In both radish and Arabidopsis, lateral root initiation involves a series of rapid divisions in pericycle cells located on the xylem radius of the root. In Arabidopsis, the number of pericycle cells that divide to form a primordium was estimated to be about 11. To determine the stage at which primordia are able to function as root meristems, primordia of different stages were excised and cultured without added hormones. Under these conditions, primordia that consist of 2 cell layers fail to develop while primordia that consist of at least 3–5 cell layers develop as lateral roots. We hypothesize that meristem formation is a two-step process involving an initial period during which a population of rapidly dividing, approximately isodiametric cells that constitutes the primordium is formed, and a subsequent stage during which meristem organization takes place within the primordium.

Formation of lateral root meristems is a two-stage process.

Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a technique widely used for bone histomorphometry. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues. Recently, an MMA embedding technique has been reported that preserves enzyme activity and antigenic determinants in bone tissue. We describe here a modification of this embedding method. For our modified MMA embedding process, commercially available methacrylates can be used without purification, and the histologic quality of bone sections is comparable to that of conventionally MMA-embedded bone specimens. The technique reported here can be employed for embedding of larger bone samples and is suitable for histochemical and immunohistological applications as well as for routine bone histomorphometry. By addition of methylbenzoate during infiltration and polymerization of the plastic, the antigenicity of the tissue was improved. As applications of this novel technique, demonstration of alkaline phosphatase and tartrate-resistant acid phosphatase as well as positive labeling of Kupffer cells and osteoclasts with the monoclonal antibody ED1 in sections of liver, tibiae, and vertebrae of 3-month-old rats was demonstrated. The method described here might be useful for the inclusion of histochemical and immunohistological methods into bone histomorphometry.

Embedding of Bone Samples in Methylmethacrylate: An Improved Method Suitable for Bone Histomorphometry, Histochemistry, and Immunohistochemistry

Summary

The plant CDK inhibitor ICK1 was identified previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauliflower mosaic virus 35S promoter. In comparison to control plants, growth was significantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modifications in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313x.2000.00899.x

Expression of the plant cyclin‐dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology

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Polyclonal antisera against a fusion protein of β-galactosidase and the 20 C-terminal amino acids of the Arabidopsis thaliana sucrose carrier AtSUC2 were used to determine the cellular localization of the AtSUC2 protein. Using fluorescence-labelling on sections from different organs of Arabidopsis the AtSUC2 protein was immunolocalized exclusively in companion cells. The presented data indicate that phloem loading in Arabidopsis may be catalyzed by the AtSUC2 sucrose carrier which transports sucrose into the companion cells. No evidence for a participation of the second Arabidopsis sucrose transporter AtSUC1 has been obtained.

The Arabidopsis thaliana AtSUC2 Gene is Specifically Expressed in Companion Cells

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5-2 μm) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa.

This work concerns an aspect of spatial regulation of cell division, the development of the preprophase band (PPB) of microtubules. The PPB is significant in plant development because its position in the dividing cell indicates where the new cell wall will be inserted[mdash]an important site for control of histogenesis. We have categorized and determined the durations of stages in the development of PPBs, and have established their timing relative to the S-, G2-, and mitotic phases of the cell cycle. Roots of wheat seedlings were supplied with bromodeoxyuridine in continuous and pulse-chase treatments. Cells that were in the S-phase were identified and changes in their microtubule arrays were monitored by double immunolabeling. PPB initiation was detectable as early as the end of the S-phase as a narrowing of the preceding interphase array of microtubules. Development continued throughout G2 to a mature, narrow PPB, which existed only briefly and then eroded during the transition to the prophase mitotic spindle. The microtubule rearrangements of PPB development showed that preparation of the future site and plane of division in higher plant cells begins just after DNA replication and is completed just before mitosis.

Rearrangements of microtubules involved in establishing cell division planes start immediately after DNA synthesis and are completed just before mitosis.

Margaret Sammut

Papers

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa.

Rearrangements of microtubules involved in establishing cell division planes start immediately after DNA synthesis and are completed just before mitosis.