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Hairy root research: recent scenario and exciting prospects.

High stability of the production of secondary metabolites is an interesting characteristic of hairy root cultures. For 25 years, hairy roots have been investigated as a biological system for the production of valuable compounds from medicinal plants. A better understanding of the molecular mechanism of hairy root development, which is based on the transfer of Agrobacterium rhizogenes T-DNA into the plant genome, has facilitated its increasing use in metabolic engineering. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for the pharmaceutical industry. In addition, hairy roots offer promise for phytoremediation because of their abundant neoplastic root proliferation. Recent progress in the scaling-up of hairy root cultures is making this system an attractive tool for industrial processes.

Biodegradation, Environmental↗

KNAT6 gene of Arabidopsis is expressed in roots and is required for correct lateral root formation.

As part of a programme to determine whether root development requires a pathway involving KNOTTED-like homeobox ( KNOX ) genes, we cloned the KNAT6 cDNA, a member of the KNOX gene family in Arabidopsis, from root tissue. The KNAT6 cDNA is represented by two isoforms, suggesting that the KNAT6 transcript is subject to alternative splicing. Promoter-reporter and RT-PCR analysis confirms expression of the KNAT6 gene in roots, and in particular in the phloem tissue close to the site of lateral root initiation, though not in the primary or lateral root meristem. The KNAT6 gene promoter activity is altered in pattern by treatment with exogenous auxin and in the rooty mutant background, with expression shifting distally in the root. In contrast, exogenous cytokinin suppresses KNAT6 promoter activity. KNAT6::GFP fusion protein is localized to the nucleus, consistent with the predicted function of KNAT6 as a transcription factor. Over-expression of the more abundant shorter isoform of the KNAT6 cDNA, but not of the less abundant longer form, leads to a lobed leaf phenotype, but no consistently aberrant root phenotype. Down-regulation of KNAT6 expression by RNA interference was associated with an increased total number of lateral roots. These data indicate a role for KNAT6 in modulating lateral root formation.

Amino Acid Sequence↗

Cellular compartmentation of ammonium assimilation in rice and barley.

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.

Cell Compartmentation↗

Experimental study of pulpotomy with calcium hydroxide-iodoform paste in dogs' immature permanent teeth.

To investigate the healing process and continuous root development following pulpotomy with improved calcium hydroxide-iodoform paste, the authors performed an experimental study of pulpotomy in immature permanent teeth. Thirty five incompletely formed root canals in dogs were used, and radiographical and histopathological observations were made. The results lead to the conclusion that pulpotomy with improved calcium hydroxide-iodoform paste is a very effective treatment for immature permanent teeth.

Animals↗

A plant regulator controlling development of symbiotic root nodules.

Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia. Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules. The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed. Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip. Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia. To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition. We show that nin is required for the formation of infection threads and the initiation of primordia. NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.

Amino Acid Sequence↗

Treatment of lower second premolar agenesis by autotransplantation: four-year evaluation of eighty patients.

The frequency of agenesis of the second lower premolar is 2.5-4%. In growing patients, early extraction of the deciduous molar and subsequent closure of the space is a common therapy, but in some cases space closure is deemed unlikely and autotransplantation is an alternative. The aim of the study was to analyze the outcome of autotransplantation in replacing missing lower second premolars and to evaluate the associated presurgical orthodontic treatment. The material consisted of records of all patients with teeth transplanted to the lower second premolar region during the period 1988-89 at the Department of Oral Surgery, Eastman Institute, Stockholm. The following variables were registered: sex, age, number of transplanted teeth, donor tooth, root development, recipient site, orthodontic treatment, persisting temporary molar, total number of congenitally missing teeth, the surgeon responsible, and clinical and radiological follow-up variables. Of 110 transplanted teeth, 99 had not completed root formation, and in 11 teeth the root formation was completed. The success rates after 4 years were 92% and 82%, respectively. Both premolars and molars served as donor teeth, but the main donor tooth was the upper second premolar. Fourteen percent had been orthodontically treated only because of the transplantation, i.e. to open the space for the donor tooth. Treatment of agenesis of the second lower premolar by autotransplantation has a good prognosis. In growing individuals the transplant not only maintains growth and development of the alveolar ridge but also provides a permanent solution to the agenesis.

Adolescent↗

Initial tree establishment on blocky quarry waste ameliorated with hydrogel or slate processing fines.

Pocket planting reclamation techniques developed in the 1970s for revegetating blocky quarrying waste have met with very limited success, often because the low water-holding capacity of the waste and limited root development within a small volume of planting pocket material result in severe drought mortality. We tested pocket planting approaches for waste tip reclamation at Europe's largest slate quarry, and compared materials for enhancing the continuity of water- and nutrient-holding down into the interior of the waste tip. When small compost-filled pocket planting bags were placed above slate processing fines (SPF) or water absorbent cross-linked polyacrylamide gel ("hydrogel"), tree growth rates increased in comparison with pocket planting bags alone. The SPF significantly improved tree survival especially during severe drought, but survival was not enhanced by the use of hydrogel. The sorption characteristics of hydrogel indicated that its presence may help to reduce nutrient leaching, but that it may have a negative effect on nitrogen availability. A more likely explanation for the poor performance of pure hydrogel is that it did not maintain sufficient available water, because of discontinuities caused by shrinkage and movement of the hydrogel, and/or degradation of water-holding capacity with environmental exposure. However, the root growth observed in the hydrogel treatments suggests that this technique, if adapted to reduce the effects of hydrogel shrinkage by using finer-grade hydrogel, mixing it with other soil-forming material, and reducing its exposure to extremes of temperature or sunlight, might have the potential to improve the growth and survival of trees planted on sites where delivery of heavy materials such as SPF is impractical. Fine mineral processing waste is freely available at active quarries and should be seen as a key resource for reclamation schemes.

Conservation of Natural Resources↗

[Outcome of wisdom tooth germ transplantation].

In 68 patients, 79 tooth germs of the third molar were transplanted. Out of this group, 43 patients with 50 transplantations under-went a follow-up examination. All surgery was performed by one surgeon. Retrospectively, we tried to establish whether the success of postoperative healing depends on the donor and receiver regions. Crossing the jaw border clearly worsens the prognosis for transplantation. Altogether 9 transplantations turned out to be failures, 7 of which had been transplanted from the upper to the lower jaw. The results of this investigation showed incomplete root development in 34%, the necessity of endodontic treatment in 6% and enlarged periodontal pockets in 8%. Ankylosed teeth were found in 10%; we did not see any root resorptions.

Adolescent↗

Replantation of an inverted lower second premolar germ.

During the initial X-ray control of a 10-year-old female orthodontic patient, a late development and an inverted position of the lower left second premolar was seen. 4.3 years later the tooth with its follicle was replanted in the upright position, preserving the second deciduous molar; at that time the root development was at an early stage. At a follow-up control 4 years after the surgical procedure the replanted tooth had erupted and the sensibility response was normal. Its root, however, although completed, could not reach the length and especially the width (in the apical half) of the contralateral premolar. This relative reduction in width seems to follow a temporary thickening of that part of the root formed during the first period after replantation. The anamnestic data suggest that physiologic exfoliation of the deciduous molar took place.

Bicuspid↗

Prediction of stem profile of Picea abies using a process-based tree growth model.

We built a simple tree growth model for Norway spruce (Picea abies (L.) Karst.) that describes the biomass and stem radial growth of one tree in a stand. Growth is controlled by an external height growth function that accounts for site quality. Crown recession is represented by an empirical function that accounts for the limitation to crown development caused by mechanical contacts with neighboring trees. The model describes biomass growth based on carbon budget (photosynthesis, respiration and senescence) and carbon partitioning between foliage, stem and root compartments. An internal regulation is introduced based on a functional balance between crown and root development. Stem annual growth is distributed along the stem by means of an empirical rule. Stem profile is the final output of the model and can be used to check the overall consistency of the model and as an aid in wood quality studies. The underlying assumptions of the model are described.

Journal Article↗

Functional significance of dauciform roots: exudation of carboxylates and acid phosphatase under phosphorus deficiency in Caustis blakei (Cyperaceae).

Caustis blakei produces an intriguing morphological adaptation by inducing dauciform roots in response to phosphorus (P) deficiency. We tested the hypothesis that these hairy, swollen lateral roots play a similar role to cluster roots in the exudation of organic chelators and ectoenzymes known to aid the chemical mobilization of sparingly available soil nutrients, such as P. Dauciform-root development and exudate composition (carboxylates and acid phosphatase activity) were analysed in C. blakei plants grown in nutrient solution under P-starved conditions. The distribution of dauciform roots in the field was determined in relation to soil profile depth and matrix. The percentage of dauciform roots of the entire root mass was greatest at the lowest P concentration ([P]) in solution, and was suppressed with increasing solution [P], while in the field dauciform roots were predominantely located in the upper soil horizons, and decreased with increasing soil depth. Citrate was the major carboxylate released in an exudative burst from mature dauciform roots, which also produced elevated levels of acid phosphatase activity. Malonate was the dominant internal carboxylate present, with the highest concentration in young dauciform roots. The high concentration of carboxylates and phosphatases released from dauciform roots, combined with their prolific distribution in the organic surface layer of nutrient-impoverished soils, provides an ecophysiological advantage for enhancing nutrient acquisition.

Acid Phosphatase↗

The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light.

We examined whether flavonoids act as endogenous auxin transport regulators during gravity vector and light intensity changes in Arabidopsis thaliana roots. Flavonoid deficient transparent testa4 [tt4(2YY6)] seedlings had elevated root basipetal auxin transport compared with the wild type, consistent with the absence of a negative auxin transport regulator. The tt4(2YY6) roots had delayed gravitropism that was chemically complemented with a flavonoid intermediate. Flavonoid accumulation was found in wild-type columella cells, the site of gravity perception, and in epidermal and cortical cells, the site of differential growth, but flavonoid accumulation was absent in tt4(2YY6) roots. Flavonoid accumulation was higher in gravity-stimulated root tips as compared with vertical controls, with maximum differences coinciding with the timing of gravitropic bending, and was located in epidermal cells. Exogenous indole-3-acetic acid (IAA) also elevated flavonoid accumulation, suggesting that flavonoid changes in response to gravity might be partly as a result of changing IAA distribution. Acropetal IAA transport was also elevated in roots of tt4(2YY6). Flavonoid synthesis was repressed in the dark, as were differences in root acropetal transport in tt4(2YY6). These results are consistent with light- and gravity-induced flavonoid stimulation that alters auxin transport in roots and dependent physiological processes, including gravitropic bending and root development.

Arabidopsis↗

Effects of radiation on the developing dentition and supporting bone.

Underdevelopment of teeth after exposure to radiation has been observed since the early experiments with X rays. Two patients had been irradiated at young ages for embryonal rhabdomyosarcomas. For the patient treated with radiation at age 4 for a lesion in the submandibular area, root development of the mandibular teeth stopped, but the teeth nevertheless erupted. In the patient treated with radiation at age 9 for a tumor in the left cheek, bone apposition in the left side of the mandible failed to continue at the same rate as on the right side. Although new megavoltage radiation machines have reduced the chances of bone complications in adults, developing bone and structures are still significantly affected by radiation.

Adolescent↗

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato.

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis beta-1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.

Amino Acid Sequence↗

Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH).

Native bacteria, Pseudomonas and filamentous bacteria were quantified and localized on wheat roots grown in the field using fluorescence in situ hybridization (FISH). Seminal roots were sampled through the season from unploughed soil in a conservation farming system. Such soils are spatially heterogeneous, and many roots grow slowly through hard soil with cracks and pores containing dead roots remnant from previous crops. Root and rhizosphere morphology, and contact with soil particles were preserved, and autofluorescence was avoided by observing sections in the far-red with Cy5 and Cy5.5 fluorochromes. Spatial analyses showed that bacteria were embedded in a stable matrix (biofilm) within 11 microm of the root surface (range 2-30 microm) and were clustered on 40% of roots. Half the clusters co-located with axial grooves between epidermal cells, soil particles, cap cells or root hairs; the other half were not associated with visible features. Across all wheat roots, although variable, bacteria averaged 15.4 x 10(5) cells per mm(3) rhizosphere, and of these, Pseudomonas and filaments comprised 10% and 4%, respectively, with minor effects of sample time, and no effect of plant age. Root caps were most heavily colonized by bacteria along roots, and elongation zones least heavily colonized. Pseudomonas varied little with root development and were 17% of bacteria on the elongation zone. Filamentous bacteria were not found on the elongation zone. The most significant factor to rhizosphere populations along a wheat root, however, was contact with dead root remnants, where Pseudomonas were reduced but filaments increased to 57% of bacteria (P < 0.001). This corresponded with analyses of root remnants showing they were heavily colonized by bacteria, with 48% filaments (P < 0.001) and 1.4%Pseudomonas (P = 0.014). Efforts to manage rhizosphere bacteria for sustainable agricultural systems should continue to focus on root cap and mucilage chemistry, and remnant roots as sources of beneficial bacteria.

Agriculture↗

Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed.

Cell Differentiation↗

Dental development and molar root length in children with cleidocranial dysplasia.

Cleidocranial dysplasia (CD) is an autosomal dominant skeletal disorder with characteristic dental findings of numerous supernumerary teeth and noneruption of permanent teeth. This investigation compared the dental development and root lengths of the mandibular first permanent molar in 11 CD patients with those of 22 healthy, normal children matched for race, age and sex. The results showed that children with CD experienced a delay in dental development of approximately 3 years compared with normal children (P < 0.05). In addition, the root lengths of the mandibular first permanent molar were significantly longer than those of the comparison children (17.8 +/- 1.6 mm vs 13.6 +/- 1.2 mm (P < 0.001). This study thus revealed two significant clinical features of CD: 1) severe delay in dental development, and 2) excessive root lengths of mandibular permanent first molars. These features may be important in the pathogenesis of delayed dental eruption observed in this disorder.

Adolescent↗

Sexual dimorphism in the development, emergence, and agenesis of the mandibular third molar.

Bilateral agenesis was encountered in about 9% of the cases, with no significant sexual difference. The right and left mandibular third molars had the same pattern of development and emergence. The slight advance of girls over boys at the crown-completion stage was similar to previous observations on other mandibular teeth, particularly the second molar. The root development course of the third molar was faster in males than in females; this sexual dimorphism was much greater for retarded cases than for advanced cases. At the apex closure, the difference between median ages of males and females was 1.5 yr. Alveolar emergence tended to occur at a lower developmental stage in advanced cases compared with retarded cases.

Adolescent↗