Aux Genes Research Articles

Efficient production of isoflavonoids by Astragalus membranaceus hairy root cultures and evaluation of antioxidant activities of extracts.

In this study, Astragalus membranaceus hairy root cultures (AMHRCs) were established as an attractive alternative source for the efficient production of isoflavonoids (IF). A. membranaceus hairy root line II was screened as the most efficient line and was confirmed by PCR amplification of rolB, rolC and aux1 genes. Culture parameters of AMHRCs were systematically optimized, and five main IF constituents were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total IF accumulation of 34 day old AMHRCs was 234.77 μg/g dry weight (DW). This yield was significantly higher compared to that of 3 year old field grown roots (187.38 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that AMHRC extracts exhibited antioxidant activities with lower IC50 values (1.40 and 1.73 mg/mL) as compared to those of field grown roots (1.96 and 2.17 mg/mL). Overall, AMHRCs may offer a promising and continuous product platform for naturally derived, high quality and valuable nutraceuticals.

Optimization of Astragalus membranaceus hairy roots induction and culture conditions for augmentation production of astragalosides

In this study, an efficient and reliable Agrobacterium rhizogenes-mediated hairy root system in A. membranaceus was developed. An optimal transformation rate of 66.84 % was obtained after 13 days by inoculating A. rhizogenes LBA9402 onto 3-week-old leaf explants followed by a co-cultivation period of 2 days with acetosyringone (100 μM). Among eight representative A. membranaceus hairy root lines (AMHRL), AMHRL VI was found to be the lead line and confirmed by PCR amplification of rolB, rolC and aux1 genes. Culture parameters of AMHRL VI were systematically optimized by central composite design and slogistic kinetic models, and six main astragalosides (AG) were quail-quantitatively determined by liquid chromatography–tandem mass spectrometry. Under optimal conditions (inoculum size 1.54 %, culture temperature 27.8 °C, sucrose concentration 3.24 % and harvest time 36 days), A. membranaceus hairy root cultures (AMHRCs) in MS medium gave the optimal biomass production of 15.79 g/L dry weight (DW) with total AG accumulation of 2.657 mg/g DW. This yield was significantly superior to that of 3-year-old field grown roots (2.435 mg/g DW). Overall, this investigation provided the generation of a high-productive AMHRCs that is capable of augmentation production of AG, offering a promising and continuous product platform for naturally derived, high quality and valuable phytochemicals.

Development of hairy root culture system of Phlogacanthus thyrsiflorus Nees

Hairy root cultures were established from leaves, shoot tips and node explants of in vitro grown plants upon co-cultivation with Agrobacterium rhizogenes Conn (strain ATCC 15834) by transformation. Hairy root induction was observed after 7d on all three explants co-cultured for different periods. Amongst different durations of co-culture (45, 75, 105, 135, 165, 195, 225 and 255min), 225min was found to be the best for all the three explants. Shoot tip explants showed maximum percentage of root induction (93.33%) as well as highest number of roots (4.80) per explant. PCR analysis of rolC, and aux1 genes was performed in transformed and non-transformed roots and confirmed. Specific amplification products of 815bp and 487bp were deserved in transformed roots by aux1 and rolC primers.

Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting

BackgroundMeyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes.ResultsWe targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping.ConclusionsWe herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.

A new biotechnological source of rosmarinic acid and surface flavonoids: Hairy root cultures of Dracocephalum kotschyi Boiss

Hairy root induction in Dracocephalum kotschyi Boiss was investigated as a method of producing rosmarinic acid and surface flavonoids, plant secondary metabolites well-known for their antioxidant and anticancer activities. The transformation of D. kotschyi hairy root lines induced by infection with Agrobacterium rhizogenes LBA 9402 was confirmed by PCR detection of rolC and aux1 genes, and their capacity to grow and biosynthesize rosmarinic acid and surface flavonoids was studied. Two types of morphology, typical hairy root and callus-like were observed in the induced root lines. The rolC and aux1 genes were detected in the genome of both morphological types of root lines, although aux1 was more frequently observed in callus-like roots. Our results showed the capacity of the obtained hairy root lines to produce rosmarinic acid and methoxylated flavonoids. Rosmarinic acid content in hairy root lines ranged from 10 to 1500μg/g DW, which at its peak was 15 times higher than in the intact control roots. Surface flavonoids were identified in most hairy root lines, some of which showed a surface flavonoid content higher than the roots of the whole plant but generally lower than the plant leaves.

Assessment of salt tolerance in transgenic tobacco (Nicotiana tobacum L.) plants expressing the AUX gene

Transformation of plants using Agrabacterium rhizogenes may affect secondary metabolite production as well as morphological changes. In this study, T-DNA from Ri plasmid in A. rhizogenes carrying pRi15834-PRT35S-GUS was introduced into tobacco leaf segments to initiate development of transformed hairy roots. Plant regeneration from transgenic roots used MS medium, and plants regenerated from transgenic roots were subjected to 300 mM NaCl. Transgenic plants showed higher levels of salt tolerance compared to non-transgenic plants. This could be due to over expression of the AUX gene in transformed hairy roots and plants regenerated from transgenic roots. © 2011 Progress in Biological Sciences, Vol. 1, No.1, 17-23.

The role of AUX1 gene and auxin content to the branching phenotype of Kenaf (Hibiscus cannabinus L.).

The objectives of this research were to identify auxin gene, AUX1, and to determine the plant auxin content and their role in conferring branching on Kenaf. PCR analysis using AUX1 primer capable to amplify the DNA of non branching (KR11) and branching kenaf mutant, resulting in 800 bp PCR product. The sequence of the PCR product showed high degree of homology with the sequence of AUX1 gene of other plants in the NCBI GenBank database, confirming kenaf possession of the gene AUX1. However, some variation on the DNA sequence was found between branching and non branching phenotype indicated allele differences of the same gene which were responsible for the variation in the type of branching. Identification of auxin content in the roots, apical shoot, and axillary branches using spectrophotometry method showed that the branching plant has higher auxin content in the apical shoot compared to the content in the branches. This indicate that AUX1 controls the formation of branches by controlling either the content of auxin in the apical shoot and branches, or the ratio of auxin content in the shoot and branches.

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxin-autotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.

Agrobacterium rhizogenes-Mediated Transformation of Hypericum tomentosum L. and Hypericum tetrapterum Fries.

This is the first evidence on successful Agrobacterium rhizogenes-mediated genetic transformation of two species from the genus Hypericum, H. tomentosum L. and H. tetrapterum Fries. Hairy root cultures were induced from root segments of both Hypericum species by two agropine wild-type strains of A. rhizogenes, ATCC 15834 and A4. The transgenic character of the hairy root cultures was proved by PCR amplification of the rolABCD genes. In some H. tetrapterum transgenic lines aux genes were detected as well.

Morphology and withanolide production of Withania coagulans hairy root cultures

AbstractInoculation of leaf sections of Withania coagulans with Agrobacterium tumefaciens strain C58C1 (pRiA4) induced transformed roots with the capacity to produce the most important bioactive compounds of Withania species, withanolide A and withaferin A. The hairy roots obtained showed two morphologies: callus‐like roots (CR) with a high capacity to produce withanolides and typical hairy roots (HR) with faster growth capacity and lower withanolide accumulation. The aux1 gene of pRiA4 was detected by PCR analyses in all roots showing callus‐like morphology. However, this gene was only detected in 12.5% of the roots showing typical hairy root morphology. This fact suggests a significant role of aux genes in the morphology of transformed roots. Time course studies of withanolide production showed that withanolide A accumulated during the first part of the culture whereas the maximum accumulation of withaferin A occurred at the end of the culture period. Some transformed root lines, such as HR112 and CR26, showed considerable potential to produce withanolides in a scaled up bioreactor system, especially the important pharmaceutical compound withanolide A.

Callus, shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in Arabidopsis

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration.

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.

Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization.

The aims of this study were to set up proliferation conditions for hairy roots of Coffea arabica regenerated after transformation by Agrobacterium rhizogenes strain A4-RS, and to carry out the morphological and molecular characterization of hairy root clones maintained over the long term. Auxin supply, light conditions and sucrose concentration were modified with the aim of establishing efficient root proliferation conditions. The morphological variability among 62 established hairy root clones was phenotyped by scanning the roots and analysing the images using 'whinRHIZO' software procedures. PCR analysis of integration in transformed root cells of rol and aux oncogenes from the T-DNA of the Ri plasmid was used to study the molecular variability among clones. Auxin supply was necessary to obtain and stimulate growth and branching, and IBA applied at 0.5 microm was the most efficient auxin. Significant differences were shown among the 62 clones for total root length and for the percentage of fine roots. These variables were stable across subcultures and could hence be used for efficient characterization of hairy root clones. The majority of hairy root clones (86 %) exhibited non-significant phenotype differences with non-transformed roots. Eight clones were significantly different from the non-transformed controls in that they possessed a low proportion of fine roots. Two other hairy root clones grew significantly faster than the other clones. The PCR analysis revealed a low variability in the integration of rol and aux oncogenes in transformed root cells. The T(R)-DNA was never integrated as aux1 and aux2 genes were not found, although rolB and rolC genes from the T(L)-DNA were always present. The discovery of low morphological variability among coffee hairy roots together with the identification of morphological variables allowing easy identification of phenotypically altered clones represent two important results. They make hairy roots a possible, and efficient, tool for functional-genomic studies of coffee root genes.

Vectorial Information for Arabidopsis Planar Polarity Is Mediated by Combined AUX1, EIN2, and GNOM Activity

Cell polarity is commonly coordinated within the plane of a single tissue layer (planar polarity), and hair positioning has been exploited as a simple marker for planar polarization of animal epithelia . The root epidermis of the plant Arabidopsis similarly reveals planar polarity of hair localization close to root tip-oriented (basal) ends of hair-forming cells . Hair position is directed toward a concentration maximum of the hormone auxin in the root tip , but mechanisms driving this plant-specific planar polarity remain elusive. Here, we report that combinatorial action of the auxin influx carrier AUX1, ETHYLENE-INSENSITIVE2 (EIN2) , and GNOM genes mediates the vector for coordinate hair positioning. In aux1;ein2;gnom eb triple mutant roots, hairs display axial (apical or basal) instead of coordinate polar (basal) position, and recruitment of Rho-of-Plant (ROP) GTPases to the hair initiation site reveals the same polar-to-axial switch. The auxin concentration gradient is virtually abolished in aux1;ein2;gnom eb roots, where locally applied auxin can coordinate hair positioning. Moreover, auxin overproduction in sectors of wild-type roots enhances planar ROP and hair polarity over long and short distances. Hence, auxin may provide vectorial information for planar polarity that requires combinatorial AUX1, EIN2, and GNOM activity upstream of ROP positioning.

Is the cost of herbicide resistance expressed in the breakdown of the relationships between characters? A case study using synthetic-auxin-resistant Arabidopsis thaliana mutants

A mutation endowing herbicide resistance is often found to induce a parallel morphological or fitness penalty. To test whether such 'cost' of resistance to herbicides is expressed through lower resource acquisition, changes in resource allocation, or both, is of ecological significance. Here, we analysed 12 morphological traits in 900 plants covering three herbicide resistance mutations at genes AUX1 , AXR1 and AXR2 in the model species Arabidopsis thaliana . Comparing these 2,4-D herbicide-resistant homozygous (RR) and heterozygous (RS) plants to homozygous susceptible (SS) plants, this analysis estimates the dominance level of the resistance allele on morphology. We also demonstrated that the herbicide resistance cost was primarily expressed as a change in resource acquisition (62.1-94% of the analysed traits). Although AUX1 , AXR1 and AXR2 genes act in the same metabolic pathway of auxin response, each resistance factor was found to have its own unique signature in the way the cost was expressed. Furthermore, no link was observed between the absolute fitness penalty and the respective modifications of resource acquisition and/or resource allocation in the resistant plants. These results and their implications for herbicide resistance spread and establishment are discussed.

Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes.

We previously isolated a partial soybean cDNA clone whose transcript abundance is increased upon infection by the sedentary, endoparasitic soybean cyst nematode Heterodera glycines. We now isolated the corresponding full-length cDNA and determined that the predicted gene product was similar to the group of cofactor-dependent phosphoglycerate mutase/bisphosphoglycerate mutase enzymes (PGM/bPGM; EC 5.4.2.1/5.4.2.4). We designated the corresponding soybean gene GmPGM. PGM and bPGM are key catalysts of glycolysis that have been well characterized in animals but not plants. Using the GmPGM cDNA sequence, we identified a homologous Arabidopsis thaliana gene, which we designated AtPGM. Histochemical GUS analyses of transgenic Arabidopsis plants containing the AtPGM promoter ::GUS construct revealed that the AtPGM promoter directs GUS expression in uninfected plants only to the shoot and root apical meristems. In infected plants, GUS staining also is evident in the nematode feeding structures induced by the cyst nematode Heterodera schachtii and by the root-knot nematode Meloidogyne incognita. Furthermore, we discovered that the AtPGM promoter was down-regulated by abscisic acid and hydroxyurea, whereas it was induced by sucrose, oryzalin, and auxin, thereby revealing expression characteristics typical of genes with roles in meristematic cells. Assessment of the auxin-inducible AUX1 gene promoter (a gene coding for a polar auxin transport protein) similarly revealed feeding cell and meristem expression, suggesting that auxin may be responsible for the observed tissue specificity of the AtPGM promoter. These results provide first insight into the possible roles of PGM/bPGM in plant physiology and in plant-pathogen interactions.

Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology, xylem structure and chemistry.

The effects of Agrobacterium pRiA4 rol and aux genes, controlled by their endogenous promoters, on tree growth and wood anatomy and chemistry were studied in 5- and 7-year-old silver birch (Betula pendula Roth) plants. Southern hybridization confirmed the following rol and aux gene combinations: control plants (no genes transferred); plants with rolC and rolD genes; plants with rolA, rolB, rolC and rolD genes; and plants with rolA, rolB, rolC, rolD, aux1 and aux2 genes. Transgene mRNA was most abundant in phloem/cambium samples and in the developing xylem, whereas no expression was detected in leaves. Plants with rolC and rolD genes or with all the rol genes were significantly shorter and had smaller leaves and a more bushy growth habit than control plants or plants with both aux and rol genes. Morphological observations and wood chemistry analyses revealed that plants with rol genes produced less xylem and broke bud later than control plants or plants with both aux and rol genes. Tension wood was detected in both control and transgenic plants irrespective of their gene combination, probably as a result of greenhouse cultivation. Xylem fibers were shorter in transgenic plants than in control plants, and plants with all the rol genes were characterized by shorter vessels compared with the control plants and a smaller proportional area of vessels compared with the other groups. In addition, silver birch plants with all the rol genes had approximately a 3.3% lower concentration of total acid soluble carbohydrates than control plants. We conclude that the rolC and rolD genes induced the typical "rol-phenotype," and that this was emphasized by concomitant expression of the rolA and rolB genes and alleviated by the presence of aux1 and aux2 genes. We observed consistent phenotypic effects of rol and aux genes on the morphology, anatomy and cell wall chemistry of the plants.

Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth.

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

Ginsenoside production in different phenotypes of Panax ginseng transformed roots

Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A. Meyer with Agrobacterium rhizogenes A4. The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium. Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching. The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A. rhizogenes transformation conditioned a higher biomass formation. When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype. The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions. This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots. The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group. From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.