Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis.

A genetic analysis of root development in Arabidopsis thaliana has identified mutants that have abnormal morphogenesis. Four of these root morphogenesis mutants show dramatic alterations in post-embryonic root development. The short-root mutation results in a change from indeterminate to determinate root growth and the loss of internal root cell layers. The cobra and lion's tail mutations cause abnormal root cell expansion which is conditional upon the rate of root growth. Expansion is greatest in the epidermal cells in cobra and in the stele cells in lion's tail. The sabre mutation causes abnormal cell expansion that is greatest in the root cortex cell layer and is independent of the root growth rate. The tissue-specific effects of these mutations were characterized with monoclonal antibodies and a transgenic marker line. Genetic combinations of the four mutants have provided insight into the regulation of growth and cell shape during Arabidopsis root development.

Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation.

Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of the anti-plasma membrane antibodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed by patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recognized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.

Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices.

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.

Cell-wall differentiation during growth of electrically polarised protoplasts of Physcomitrella.

Protoplasts of Physcomitrella patens have been grown in continuous electric field of 50 V cm(-1), resulting in a predictable pattern of filament emergence. The events preceding the visible formation of a polar axis have been examined by electron microscopy. The first sign of polarity is the formation of a thickened inner wall layer over the potential growth site. Elongation of the filament is preceded by the appearance of a layer of heavily stained amorphous material at the external surface of the thickened wall. This material marks the region of initial extension of the filament, but it is not produced once extension has begun, and further growth of the filament results in the retention of the material as an annular ring at its base. The wall of the filament has a complex thickened structure which is a result of the osmotic conditions under which the protoplasts are grown. These results are discussed in terms of the development of the polar axis.

Studies on the growth and development of protoplasts of the moss, Physcomitrella patens, and its control by light.

Protoplasts prepared from protonemal cultures of the moss Physcomitrella patens begin to regenerate a new cell wall within 1 h of removal from cellulase. The wall is seen as a gradually thickening mat of fibres when examined by scanning electron microscopy. Development of filaments from protoplasts takes place in the majority of cases only after one or more cell divisions have occurred. The direction of emergence of filaments is random in uniform light, but strongly negatively phototropic in bright unidirectional horizotal light. Filament growth is also strongly negatively phototropic. The influence of unidirectional light can be destroyed by incubating protoplasts in the presence of colchicine. Filaments growing in unidirectional light have cytoplasmic microtubules running along their long axes and in close association with large organelles. These results are discussed in terms of the potential for this system for the study of polarity in plants.

Structure and association of wall fibrils produced by regenerating tobacco protoplasts.

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.

Observations on the time course of wall development at the surface of isolated protoplasts.

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.

Membrane mobility and the Concanavalin A binding system of the plasmalemma of higher plant protoplasts.

The binding of a colloidal gold-Concanavalin A (ConA) complex to the plasmalemma of tobacco leaf protoplasts has been investigated using scanning electron microscopy. At 5° C the particles of gold-ConA appear to be randomly distributed over the surface of the protoplast. If the temperature is raised, the particles associate into clusters. Saturation of the membrane with particles can only occur when the weight of ConA in solution exceeds 1 µg/10(4) protoplasts in suspension, and when its concentration exceeds 15 µg/ml. These results are discussed in terms of the properties of the ConA binding site and the mobility of such sites within the membrane surface.

Coumarin inhibition of microfibril formation at the surface of cultured protoplasts.

The effect of including coumarin in the culture medium of isolated tobacco leaf protoplasts has been studied by scanning microscopy. Fibre formation is completely suppressed for 48 h in concentrations of 200 and 250 mg/l of coumarin. Continuous culture in these concentrations of coumarin does not result in cell division or colony formation, although an atypical wall is visible after 4 days. The effect of coumarin is reversible. Viability of protoplasts exposed for 48 h to 200 mg/l coumarin is unaffected in terms of plating efficiency and infectability with virus. These results are discussed in relation to cell wall synthesis and the possible use of coumarin in prolonging the protoplast state.

Ultrastructural studies of the binding of concanavalin A to the plasmalemma of higher plant protoplasts.

The binding of concanavalin A to the plasmalemma of higher plants has been studied using protoplasts of two species. The lectin aggregates both tobacco (Nicotiana tabacum L.) leaf protoplasts and protoplasts prepared from a suspension cell culture of grapevine (Vitis vinifera L.). Differences in lectin binding have been investigated using concanavalin A conjugated to ferritin or bound to colloidal gold. Tobacco protoplasts exhibit continuous and saturated labelling of the plasmalemma surface with gold-concanavalin A mixtures. Vine protoplasts under the same conditions show a discontinuous and patchy distribution of label. These results are discussed in terms of a possible binding mechanism.

Scanning electron microscopy of cell wall formation around isolated plant protoplasts.

The process of cell wall regeneration around two species of higher plant protoplasts has been studied using reflection scanning electron microscopy. The first stage in the process is the formation of short fibres from randomly spaced centres. With protoplasts of tobacco leaf (Nicotiana tabacum L., cv White Burley) these fibres then elongate and interlace apparently at random to give rise to a matted continuous layer of wall. Protoplasts of a suspension culture of grapevine cells (Vitis vinifera L. cv Müller Thurgau) produce short fibres but these fail to elongate. Budding is observed during wall regeneration around vine protoplasts. The results are discussed in terms of the mechanical properties of the wall and its relationship to changes in plasmalemma morphology which are observed during wall formation.

Effect of calcium on the potassium flux into the exudate of excised maize roots.

The effect of varying calcium concentration in the medium on the potassium flux into the exudate has been studied. In media of low ionic strength (o.1 mM KCl) the potassium flux, J K, was significantly increased by increasing the calcium concentration of the medium. But in higher ionic strength media (10 mM) KCl) there was no increase in J K as the calcium concentration of the medium was increased. The effect of external sodium concentration on J K was also studied. These results are discussed in relation to present theories of salt and water movement into the plant root. It is concluded that two pathways potentially exist for movement of salts to the exudate stream: firstly, via a symplasm and secondly, through the cell wall pathway. But is is further concluded that the cell wall pathway, at normal physiological ionic strengths, is not available for salt transport due to co-ion exclusion by the fixed negative charges.