Structure and function at the root apex - phylogenetic and ontogenetic perspectives on apical cells and quiescent centres

The summit of roots of various plant species may be occupied by a single, rapidly proliferating tetrahedral apical cell (as in ferns), or by a multicellular and slowly proliferating quiescent centre (as in angiosperms), or by intermediate types of cellular organizations. The present paper attempts to deduce the phylogeny of these various types of cellular patterning at the root apex.     

Development of an in vitro culture technique for conservation of Saccharum spp. hybrid germplasm

An in vitro method for the establishment and storage of over 200 Saccharum spp. hybrid clones was developed that involved only 1 medium for shoot development and multiplication, and no decontamination procedures. Apical buds, from the leaf axils of developing leaves surrounding the apical meristem, were cultured on medium containing the plant growth regulators 6-benzylaminopurine (BAP) and 6-furfurylaminopurine (kinetin), and regenerated multiple shoots. Shoots transferred to medium containing naphthaleneacetic acid (NAA) developed roots. In vitro plants transferred to a medium containing half strength salts and vitamins without plant growth regulators were placed in storage at 18°C. After 12 months of storage plants were transferred to fresh medium and returned to storage. The genetic integrity of clones (based on phenotype assessment) was not affected by the in vitro culture method and up to 14 months of low-maintenance storage conditions. These in vitro plants will be further tested for genetic stability using biochemical and molecular techniques.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

Low temperature compartment formation in feline immunodeficiency virus-infected and uninfected feline kidney cells

Summary This study was to determine if feline immunodeficiency virus (FIV)-infected and uninfected Crandall feline kidney (CRFK) cells exhibited a low temperature (16°C) block in membrane trafficking between transitional endoplasmic reticulum and Golgi apparatus represented by intermediate compartment formation. Cells were cultured at different temperatures and membrane changes involving the Golgi apparatus and Golgi apparatus-associated membrane structures were monitored by electron microscopy and quantitated. With 30 min of incubation, membranes of the Golgi apparatus stack increased in amount at temperatures of 16°C and below compared to temperatures above 18°C. The increase was greatest along the major polarity axis as evidenced by an increased stack height. Neither the number of cisternae per stack nor the average stack diameter (width) was affected by temperature. The response was maximal between 15 and 30 min of low temperature treatment of the cells. Results with cells infected and uninfected with feline immunodeficiency virus were similar. The increase in stack height was due primarily to an increase of membranes at the cis face (cis Golgi apparatus network). At 18°C, membranes of the trans Golgi apparatus network accumulated suggesting that import from the cis Golgi network could proceed at this temperature, whereas exit from the trans Golgi network was still at least partially blocked. Also increased at 16°C and below were numbers of transition vesicles in the space between the Golgi apparatus and the transitional endoplasmic reticulum associated with the cis Golgi apparatus face. The results suggested interruption of the orderly flux of membranes into the Golgi apparatus at 16°C and below. Moreover, the block appeared to be reversible. Upon transfer from 16°C to 37°C, there was a time-dependent decrease in the accumulations of cis compartment membrane accompanied by a corresponding equivalent increase in the membranes of the trans Golgi apparatus compartment.     

Establishing a new genus,Chiharadinium gen. nov. (Peridiniales,Dinophyceae) for a tidal pool dinoflagellate formerly known as Scrippsiella hexapraecingula

To determine its accurate taxonomic position, a tidal pool bloom-forming dinoflagellate, Scrippsiella hexapraecingula was re-investigated using light, scanning and transmission electron microscopy together with a phylogenetic analysis based on concatenated ribosomal DNA sequences. The culture strains used in this study were established from intertidal rock pool samples taken from Jogashima, Kanagawa prefecture and Heisaura, Chiba prefecture, Japan and were identified as S. hexapraecingula originally described by Horiguchi and Chihara from a tidal pool in Hachijo Island, Tokyo, Japan in 1983. The thecal plate arrangement was determined as Po, X, 4′, 3a, 6″, 6c, 5s, 5″′, 2″″. The internal structure was investigated for the first time. The organism has typical dinoflagellate cellular organelles such as a dinokaryotic nucleus, mitochondria with tubular cristae, trichocysts and pusule. The chloroplast was single and connected to the central pyrenoid (stalked type). The eyespot found in the sulcus is of the B type with two rows of superficial intraplastidal lipid globules directly overlain by an extraplastidal single layer of crystalline bricks enveloped by a common membrane. The apical pore is plugged by a double-layered stub-like structure. Stalk building material for attachment covered the apical pore. Phylogenetic analysis indicated that S. hexapraecingula was most closely related to a freshwater dinoflagellate, Peridiniopsis borgei, the type species of the genus Peridiniopsis. However, clear differences exist between these two organisms, including their thecal plate arrangement, habitat and habit. As a result, a new genus, Chiharadinium Dawut & T. Horiguchi gen. nov. has been proposed rather than attempting to accommodate S. hexapraecingula in the genus Peridiniopsis. The new combination, Chiharadinium hexapraecingulum (T. Horiguchi & Chihara) Dawut & T. Horiguchi comb. nov. has been proposed.     

Impacts of iron on phosphate starvation-induced root hair growth in Arabidopsis

In Arabidopsis, phosphate starvation (-Pi)-induced responses of primary root and lateral root growth are documented to be correlated with ambient iron (Fe) status. However, whether and how Fe participates in -Pi-induced root hair growth (RHG) remains unclear. Here, responses of RHG to different Fe concentrations under Pi sufficiency/deficiency were verified. Generally, distinct dosage effects of Fe on RHG appeared at both Pi levels, due to the generation of reactive oxygen species. Following analyses using auxin mutants and the phr1 mutant revealed that auxin and the central regulator PHR1 are required for Fe-triggered RHG under −Pi. A further proteomic study indicated that processes of vesicle trafficking and auxin synthesis and transport were affected by Fe under −Pi, which were subsequently validated by using a vesicle trafficking inhibitor, brefeldin A, and an auxin reporter, R2D2. Moreover, vesicle trafficking-mediated recycling of PIN2, an auxin efflux transporter, was notably affected by Fe under -Pi. Correspondingly, root hairs of pin2 mutant displayed attenuated responses to Fe under -Pi. Together, we propose that Fe affects auxin signalling probably by modulating vesicle trafficking, chiefly the PIN2 recycling, which might work jointly with PHR1 on modulating -Pi-induced RHG.     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5 ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.