Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations.

Overexpression of Arabidopsis thaliana genes for the fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, improved the tolerance of A. thaliana hairy roots to low oxygen conditions. Whereas the specific growth rate of untransformed hairy roots in shake flasks and in a multiple-tube recirculation bioreactor declined significantly with decreasing oxygen tension down to 25% air saturation, growth of the transformant root lines was maintained at rates similar to those achieved with full aeration. This work demonstrates that altering the expression of selected genes involved in anaerobic metabolism can alleviate the problems of oxygen deficiency in hairy root cultures caused by poor mixing and mass transfer conditions.     

Investigation of liquid-solid hydrodynamic boundary layers and oxygen requirements in hairy root cultures.

Rates of oxygen uptake, growth and alkaloid production by hairy roots in submerged culture were investigated using a recirculation reactor allowing operation at high liquid velocities for removal of hydrodynamic boundary layers. Measurements were performed at dissolved oxygen tensions of 31-450% air saturation. Critical oxygen concentrations for Atropa belladonna hairy roots were above air saturation, viz. 100-125% air saturation for oxygen uptake and 150% air saturation for growth, demonstrating that these roots cultivated in reactors with air sparging are oxygen-limited. The critical oxygen tension for oxygen uptake by Solanum aviculare hairy roots was 75% air saturation. Both the specific oxygen uptake rate and specific growth rate of A. belladonna hairy roots were dependent on the mass (g dry weight) of roots present; even in the absence of boundary layers, growth did not remain exponential over the entire culture period. Cryo-scanning electron microscopy showed that hairy roots grown submerged in liquid medium were covered with thick layers of hydrated mucilage and root hairs, representing a significant additional barrier to oxygen transfer. Roots protruding out of the liquid medium showed no evidence of mucilage accumulation. The specific oxygen demand of A. belladonna root tips was 3.3-11.5 times higher than for the remainder of the roots, the ratio increasing as the dissolved oxygen tension was reduced. Specific growth rates, biomass yields from sugar, and atropine levels were maximum at around 150% air saturation, but decreased significantly with oxygen concentrations above ca. 200%.     

Inhibitory role of root hairs on transport within root culture bioreactors

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture. These root cultures were used to demonstrate decreased flow resistance in a tubular reactor as a result of root hair removal. To assess the impact on bioreactor performance, hairy and hairless root cultures were grown in a highly characterized 15-L bubble column bioreactor. In the absence of root hairs, the mixing was greatly enhanced; mixing times became shorter for the hairless culture at roughly 100 g (fresh weight)/L. By the end of the 3-week culture period, the mixing time of the hairy culture was 29 times longer than that of the hairless culture. The growth rate of the hairless culture in the bioreactor was as much as 2.4 times greater than growth of the hairy culture under the same conditions. The improved reactor performance was reflected in greater biomass accumulation and respiratory activity. These results show that the root hairs-which facilitate nutrient uptake in a static soil environment-are detrimental to growth in a liquid environment as an effect of their stagnating fluid flow and limiting oxygen availability.     

Oxygen requirements and mass transfer in hairy-root culture

Oxygen mass transfer in clumps of Atropa belladonna hairy roots was investigated as a function of root density and external flow conditions. Convection was the dominant mechanism for mass transfer into root clumps 3.5 to 5.0 cm in diameter; Peclet numbers inside the clumps ranged from 1.4 x 10(3) to 7.1 x 10(4) for external superficial flow velocities between 0.4 and 1.4 cm s(-1). Local dissolved-oxygen levels and rates of oxygen uptake were measured in aflow chamber and in bubble column and stirred bioreactors. When air was used as oxygen source, intraclump dissolved-oxygen tensions ranged from90% to 100% air saturation at high external flow velocity andlow root density, to less than 20% air saturation in dense root clumps. Specific oxygen-uptake rate declined with increasing root density. When external boundary layers around individual roots were eliminated byforcing liquid through the clumps at superficial velocities between 0.2 and1.0 cm s(-1), internal dissolved-oxygen tension was maintained at 95% to 100% air saturation and rate of oxygen uptake at 1.6 x 10(-6) g g(-1) s(-1) dry weight. Liquid culture of single A. belladonna hairy roots was used to investigate the effect of dissolved-oxygen tensionon root growth and morphology. Total root length and number of root tips increased exponentially at oxygen tensions between 70% and 100%air saturation. Specific growth rate increased with oxygen tension up to 100% air saturation; this result demonstrates that hairy roots aeratedwithout oxygen supplementation are likely to be oxygenlimited. No growth occurred at 50% air saturation. Growth of hairy roots proceeded with an average length per tip of about 1 cm; this value was essentially independent of dissolved-oxygen tension between 70% and 100% air saturation. (c) 1994 John Wiley & Sons, Inc.     

The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in phosphorus acquisition

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. In addition, plants with the root hair response acquire more phosphorus than mutants that have root hairs that do not respond to phosphorus limiting conditions. The purpose of this experiment was to determine the efficiency of root hairs in phosphorus acquisition at high- and low-phosphorus availability. Root hair growth, root growth, root respiration, plant phosphorus uptake, and plant phosphorus content of 3-wk-old wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under high (54 mmol/m) and low (0.4 mmol/m) phosphorus availability. A cost-benefit analysis was constructed from the measurements to determine root hair efficiency. Under high-phosphorus availability, root hairs did not have an effect on any of the parameters measured. Under low-phosphorus availability, wild-type Arabidopsis had greater total root surface area, shoot biomass, phosphorus per root length, and specific phosphorus uptake. The cost-benefit analysis shows that under low phosphorus, wild-type roots acquire more phosphorus for every unit of carbon respired or unit of phosphorus invested into the roots than the mutants. We conclude that the response of root hairs to low-phosphorus availability is an efficient strategy for phosphorus acquisition.     

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.     

Computational fluid dynamics modeling of mass-transfer behavior in a bioreactor for hairy root culture. II. Analysis of ultrasound-intensified process

Recently, cichoric acid production from hairy roots of Echinacea purpurea was significantly improved by ultrasound stimulation in an airlift bioreactor. In this article, the possible mechanism on ultrasound-intensified hairy root culture of E. purpurea in the bioreactor was elucidated with the help of computational fluid dynamics (CFD) simulation, membrane permeability detection, dissolved oxygen concentration detection, confocal laser-scanning microscopy (LSM) observation, and phenylalanine ammonium lyase (PAL) activity analysis. The CFD model developed in Part I was used to simulate the hydrodynamics and oxygen mass transfer in hairy root bioreactor culture stimulated by ultrasound. A dynamic mesh model combined with a changing Schmidt number method was used for the simulation of the ultrasound field. Simulation results and experimental data illustrated that ultrasound intensified oxygen mass transfer in the hairy root clump, which subsequently stimulated root growth and cichoric acid biosynthesis. Ultrasound increased the hairy root membrane permeability, and a high root membrane permeability of 0.359 h(-1) was observed at the bottom region in the bioreactor. LSM observation showed that the change in the membrane permeability recovered to normal in the further culture after ultrasound stimulation. PAL activity in the hairy roots was stimulated by ultrasound increase and was correlated well to cichoric acid accumulation in the hairy roots of E. purpurea.     

Infection of turnip and radish storage roots with Agrobacterium rhizogenes

Within about 10 days after inoculation with Agrobacterium rhizogenes, the vascular bundles of storage root disks of turnip or radish developed small outgrowths with numerous root hairs. Thereafter, adventitious roots (hairy roots) emerged extensively from these outgrowths. The hairy roots which emerged fully supported the growth of host plants, though they lacked geotropism. An excised hairy root could be subcultured as an axenic root culture in the absence of phytohormones. Hairy root cultures with extensive lateral branches grew much more rapidly than those with few lateral branches or ordinary roots. Calli were induced from hairy root cultures in the presence of 2,4-D, and root proliferation from these calli occurred in the absence of 2,4-D. Both the primary hairy roots and the roots which grew from them synthesized agropine and mannopine.     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

Heterogeneous nutrient supply promotes maize growth and phosphorus acquisition: additive and compensatory effects of lateral roots and root hairs

Background and AimsRoot proliferation is a response to a heterogeneous nutrient distribution. However, the growth of root hairs in response to heterogeneous nutrients and the relationship between root hairs and lateral roots remain unclear. This study aims to understand the effects of heterogeneous nutrients on root hair growth and the trade-off between root hairs and lateral roots in phosphorus (P) acquisition.MethodsNear-isogenic maize lines, the B73 wild type (WT) and the rth3 root hairless mutant, were grown in rhizoboxes with uniform or localized supply of 40 (low) or 140 (high) mg P kg⁻¹ soil.ResultsBoth WT and rth3 had nearly two-fold greater shoot biomass and P content under local than uniform treatment at low P. Significant root proliferation was observed in both WT and rth3 in the nutrient patch, with the WT accompanied by an obvious increase (from 0.7 to 1.2 mm) in root hair length. The root response ratio of rth3 was greater than that of WT at low P, but could not completely compensate for the loss of root hairs. This suggests that plants enhanced P acquisition through complementarity between lateral roots and root hairs, and thus regulated nutrient foraging and shoot growth. The disappearance of WT and rth3 root response differences at high P indicated that the P application reduced the dependence of the plants on specific root traits to obtain nutrients.ConclusionsIn addition to root proliferation, the root response to a nutrient-rich patch was also accompanied by root hair elongation. The genotypes without root hairs increased their investment in lateral roots in a nutrient-rich patch to compensate for the absence of root hairs, suggesting that plants enhanced nutrient acquisition by regulating the trade-off of complementary root traits.     

Root hair defective4 encodes a phosphatidylinositol-4-phosphate phosphatase required for proper root hair development in Arabidopsis thaliana

Polarized expansion of root hair cells in Arabidopsis thaliana is improperly controlled in root hair-defective rhd4-1 mutant plants, resulting in root hairs that are shorter and randomly form bulges along their length. Using time-lapse fluorescence microscopy in rhd4-1 root hairs, we analyzed membrane dynamics after labeling with RabA4b, a marker for polarized membrane trafficking in root hairs. This revealed stochastic loss and recovery of the RabA4b compartment in the tips of growing root hairs, consistent with a role for the RHD4 protein in regulation of polarized membrane trafficking in these cells. The wild-type RHD4 gene was identified by map-based cloning and was found to encode a Sac1p-like phosphoinositide phosphatase. RHD4 displayed a preference for phosphatidylinositol-4-phosphate [PI(4)P] in vitro, and rhd4-1 roots accumulated higher levels of PI(4)P in vivo. In wild-type root hairs, PI(4)P accumulated primarily in a tip-localized plasma membrane domain, but in rhd4-1 mutants, significant levels of PI(4)P were detected associated with internal membranes. A fluorescent RHD4 fusion protein localized to membranes at the tips of growing root hairs. We propose that RHD4 is selectively recruited to RabA4b-labeled membranes that are involved in polarized expansion of root hair cells and that, in conjunction with the phosphoinositide kinase PI-4Kbeta1, RHD4 regulates the accumulation of PI(4)P on membrane compartments at the tips of growing root hairs.     

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.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.     

Biological and environmental degradation of gorilla hair and microsatellite amplification success

Naturally shed hairs are an important source of genetic material for both conservation and forensics but are notoriously poor sources of DNA. DNA degradation in hair roots is caused by apoptosis as part of the cycle of hair growth and by autolysis in decomposing animals. Shed hairs are additionally exposed to degenerative environmental processes. However, genetic studies rarely examine hair root morphologies or refer to root growth phases prior to analysis, and detailed knowledge of the rapidity of DNA degradation amongst shed hairs is lacking. We examined the effects of biological and environmental processes on western lowland gorilla ( Gorilla gorilla gorilla Savage and Wyman) hair roots with respect to morphological characteristics and polymerase chain reaction (PCR) success at eight nuclear loci. Root type frequencies indicate that gorilla body hairs may exhibit a longer telogen phase than human head hairs. All plucked hair root types amplified more efficiently than shed hairs, and only 41% of shed hairs had root types considered suitable for genotyping. Telogen hairs from fresh nests were four-fold more useful for genotyping if the roots were associated with translucent epithelial tissue, and preselection of these root types doubled the overall data-yield to 58%. Nest age correlated with root morphology and PCR success, and PCR success was almost halved after 3 days of exposure. Finally, an association between postmortem interval, root morphology, and PCR success was observed that was consistent with postmortem changes reported in human head hairs.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 281–294.     

Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures.

Various strategies aimed at improving IgG(1) antibody accumulation in transgenic tobacco cell and organ cultures were tested. The form of tissue had a significant effect on antibody levels; shooty teratomas were less productive than hairy roots or suspended cells. Although there were several disadvantages associated with hairy roots compared with suspensions, such as slower growth, slower antibody production, and formation of a greater number of antibody fragments, the roots exhibited superior long-term culture stability. Antibody accumulation in hairy root cultures was improved by increasing the dissolved oxygen tension to 150% air saturation, indicating the need for effective oxygen transfer in root reactors used for antibody production. Preventing N-linked glycosylation using tunicamycin or inhibition of subsequent glycan processing by castanospermine reduced antibody accumulation in the biomass and/or medium in cell suspensions. Loss of antibody from the cultures after its secretion and release into the medium was identified as a major problem. This effect was minimized by inhibiting protein transport in the secretory pathway using Brefeldin A, resulting in antibody accumulation levels up to 2.7 times those in untreated cells. Strategies for protecting secreted antibody, such as addition of poly(vinylpyrrolidone) and periodic harvesting from the medium using hydroxyapatite resin, also increased antibody titers. The mechanisms responsible for the disappearance of antibody from plant culture media were not clearly identified; degradation by proteases and conformational modification of the antibody, such as formation of aggregates, provided an explanation for some but not all the phenomena observed. This work demonstrates that the manipulation and control of culture conditions and metabolic processes in plant tissue cultures can be used to improve the production of foreign proteins. However, loss of secreted antibody from plant culture medium is a significant problem that may limit the feasibility of using product recovery from the medium to reduce downstream processing costs relative to agricultural systems.     

OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.     

Agrobacterium tumefaciens-mediated hairy root production from seedlings of Chinese cabbage

Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.