Cell type-specific protoberberine alkaloid accumulation inThalictrum flavum

Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.     

An easy method for cutting and fluorescent staining of thin roots

Background and Aims

Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here.

Methods

To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light.

Key Results

Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots.

Conclusions

This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.     

The pulvinus endodermal cells and their relation to leaf movement in legumes of the Brazilian cerrado

Legume pulvini have a clearly delimited endodermis, whose variable content has been associated with the velocity and type of leaf movement: pulvini in leaves with fast nastic movement contain starch grains; pulvini in leaves with slow nastic movements have calcium oxalate crystals as well as starch grains in the endodermis. However, the studies carried out to date have involved few legume species. This study therefore purported to examine the consistency of this hypothesis in other legumes. Thus, the structure and content of the pulvinus endodermal cells of nine legumes of the Brazilian cerrado, with different types and velocities of leaf movement, were investigated: slow nyctinastic and heliotropic movements ( BAUHINIA RUFA, COPAIFERA LANGSDORFFII, SENNA RUGOSA - Caesalpinioideae; ANDIRA HUMILIS and DALBERGIA MISCOLOBIUM - Faboideae; STRYPHNODENDRON POLYPHYLLUM - Mimosoideae), slow heliotropic movement ( ZORNIA DIPHYLLA - Faboideae), and fast seismonastic and slow nyctinastic and heliotropic movements ( MIMOSA RIXOSA and MIMOSA FLEXUOSA - Mimosoideae). Samples were prepared following standard plant anatomy and ultrastructure techniques. The endodermis of all the species contains starch grains. In the species displaying only slow movements, calcium oxalate prismatic crystals were observed in addition to starch grains, except in ZORNIA DIPHYLLA. In conclusion, oxalate crystals occur only in endodermal cells of pulvini that display slow movements, while starch grains are always present in pulvinus endodermal cells of plants with any kind of movement.     

Structure and chemical composition of endodermal and rhizodermal/hypodermal walls of several species

CWM, isolated cell wall material
ECW, isolated endodermal cell walls
G, guaiacyl monomer
H, p-hydroxyphenyl monomer
HCW, isolated hypodermal cell walls
RHCW, isolated rhizodermal and hypodermal cell walls
S, syringyl monomer
XV, isolated xylem vessels

Endodermal cell walls of the three dicotyledoneous species Pisum sativum L., Cicer arietinum L. and Ricinus communis L. were isolated enzymatically and analysed for the occurrence of the biopolymers lignin and suberin. From P. sativum, endodermal cell walls in their primary state of development (Casparian strips) were isolated. Related to the dry weight, these isolates contained equal amounts of suberin (2·5%) and lignin (2·7%). In contrast, the endodermal cell walls of C. arietinum and R. communis, which were nearly exclusively in their secondary state of development, contained significantly higher proportions of suberin (10–20%) and only traces of lignin (1–2%). The results of the chemical analyses were supported by a microscopic investigation of Sudan III-stained root cross-sections, showing a Casparian strip restricted to the radial walls of the endodermis of P. sativum and well-pronounced red suberin lamellae in C. arietinum and R. communis roots. Compared with recently investigated monocotyledoneous species, higher amounts of suberin by one order of magnitude were detected with the secondary state of development of dicotyledoneous species. Furthermore, the carbohydrate and protein contents of primary (Clivia miniata Reg. and Monstera deliciosa Liebm.), secondary (C. arietinum and R. communis) and tertiary endodermal cell walls (Allium cepa L. and Iris germanica L.) were determined. The relative carbohydrate content of secondary endodermal cell walls was low (14–20%) compared with the content of primary (42–50%) and tertiary endodermal cell walls (60%), whereas the protein content of isolated endodermal cell walls was high in primary (13%) and secondary (8%) and low in tertiary endodermal cell walls (0·9–2%). The results presented here indicate that the quantitative chemical composition of primary, secondary, and tertiary endodermal cell walls varies significantly. Finally, cell wall proteins are described as an additional important constituent of endodermal cell walls, with the highest concentrations occurring in primary (Casparian strips) and secondary endodermal cell walls.     

Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root

Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.     

Tracing the Path of Oxygen into Birdsfoot Trefoil and Alfalfa Nodules using Iodine Vapor

Although physiological control of nodule 0₂ permeability is an active area of research, the gas diffusion pathway between the atmosphere and the infected zone has not been firmly established. Previous studies have used infiltration of ink or dyes to identify points of entry, but such water-soluble tracers could give a misleading picture of gas diffusion pathways. We therefore used iodine vapor (and its reaction with starch) to trace gas-phase pathways into the infected zone of determinate birdsfoot trefoil (Lotus corniculatus) and indeterminate alfalfa (Medicago sativa) nodules. We also used histochemical methods to identify suberized or lignified layers that could act as barriers to gas diffusion. Birdsfoot trefoil nodules were surrounded by a suberized periderm, but nonsuberized cells and intercellular spaces were observed in the periderm between lenticels and their associated vascular bundles. Iodine entered birdsfoot trefoil nodules only through lenticels. The periderm appears to provide a significant barrier to gas diffusion. Although airspaces were rare in the nodule parenchyma (also referred to as the “inner cortex”), we found some evidence that a few air-filled pathways cross this secondary barrier, also in the vicinity of vascular bundles. Alfalfa nodules were cylindrically surrounded by a suberized endodermis which ended near the meristematic tip; iodine entered principally at the end of the endodermis near the meristem. Future research on physiological control of nodule O₂ permeability should concentrate on strategic “choke points”, associated with lenticels in determinate nodules, or in the zone proximal to the meristem in indeterminate nodules.     

荷叶铁线蕨孢子体解剖结构和组织化学特征研究

荷叶铁线蕨为岩生珍稀蕨类植物,分布在中国重庆市万州、涪陵等极少地区.为揭示荷叶铁线蕨生长特性,采集栽培在种质资源圃中的荷叶铁线蕨根样、根状茎、在阳光下生长和在阴暗环境下生长的叶片,固定于甲醛-酒精-乙酸溶液中,用双面刀片进行徒手切片,分别用三种0.1％苏丹红、0.1％硫酸氢黄连素-苯胺兰、0.05％甲苯胺蓝染色剂染色,...