Silicification of the adaxial epidermis of leaves of a panicoid grass in relation to leaf position and section and environmental conditions

Many studies relate silica content in plants with internal or external factors; however, few works analyse the effect of these factors on the silicification of different cell types. In this study, we examined the effect of leaf section and leaf position, and environmental conditions on the percentages of silicified epidermal cells of a native Pampean panicoid grass, Bothriochloa laguroides D. C. Pilger. Two different environmental situations were selected for the collection of plants: a natural wetland and a quartzite quarry, located in the southeast Buenos Aires province, Argentina. Clarification and staining methodologies were applied so as to study the distribution of silicified cells in different sections of leaves of the plants collected. Two and three-factor anovas were applied to the data. Between 13% and 19% of total cells of the adaxial epidermis of leaf blades were silicified. Typical silica short cells were the largest contributor to total silicified cells (53-98%), while the second largest contributor was bulliform cells (0-30%). Percentages of total silicified cells were higher in superior than in inferior leaves, while values from leaf sections varied. When collection sites were compared, plants growing in Los Padres pond, where the silica content in soils is higher, had the higher percentage of silicified cells. Among all types of cell, bulliform cells showed differences in the proportion of silicified cells between leaf position and section and collection site. These results show that silica availability in soils is an important factor that conditions silica accumulation and overlaps with the transpiration effect.     

Some Factors in Relation to Bulliform Cell Silicification in the Grass Leaf

The formation of discrete ‘tablets’ of hydratedsilica in the bulliform cells of the leaf blade was followedover a 16-day period in three species of the Gramineae representingdifferent habitats. Seedlings of Oryza sativa (rice) and Cynodondactylon (Bermuda Grass) were cultured under growth-cabinetconditions at levels of 50 and 500 ppm dissolved silica (SiO₂)in the nutrient solution. In addition, bulliform depositionwas studied in mature tiller leaves of Sieglingia decumbens(Heath Grass). Attached leaves, as well as those excised fromthe culm, were used. Initial stages of tablet formation were observed by the 2-dayharvest in the central and basal zones of the fully expandedseedling blades. Deposition did not occur at a stage when bulliformturgor changes might influence blade evolvement. At the 16-dayharvest, deposition was heaviest in the tip zone, and decreasedprogressively towards the base of the blade. In addition, proportionatelyhigher tablet counts (P = 0.05) generally were absent from theleaves grown at the higher silica level. This indicated a limitedavailability of deposition sites. These results are discussed in relation to (i) cellular maturation;(ii) internal leaf anatomy; (iii) leaf expansion; (iv) a basipetalsenescence gradient within the leaf blade. Certain of theseare considered to be possible limiting factors to silica depositionin the grass leaf.     

Subcellular localization of silicon and germanium in grass root and leaf tissues by SIMS: evidence for differential and active transport

Silicon transport and incorporation into plant tissue is important to both plant physiological function and to the influence plants have on ecosystem silica cycling. However, the mechanisms controlling this transport have only begun to be explored. In this study, we used secondary ion mass spectrometry (SIMS) to image concentrations of Si in root and shoot tissues of annual blue grass (Poa annua L.) and orchard grass (Dactylis glomerata L.) with the goal of identifying control points in the plant silica uptake pathway. In addition, we used SIMS to describe the distributions of germanium (Ge); the element used to trace Si in biogeochemical studies. Within root tissue, Si and Ge were localized in the suberized thick-walled region of endodermal cells, i.e. the proximal side of endodermal cells which is in close association to the casparian strip. In leaves, Si was present in the cell walls, but Ge was barely detectable. The selective localization of Si and Ge in the proximal side of endodermal cell walls of roots suggests transport control is exerted upon Si and Ge by the plant. The absence of Si in most root cell walls and its presence in the cell walls of leaves (in areas outside of the transpiration terminus) suggests modifications in the chemical form of Si to a form that favors Si complexation in the cell walls of leaf tissue. The low abundance of Ge in leaf tissue is consistent with previous studies that suggest preferential transport of Si relative to Ge.     

Function of silica bodies in the epidermal system of rice (Oryza sativa L.): testing the window hypothesis

Silicon has been considered to be important for normal growthand development of the rice plant (Oryza sativa L.). To investigatethe physiological function of deposited silica in rice leaves,the hypothesis that silica bodies in the leaf epidermal systemmight act as a ‘window’ to facilitate the transmissionof light to photosynthetic mesophyll tissue was tested. Thesilica content of leaves increased with supplied silicon andwas closely correlated with the number of silica bodies perunit leaf area in the epidermal system. There was a significantdifference in silica deposition and formation of silica bodiesbetween Si-treated and non-treated leaves; silicon was polymerizedinside the silica cells and bulliform cells of the epidermis,in Si-treated leaves. Although the ‘windows’ wereonly formed in leaves with applied silicon, optical propertiesof leaf transmittance, reflectance and absorptance spectra inSi-treated and non-treated leaves were almost equal. Furthermore,light energy use efficiency and quantum yield of Si-treatedleaves were less than in leaves not containing silica bodies.Thus, silica bodies, at least based on the data, do not functionas windows in rice leaves. Key words: Silicon, window hypothesis, rice, optical property, quantum yield     

Distribution of Silicified Cells in the Leaf Blades of Pleioblastus chino(Franchet et Savatier) Makino (Bambusoideae)

Distribution of silicified cells in the leaf blades of Pleioblastuschino was investigated using a light microscope and a scanningelectron microscope equipped with an energy dispersive X-raymicroanalyser. The most dense accumulation of silica was foundin epidermal tissues. Little silica was deposited in vascularbundles and chlorenchyma, while more was deposited in bundlesheath and fusoid cells. In the epidermis, silica density andfrequency of silicified cells differed depending on cell type,although silica deposition was observed in most cell types.Heavy deposition was found in silica cells, bulliform cells,micro hairs and prickle hairs. Silica cells were the cell typemost frequently silicified (96.9–99.7%) in the adaxialand abaxial epidermis. Silica may be deposited as leaf tissuesage.Copyright 2000 Annals of Botany Company Pleioblastus chino(Franchet et Savatier) Makino, bamboo, silicified cells, leaf blade, epidermis, chlorenchyma, silica, clearing method, freeze-fracturing, freeze-drying, light microscopy, scanning electron microscopy, X-ray microanalysis     

Function of silica bodies in the epidermal system of rice (Oryza sativa L.): testing the window hypothesis

Silicon has been considered to be important for normal growth and development of the rice plant (Oryza sativa L.). To investigate the physiological function of deposited silica in rice leaves, the hypothesis that silica bodies in the leaf epidermal system might act as a 'window' to facilitate the transmission of light to photosynthetic mesophyll tissue was tested. The silica content of leaves increased with supplied silicon and was closely correlated with the number of silica bodies per unit leaf area in the epidermal system. There was a significant difference in silica deposition and formation of silica bodies between Si-treated and non-treated leaves; silicon was polymerized inside the silica cells and bulliform cells of the epidermis, in Si-treated leaves. Although the 'windows' were only formed in leaves with applied silicon, optical properties of leaf transmittance, reflectance and absorptance spectra in Si-treated and non-treated leaves were almost equal. Furthermore, light energy use efficiency and quantum yield of Si-treated leaves were less than in leaves not containing silica bodies. Thus, silica bodies, at least based on the data, do not function as windows in rice leaves.     

Silicification of bamboo (Phyllostachys heterocycla Mitf.) root and leaf

Bamboo is a silicon accumulating plant. In leaves, the major place of silicon (Si) deposition is the epidermis, with the highest concentration of Si in silica cells. In bamboo roots, the deposition of Si is found only in endodermal cell walls. The silicification of leaves and roots was examined in the economically important bamboo plant Phyllostachys heterocycla, using an environmental scanning electron microscope coupled with X-ray microanalysis, as well as gravimetric quantification. The content of Si on a dry weight basis measured by gravimetric quantification was 7.6% in leaves and 2.4% in roots, respectively. Moreover, quantification of EDX data showed high Si impregnation of the inner tangential endodermal walls. Si content in this part of the root endodermal cell walls was even higher than that in the outer leaf epidermal walls, where conspicuous deposition of Si often occurs in grass plants.     

Observations on the Distribution of Mineral Elements in the Leaf of Wheat (Triticum aestivum L.), with Particular Reference to Silicon

Wheat (Triticum aestivum L. cv. Wheaton) plants were grown inwater culture or in soil. Basal leaves (B) were harvested after3 weeks from the water culture plants, while flag leaves werecollected from soil-grown material at the time of inflorescenceemergence (E0) and 7 d after emergence (E + 7). Mineral distributionin bulk frozen leaves was investigated using SEM and X-ray microanalysis.The elements detected were silicon, phosphorus, chlorine, sulphur,potassium and calcium. Potassium was present in all cell typesat all harvests, chlorine was almost entirely confined to theadaxial and abaxial epidermi, while sulphur was only rarelydetected in the E0 and E + 7 leaves. Phosphorus was presentat higher levels in the E + 7 leaves than in the B or E0 leaves.At the B harvest calcium was confined to the adaxial epidermalcells, but in the E0 and E + 7 leaves it was present in bothepidermi. Silicon was, initially, mainly detected in the abaxialepidermal cells, but in older (E + 7) leaves it was presentin both epidermi and in some internal tissues. Mineral transportwithin the leaf and ionic environment at silica deposition sitesare discussed. Wheat, Triticum aestivum L., leaf, mineral distribution, X-ray microanalysis, silicon, calcium     

Defense Responses in Rice Induced by Silicon Amendment against Infestation by the Leaf Folder Cnaphalocrocis medinalis

Silicon (Si) amendment to plants can confer enhanced resistance to herbivores. In the present study, the physiological and cytological mechanisms underlying the enhanced resistance of plants with Si addition were investigated for one of the most destructive rice pests in Asian countries, the rice leaf folder, Cnaphalocrocis medinalis (Guenée). Activities of defense-related enzymes, superoxide dismutase, peroxidase, catalase, phenylalanine ammonia-lyase, and polyphenol oxidase, and concentrations of malondialdehyde and soluble protein in leaves were measured in rice plants with or without leaf folder infestation and with or without Si amendment at 0.32 g Si/kg soil. Silicon amendment significantly reduced leaf folder larval survival. Silicon addition alone did not change activities of defense-related enzymes and malondialdehyde concentration in rice leaves. With leaf folder infestation, activities of the defense-related enzymes increased and malondialdehyde concentration decreased in plants amended with Si. Soluble protein content increased with Si addition when the plants were not infested, but was reduced more in the infested plants with Si amendment than in those without Si addition. Regardless of leaf folder infestation, Si amendment significantly increased leaf Si content through increases in the number and width of silica cells. Our results show that Si addition enhances rice resistance to the leaf folder through priming the feeding stress defense system, reduction in soluble protein content and cell silicification of rice leaves.     

A transporter regulating silicon distribution in rice shoots

Rice (Oryza sativa) accumulates very high concentrations of silicon (Si) in the shoots, and the deposition of Si as amorphous silica helps plants to overcome biotic and abiotic stresses. Here, we describe a transporter, Lsi6, which is involved in the distribution of Si in the shoots. Lsi6 belongs to the nodulin-26 intrinsic protein III subgroup of aquaporins and is permeable to silicic acid. Lsi6 is expressed in the leaf sheath and leaf blades as well as in the root tips. Cellular localization studies revealed that Lsi6 is found in the xylem parenchyma cells of the leaf sheath and leaf blades. Moreover, Lsi6 showed polar localization at the side facing toward the vessel. Knockdown of Lsi6 did not affect the uptake of Si by the roots but resulted in disordered deposition of silica in the shoots and increased excretion of Si in the guttation fluid. These results indicate that Lsi6 is a transporter responsible for the transport of Si out of the xylem and subsequently affects the distribution of Si in the leaf.     

Silica deposition in abaxial epidermis before the opening of leaf blades of Pleioblastus chino (Poaceae, Bambusoideae)

BACKGROUND AND AIMS Silica deposition is one of the important characteristics of the family Poaceae. The distribution, deposition process and physiology of silica in this family have been extensively investigated. Bamboos among members of Poaceae have leaves with a fairly long life span, and the leaves continuously accumulate silica in their tissues throughout their life, not only during the course of leaf opening, but also after opening. It has been revealed that the silica deposition process in relation to ageing of the bamboo leaf after opening differed depending on the cell types comprising the tissues. However, silica deposition has never been examined during the development and maturation periods of bamboo leaves. Hence, to clarify the silica deposition process in a developmental stage of the bamboo leaf, distribution of silica was observed in the abaxial epidermis before the opening of the leaf blades of Pleioblastus chino. METHODS: Abaxial epidermal tissues of leaves were examined using a scanning electron microscope equipped with an energy dispersive X-ray microanalyser. KEY RESULTS: Among seven cell types comprising the abaxial epidermis, three types of cells, guard cells, prickle hairs and silica cells, deposited silica conspicuously, and another four types, cork cells, long cells, micro hairs and subsidiary cells, deposited only a little silica. Among the former group of cell types, silica cells and guard cells deposited silica over their entire surfaces, while prickle hairs deposited silica only in the point-tips. Silica deposition was detected firstly in prickle hairs, and then in silica cells and guard cells. Only silica cells were assumed to deposit silica conspicuously before leaf opening but not conspicuously after opening. CONCLUSIONS: Cell types in leaf epidermis of bamboo are classified into three groups according to the silica deposition pattern. Silica deposition in silica cells may be positive as a part of the physiological activities of leaves.     

Availability and toxicity of cadmium to forage grasses grown in contaminated soil

It is important to know the mechanisms for forage development, especially those related to the tolerance of potentially toxic elements, when considering their use in phytoremediation in heavy metal contaminated areas. In this study, we evaluated plant growth, concentration, and the availability of cadmium (Cd) for forage grasses (Panicum maximum Jacq. cv. Aruana and cv. Tanzânia; Brachiaria decumbens cv. Basilisk; Brachiaria brizantha cv. Xaraés and cv. Marandu) cultivated in Cd contaminated soils. The experiments were performed under greenhouse conditions over a 90-day evaluation period, and the Cd rates were 2, 4, and 12 mg/kg of soil. The relative growth rate of the forage grasses decreased as Cd rates increased, and the following descending order of susceptibility was observed: Marandu > Xaraés > Aruana > Tanzânia > Basilisk, with regard to phytotoxicity in these plants. The forage Cd concentration increased in line with increases in the Cd rates. Cd contents extracted by Mehlich-1 and by diethylenetriaminepentaacetic acid presented high positive correlation with forage relative growth. The forage plants did not block Cd entry into the food chain because they were not capable of limiting Cd absorption.     

Effect of silicon deficiency on secondary cell wall synthesis in rice leaf

Rice (Oryza sativa L.) is a typical Si-accumulating plant and is able to accumulate Si up to >10?% of shoot dry weight. The cell wall has been reported to become thicker under Si-deficient condition. To clarify the relationship between Si accumulation and cell wall components, the physical properties of, and macromolecular components and Si content in, the pectic, hemicellulosic, and cellulosic fractions prepared from rice seedlings grown in hydroponics with or without 1.5?mM silicic acid were analyzed. In the absence of Si (the ?Si condition), leaf blades drooped, but physical properties were enhanced. Sugar content in the cellulosic fraction and lignin content in the total cell wall increased under ?Si condition. After histochemical staining, there was an increase in cellulose deposition in short cells and the cell layer just beneath the epidermis in the ?Si condition, but no significant change in the pattern of lignin deposition. Expression of the genes involved in secondary cell wall synthesis, OsCesA4, OsCesA7, OsPAL, OsCCR1 and OsCAD6 was up-regulated under ?Si condition, but expression of OsCesA1, involved in primary cell wall synthesis, did not increase. These results suggest that an increase in secondary cell wall components occurs in rice leaves to compensate for Si deficiency.     

Do lignification and silicification of the cell wall precede silicon deposition in the silica cell of the rice (Oryza sativa L.) leaf epidermis?

Aims

Rice is a well-known silica-accumulating plant. The dumbbell-shaped silica bodies in the silica cells in rice leaf epidermis are formed via biosilicification, but the underlying mechanisms are largely unknown.

Methods

Leaves at different developmental stages were collected to investigate silica cell differentiation by analyzing structures and silicon localization in the silica cells.

Results

Exogenous silicon application increased both shoot and root biomass. When silicon was supplied, silica cells in the leaf epidermis developed gradually into a dumbbell-shape and became increasingly silicified as leaves aged. Silicon deposition in the silica cells was not completed until the leaf was fully expanded. Multiple lines of evidence suggest that lignification of silica cell walls precedes silicon deposition in the lumen of silica cells. The organized needle-like silica microstructures were formed by moulding the inner cell walls and filling up the lumen of the silica cell following leaf maturation.

Conclusions

Two processes were involved in silicon deposition: (1) the silica cell wall was lignified and silicified, and then (2) the silicon was deposited gradually in silica cells as leaves aged. Silica body formation was not completed until the leaf was fully mature.     

Silicon Deposition in the Roots, Culm and Leaf of Phalaris canariensis L

Silicon deposition in the roots, culm and leaf of canary grass(Phalaris canariensis L.) was investigated using light microscopy,scanning electron microscopy and electron probe microanalysis. In adventitious roots grown in solution silicon was concentratedin four endodermal walls. Silicon was not detected in the endodermisof aerial adventitious roots, but was present in the epidermisand outer cortical cell layers. Silicon deposition in the culm mainly took place in the epidermis,and particularly in epidermal papillae. The silica deposition pattern in the leaf was typical of thesub-group Festucoideae. The leaf blade showed deposits in costalprickle hairs and wavy rods, but few intercostal deposits. Inthe ligule deposition was confined to isolated groups of pricklehairs on the abaxial surface. The major sites of silica depositionin the leaf sheath were the stomatal subsidiary cells, papillaeand intercostal idioblasts. Prickle hairs were much less commonin the sheath than the blade, and costal wavy rods appearedto be absent in the sheath. Phalaris canariensis L., canary grass, silicification, root, culm, leaf, electron probe microanalysis     

Studies on the feeding behaviour of the eriophyid mite Abacarus hystrix, a vector of grass viruses

On Italian ryegrass (Lolium multiflorum cv. S 22), the eriophyid mite Abacarus hystrix usually fed on the bulliform epidermal cells at the base of the grooves on the adaxial leaf surface. Scanning electron micrographs illustrate (1) the external morphology of the mouthparts of the mites, (2) how, prior to feeding, they remove cuticular leaf wax with their rostra and (3) feeding punctures in epidermal cells made by the styliform chelicerae. Of eleven graminaceous species tested, A. hystrix only settled on Festuca pratensis, L. perenne and L. multiflorum. On L. multiflorum, the mites preferred the youngest leaves and on mature leaves, preferred the youngest part.     

Intracellular Silica Deposition in Mature and Senescent Leaves of Sieglingia decumbens (L.) Bernh

Following 1, 2, 4, or 8 weeks of growth in a silica-minimalsolution, tillers of Sieglingia decumbens (Heath Grass) weretransferred for 8 days to a nutrient solution which contained50 or 100 ppm silica. The resultant formation of intracellularopal phytoliths (silica bodies) was compared for the four developmentalstages of leaf 3. Characteristically different phytolith typesoccurred in leaves of different ages. Senescent, in contrastto younger, mature leaves, typically exhibited extensive extracellularsilicification of the mesophyll, in addition to deposits instomatal, long, and bulliform cells of adaxial epidermis; depositionin abaxial silica cells and long cells, characteristic of youngleaves, was much reduced, or absent. Physico-chemical factors and cytoplasmic changes associatedwith senescence are discussed in relation to intracellular opalphytolith formation in mature leaves of S. decumbens. In thisrespect, the tendency of silicic acid sols to polymerize inthe presence of an organic matrix and mineral cations is consideredto be significant.     

Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.     

Detection of Biogenic Silica in Leaf Blade, Leaf Sheath, and Stem of Bermuda Grass (Cynodon dactylon) Using LIBS and Phytolith Analysis

Laser induced breakdown spectroscopy (LIBS) has been used to perform in situ analysis of major and minor elements present in the different parts of the Bermuda grass (Cynodon dactylon). In situ, point detection/analysis of the elements in plants without any sample preparation has been demonstrated. LIBS spectra of the different parts (leaf blade, leaf sheath and stem) of fresh C. dactylon plant have been recorded to study the pattern of silica deposition in its different parts. Atomic lines of Si, Mg, Ca, C, Al, Zn, N, Sr, etc. have been observed in the LIBS spectra of the C. dactylon. A close observation of LIBS spectra of the different parts of the plants shows that silica concentration is greater in leaf blades than leaf sheaths and stems. The results obtained with LIBS analysis are also compared with the number density of phytoliths deposited in different parts of C. dactylon. It is observed that the highest silicified cell frequency is present in leaf blades followed by leaf sheaths and stems which is in close agreement with LIBS analysis.