves in the transpiration stream, it also was taken up most strongly by the same phloem parenchyma and epithem cells. It is proposed that one function of the hydathodes in leaf teeth is the retrieval of solutes from the transpiration stream.
Changes of view on the course of the transpiration stream beyond the veins in leaves are followed from the imbibition theory of Sachs, through the (symplastic) endosmotic theory of Pfeffer (which prevailed almost unquestioned until the late 1930s), to Strugger's experiments with fluorescent dye tracers and the epifluorescence microscope. This latter work persuaded many to return to the apoplastic‐(wall)‐path viewpoint, which, despite early and late criticisms that were never rebutted, is still widely held. Tracer experiments of the same kind are still frequently published without consideration of the evidence that they do not reveal the paths of water movement. Experiments on rehydration kinetics of leaves have not produced unequivocal evidence for either path. The detailed destinies of the solutes that reach the leaf in the transpiration stream have received little attention.
Consideration of physical principles governing flow and evaporation in a transpiring leaf emphasizes that: (1) Diffusion over interveinal distances at the rates in water will account for substantial solute movement in a few minutes, even in the absence of flow. (2) Diffusion can occur also against opposing now. (3) Volume fluxes in veins are determined by the diameter of the largest leaves examined contain high conductance supply veins which are tapped into by low‐conductance distributing veins. (4) Edges and teeth of leaves will be places of especially rapid evaporation, and they often have high‐conductance veins leading to them. (5) Solutes in the stream will tend to accumulate at leaf margins.
On the basis of recent work, the view is maintained that the water of the stream enters the symplast through cell membranes very close to tracheary elements. Also, that this occurs locally over a small area of membrane. Many solutes in the stream are left outside in the apoplast. This produces regions of high solute concentration in the apoplast and an enrichment of solutes in the stream as it perfuses the leaf. Solutes that enter the symplast are not so easily tracked. Suggestions about where some of them may go can be gained from a fluorescent probe that identifies particular cells (scavenging cells) as having H ⁺ ‐ATPase porter systems to scrub selected solutes from the stream.
Unpublished case‐histories are presented which illustrate many aspects of these processes and principles. These are: (1) Maize leaf veins, where the symplastic water path starts at the parenchyma sheath; (2) Lupin veins, where the symplastic path starts at the bundle sheath and where solutes are concentrated in blind terminations; (3) The edges of maize leaves where flow is enhanced by a large vein (open to the apoplast), and solutes are deposited in the apoplast by evaporation; (4) Poplar leaf teeth, which receive strong flows, and where the epithem cells are scavenging cells; (5) Mimosa leaf marginal hairs, which have scavenging cells at their base; (6) Active hydathodes, whose epithem cells are scavenging cells; (7) Pine needle transfusion tissue, which is a site of both solute enrichment (in the tracheids), and scavenging (in the parenchyma); (8) Estimates are made of diffusion coefficients of a solute both along and at right angles to the major diffusive pathway in wheat leaves. The first is 1000 times the second, but is 1/100 of free diffusion in water.
Five general themes of the behaviour and organization of the transpiration stream are induced from the facts reviewed. These are: (1) The stream is channelled into courses of graded intensities by the interplay of the physical forces with the anatomical features, each course with a distinct contribution to the processing of the stream. (2) Water enters the symplast at precise locations as close as possible to the tracheary elements. (3) As the stream moves through the leaf its solute concentration is enriched many‐fold at predictable sites. (4) Solutes excluded from the symplast diffuse from these sources of high concentration in specially formed wall paths, in precise patterns, at rates which can be measured, and which are low compared with diffusion in water. (5) Other solutes permeate the symplast, often over the surfaces of groups of cells which are organized into recognized structural features.
CONTENTS
Summary
341
I.
What becomes of the transpiration stream ?
342
II.
Review
343
III.
Preview
355
IV.
Overview
361
Acknowledgements
365
References
365
Guttating leaf teeth of Potentilla palustris plants from Wisconsin, USA, were cleared or processed for plastic sectioning or scanning electron microscopy. Anatomical features include: 1) long slender hydathode area occupying most of the tooth, 2) adaxial pad of small, flat epidermal cells with 50 or more sunken water pores about the size of ordinary abaxial stomates, 3) three converged bundles that extend distally, where their tracheary files are separated by intervening files of xylem parenchyma cells with sinuous walls, 4) adaxial mass of small, loosely arranged epithem cells above the xylem, 5) one slender phloem strand that extends only about a third of the way into the hydathode, and 6) bundle sheath extending distally only abaxially and along the flanks of the hydathode. Potentilla hydathodes differ significantly from non-guttating ones described earlier in Physocarpus (Rosaceae).
The vacuoles of three “resurrection” plants, Myrothamnus flabellifolia, Anastatica hierochuntica and Selaginella dregei were found to contain large quantities of osmiophilic material which may be part of the “resurrection” mechanism. Myrothamnus differed from the others by having mitochondria, and possibly plastids, which are separated from the remainder of the cytoplasm by sheaths or membranes during desiccation. Upon “resurrection” these barriers appear to be perforated and explain in part the faster rate of “resurrection” in Myrothamnus than in other “resurrection” plants. The chloroplasts of Myrothamnus are remarkable in that they possess “staircase” granum stacks of a type not previously described in any other plant tissue.
The resurrection plant Myrothamnus flabellifolia has the ability to recover from repeated prolonged and extreme desiccation cycles. During the dry state the inner walls of the xylem vessels seemed to be covered, at least partly, by a lipid film as shown by Sudan III and Nile Red staining. The lipid film apparently functioned as an ‘internal cuticle’ which prevented the adjacent parenchyma ray cells from complete water loss. The hydrophobic nature of the inner xylem walls was supported by the finding that benzene ascended as rapidly as water in the xylem of dry Myrothamnus branches. On watering, numerous lipid bodies were found in the water-conducting vessels, presumably formed from the lipid film and/or from lipids excreted from the adjacent living cells into the vessels. The presence of lipid bodies within the vessels, as well as the hydrophobic properties of the inner xylem walls, could explain the finding that the xylem pressure of hydrated, well watered plants (measured both under laboratory and field conditions with the xylem pressure probe) never dropped below c. −0.3 MPa and that cavitation occurred frequently at low negative xylem pressure values (−0.05 to −0.15 MPa). The xylem pressure of M. flabellifolia responded rapidly and strongly to changes in relative humidity and temperature, but less obviously to changes in irradiance (which varied between 10 and c. 4000 μmol m m−2 s−1). The morphological position of the stomata in the leaves could explain the extremely weak and slow response of the xylem pressure of this resurrection plant to illumination changes. Stomata were most abundant in the furrows, and were thus protected from direct sunlight. Simultaneous measurements of the cell turgor pressure in the leaf epidermal cells (made by using the cell turgor pressure probe) revealed that the xylem and the cell turgor pressure dropped in a ratio of 1:0.7 on changes in the environmental parameters, indicating a quite close hydraulic connection and, thus, water equilibrium between the xylem and cellular compartments. An increase in irradiance of c. 700 μmol m−2 s−1 resulted in a turgor pressure decrease from 0.63 to 0.48 MPa. Correspondingly, the cell osmotic pressure increased from 1.03 to 1.22 MPa. From these values and by assuming water equilibrium, the osmotic pressure of the xylem sap was estimated to be 0.25–0.4 MPa. This value seems to be fairly high but may, however, be explained by the reduction of the water volume within the vessels due to the floating lipid bodies.
The earlier observation that the fluorochrome sulphorhodamine G selectively enters cells of the extended bundle sheath system (paraveinal mesophyll) of soybean leaves from the transpiration stream, is extended to all the 26 species of legumes so far tested. The species examined were selected to include the three types of the system previously identified: Type 1 with complete systems, Type 2 with attenuated systems, and Type 3 with ordinary bundle sheaths. In Type 3 the dye selectively entered the bundle sheath cells. The hypothesis that the entry of the dye to a cell is caused by local low external pH, which suppresses its ionization, is confirmed by tests of the response of sulphorhodamine uptake to changing external pH, and of the inhibition of uptake by the ionophore dinitrophenol. An extension of this hypothesis identifies the local pH gradient as an energy source driving influx pumps to scavenge solutes from the transpiration stream and store them in special cells. It is proposed that this may be broadened to include many legumes. The activity of the proton extrusion pumps in this storage network is shown to be correlated with the flowering and fruiting of soybean, showing high activity before flowering, and disappearing during pod formation when nitrogenous materials are exported from the network.
The fluorescent probe sulphorhodamine G (SR) has been previously used as an indicator of low extra-cellular pH and, by inference, of proton extrusion activity in living leaves. In legumes the SR uptake and proton extrusion was characteristic of the extended bundle sheath system (EBS) or paraveinal mesophyll, composed of bundle sheath cells and the related network of bridging cells between veins. This system has been identified as a site of temporary storage of amino carbon in soybean. A tree species. Populus deltoides Bartr. ex Marsh, was known both to have the EBS system in its leaves and to carry organic nitrogen in its xylem sap. It is now shown that P. deltoides also accumulates the SR probe in the EBS system. This association has been explored in 8 other broad-leaved tree species. Seven of the 8 species have EBS systems and accumulate SR in them in early summer. The 8th species, Tilia americana L. has no EBS system and shows weak SR accumulation. The capacity to accumulate SR (and by inference to scavenge solutes from the transpiration stream) disappeared in all species at various stages in late summer. In two species, in addition, SR accumulation is interrupted for several weeks during fruit growth. It is proposed that EBS systems will be found in many dicotyledonous leaves, and will be found to scavenge solutes, especially organic nitrogen, from the xylem sap.
Abstract Stabilized microscopic preparations of an apoplastic fluorescent tracer, sulphorhodamine G (SR), have previously shown it confined to leaf cell walls. SR has a pK of 3.2, is dissociated at normal wall pH, and therefore does not enter cells. In transpiring soybean leaves, the SR showed a major internal water pathway in the walls of the paraveinal mesophyll (PVM), which has been implicated in the temporary storage of protein. Also the SR penetrated the PVM and bundle sheath cells, staining organelles and vacuoles, but not other leaf cells. This implies that sufficient SR is undissociated in these walls to allow penetration, and that the pH of the PVM walls is lower than that of most other cells. It is proposed that proton extrusion pumps are revealed by the low wall pH, and that these pumps are probably involved in collecting ammo acids from the transpiration stream.
Why the leaves of cold temperate deciduous and moisture-loving angiosperms are so often toothed has long puzzled biologists because the functional consequences of teeth remain poorly understood. Here we provide functional and structural evidence that marginal leaf teeth of Chloranthus japonicus, an understory herb, enable the release of guttation sap during root pressure. When guttation from teeth hydathodes was experimentally blocked, we found that the leaf intercellular airspaces became flooded. Measurements of chlorophyll a fluorescence revealed that internal flooding resulted in an inhibition of photosynthesis, most likely through the formation of a film of water within the leaf that reduced CO2 diffusion. Comparing a developmental series of leaves with and without teeth experimentally covered with wax, we found that teeth did not affect overall leaf stomatal conductance and CO2 uptake. However, maximal and effective light-saturation PSII quantum yields of teeth were found to be lower or equal to the surrounding lamina throughout leaf ontogeny. Collectively, our results suggest hydathodes and their development on teeth apices enable the avoidance of mesophyll flooding by root pressure. We discuss how these new findings bear on the potential physiological interpretations of models that apply leaf marginal traits to infer ancient climates.
Drought tolerance limits are given for 36 new resurrection plants, sufficient to double the number of desiccation tolerant plants reported from southern Africa. Tolerance limits for angiosperm examples are usually better than those for ferns. Air-dry foliage survives for 1/2 to 5 years or more, unless stored in humidities above 50% RH1. Dehydration is sufficiently slow (usually 2–3 days) to allow the possibility of a tolerance induction process, like that found in Borya nitida. Rehydration after rain is usually complete in 1/2 to 1 day. A significant proportion of rain is absorbed through the leaf surface, but there is no evidence of appreciable rehydration from dew.Resurrection plants are usually pioneers in xeroseres, but they often lack xeromorphic characteristics. Anthocyanin pigmentation during drying is a reliable indicator of viability in some species.
The effect of drying rate on the survival of three angiosperm resurrection plants, Craterostigma wilmsii (homoiochlorophyllous), Xerophyta humilis (poikilochlorophyllous) and Myrothamnus flabellifolius (homo-iochlorophyllous) was examined. All species survived slow drying, but only C. wilmsii was able to survive rapid drying. C. wilmsii was rapidly able to induce protection mechanisms such as folding of cell walls to prevent mechanical stress and curling of leaves to minimize light stress, and thus survived fast drying. Rapid drying of X. humilis and M. flabellifolius appeared to allow insufficient time for complete induction of protection mechanisms. In X. humilis, there was incomplete replacement of water in vacuoles, the photosynthetic apparatus was not dismantled, plasma membrane disruption occurred and quantum efficiency of photosystem II (F V \\F M) did not recover on rehydration. Rapidly dried leaves of M. flabellifolius did not fold tightly against the stem and F V \\F M did not recover. Ultrastructural studies showed that subcellular damage incurred during drying was exacerbated on rehydration. The three species co-occur in environments in which they experience high desiccation pressures. C. wilmsii has few features to retard water loss and thus the ability for rapid induction of subcellular protection is vital to survival. X. humilis and M. flabellifolius are able to retard water loss and protection is acquired relatively slowly. # 1999 Annals of Botany Company
Under water-limiting conditions excitation energy har-nessed by chlorophyll can lead to the formation of reactive oxygen species (ROS). Resurrection plants minimize their formation by preventing the opportunity for light–chloro-phyll interaction but also quench them via antioxidants. Poikilochlorohyllous species such as X erophyta humilis break down chlorophyll to avoid ROS formation. Homoio-chlorophyllous types retain chlorophyll. We proposed that leaf folding during drying of Craterostigma wilmsii and Myrothamnus flabellifolius shades chlorophyll to avoid ROS (Farrant, Plant Ecology 151, 29–39, 2000). This was tested by preventing leaf folding during drying in light. As controls, plants were dried without light, and X. humilis was included. Craterostigma wilmsii did not survive drying in light if the leaves were prevented from folding, despite protection from increased anthocyanin and sucrose and ele-vated antioxidant enzyme activity. Membranes were dam-aged, electrolyte leakage was elevated and plastoglobuli (evidence of light stress) accumulated in chloroplasts. Restrained leaves of M. flabellifolius survived drying in light. Leaf folding allows less shading, but the extent of chemical protection (anthocyanin content and antioxidant activity) is considerably higher in this species compared with C. wilmsii . Chemical protection appears to be light regulated in M. flabellifolius but not in C. wilmsii . Drying in the dark resulted in loss of viability in the homoiochlo-rophyllous but not the poikilochlorophyllous species. It is hypothesized that some of the genes required for protec-tion are light regulated in the former.
An optical brightener Calcofluor White M2R New has been used to stain cell walls of higher plants. It can he used either as a vital stain for intact plants or for hand sections and plastic-embedded thin sections. Walls are brilliantly fluorescent while most cytoplasmic components are normally unstained. The brightener binds strongly to cellulose, carborylated polysaccbaridcs, and callose. Staining for 20 sec to 2 min in a 0.01% solution of the brightener is preferred for most purposes. © 1975 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
A low-viscosity embedding medium based on ERL-4206 is recommended for use in electron microscopy. The composition is: ERL-4206 (vinyl cyclohexene dioxide) 10 g, D.E.R. 736 (diglycidyl ether of polypropylene glycol) 6 g, NSA (nonenyl succinic anhydride) 26 g, and S-1 (dimethylaminoethanol or DMAE) 0.4 g. The medium is easily and rapidly prepared by dispensing the components, in turn by weight, into a single flask. The relatively low viscosity of the medium (60 cP) permits rapid mixing by shaking and swirling. The medium is infiltrated into specimens after the use of any one of several dehydrating fluids, such as ethanol, acetone, dioxan, hexylene glycol, isopropyl alcohol, propylene oxide, and tert.-butyl alcohol. It is compatible with each of these in all proportions. After infiltration the castings are polymerized at 70°C in 8 hours. Longer curing does not adversely affect the physical properties of the castings. Curing time can be reduced by increasing the temperature or the accelerator, S-1, or both; and the hardness of the castings is controlled by changes in the D.E.R. 736 flexibilizer. The medium has a long pot life of several days and infiltrates readily because of its low viscosity. The castings have good trimming and sectioning qualities. The embedding matrix of the sections is very resistant to oxidation by KMnO4 and Ba(MnO4)2, compared with resins containing NADIC methyl anhydride. Sections are tough under the electron beam and may be used without a supporting membrane on the grids. The background plastic in the sections shows no perceptible substructure at magnifications commonly used for biological materials. The medium has been used successfully with a wide range of specimens, including endosperms with a high lipid content, tissues with hard, lignified cell walls, and highly vacuolated parenchymatous tissues of ripe fruits.
The acropetal water refilling kinetics of the dry xylem of branches (up to 80 cm tall) of the resurrection plant Myrothamnus flabellifolia were determined with high temporal resolution by observation of light refraction at the advancing water front and the associated recurving of the folded leaves. To study the effect of gravity on water rise, data were acquired for cut upright, horizontal and inverted branches. Water rise kinetics were also determined with hydrostatic and osmotic pressure as well as at elevated temperatures (up to 100 degrees C) under laboratory conditions and compared with those obtained with intact (rooted) and cut branches under field conditions. Experiments in which water climbed under its capillary pressure alone, showed that the axial flow occurred only in a very few conducting elements at a much higher rate than in many of the other ones. The onset of transpiration of the unfolded and green leaves did not affect the rise kinetics in the 'prominent' conducting elements. Application of pressure apparently increased the number of elements making a major contribution to axial xylem flow. Analysis of these data in terms of capillary-pressure-driven water ascent in leaky capillaries demonstrated that root pressure, not capillary pressure, is the dominant force for rehydration of rooted, dry plants. The main reasons for the failure of capillary forces in xylem refilling were the small, rate-limiting effective radii of the conducting elements for axial water ascent (c. 1 micrometer compared with radii of the vessels and tracheids of c. 18 micrometers and 3 micrometers, respectively) and the very poor wetting of the dry walls. The contact (wetting) angles were of the order of 80 degrees and decreased on root or externally applied hydrostatic pressure. This supported our previous assumption that the inner walls of the dry conducting elements are covered with a lipid layer that is removed or disintegrates upon wetting. Consistent with this, potassium chloride and, particularly, sugars exerted an osmotic pressure effect on axial water climbing (reflection coefficients > zero, but small). Although the osmotically active solutes apparently suppressed radial water spread through the tissue to the leaf cells, they reduced the axial water ascent rather than accelerating it as predicted by the theory of capillary-driven water rise in leaky capillaries. Killing cells by heat treatment and removal of the bark, phelloderm, cortex and phloem also resulted in a reduction of the axial rise rate and final height. These observations demonstrated that radial water movement driven by the developing osmotic and turgor pressure in the living cells was important for the removal of the lipid layer from the walls of those conducting elements that were primarily not involved in water rise. There is some evidence from field measurements of the axial temperature gradients along rooted branches that interfacial (Marangoni) streaming facilitated lipid removal (under formation of vesicle-like structures and lipid bodies) upon wetting. Grant numbers: 50WB 9643, Zi 99/9-1.
Contributions from the Bolus Herbarium No. 8 Part 1. A Revision of the Genus Crassula in Southern Africa. The Bolus Herbarium, Rondebosch Life Strategies of Succulents in Deserts with Special Reference to the Namib Desert
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- B M Eller
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- H.-D Ihlenfeldt
Toï, H.R., 1977. Contributions from the Bolus Herbarium No. 8 Part 1. A Revision of the Genus Crassula in Southern Africa. The Bolus Herbarium, Rondebosch. von Willert, D.J., Eller, B.M., Weger, M.J.A., Brinckmann, E., Ihlenfeldt, H.-D., 1992. Life Strategies of Succulents in Deserts with Special Reference to the Namib Desert. Cambridge University Press, Cambridge.
Desiccation tolerance in Myrothamnus flabellifolia Welw
- D.-A Goldsworthy
Goldsworthy, D.-A., 1992. Desiccation tolerance in Myro-thamnus flabellifolia Welw. MSc thesis, University of Natal, Pietermaritzburg, South Africa.
Plant Anatomy, Part I, Cells and Tissues
- E G Cutter
Cutter, E.G., 1978. Plant Anatomy, Part I, Cells and Tissues. Edward Arnold, London.
A comparison of the structure of the hydathodes of selected species of Crassula (Crassulaceae)
- Buswell
Buswell, A., Drennan, P.M., 1991. A comparison of the structure of the hydathodes of selected species of Crassula (Crassulaceae). Commun. Electron Microsc. Soc. S. Afr. 21, 77–78.
Anatomie von Myrothamnus flabellifolius Welw
- Grundell
Grundell, R., 1933. Anatomie von Myrothamnus flabellifolius Welw. Symb. Bot. Ups. 2, 3–17.
What becomes of the transpiration stream? New Phytol
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Anhydrous fixa-tion of desiccated leaves of Myrothamnus flabellifolius (Welw.). Commun. Electron Microsc
- D.-A Goldsworthy
- P M Drennan
Goldsworthy, D.-A., Drennan, P.M., 1991. Anhydrous fixa-tion of desiccated leaves of Myrothamnus flabellifolius (Welw.). Commun. Electron Microsc. Soc. S. Afr. 21, 105–106.