Auxin Quantification Research Articles

A rapid and robust colorimetric method for measuring relative abundance of auxins in plant tissues.

Auxin estimation in plant tissues is a crucial component of auxin signaling studies. Despite the availability of various high-throughput auxin quantification methods like LC-MS, GC-MS, HPLC, biosensors, and DR5-gus/gfp-based assays, auxin quantification remains troublesome because these techniques are very expensive and technology intensive and they mostly involve elaborate sample preparation or require the development of transgenic plants. To find a solution to these problems, we made use of an old auxin detection system to quantify microbe derived auxins and modified it to effectively measure auxin levels in rice plants. Auxins from different tissues of rice plants, including root samples of seedlings exposed to IAA/TIBA or subjected to different abiotic stresses, were extracted in ethanol. The total auxin level was measured by the presently described colorimetric assay and counterchecked by other auxin estimation methods like LC-MS or gus staining of DR5-gus overexpressing lines. The presented colorimetric method could measure (1) the auxin levels in different tissues of rice plants, thus identifying the regions of higher auxin abundance, (2) the differential accumulation of auxins in rice roots when auxin or its transport inhibitor was supplied exogenously, and (3) the levels of auxin in roots of rice seedlings subjected to various abiotic stresses. The thus obtained auxin levels correlated well with the auxin levels determined by other methods like LC-MS or gus staining and the expression pattern of auxin biosynthesis pathway genes. The auxin estimation method described here is simple, rapid, cost-effective, and sensitive and allows for the efficient detection of relative auxin abundances in plant tissues.

Influence of Leaf Number, Rooting Trait, and Cutting Size in Vegetative Propagation of Aquilaria cumingiana [Decne] Ridl.

A low-cost technique of propagation study to determine the influence of leaf number and relationship among rooting trait and cutting size was carried out on cuttings from seedlings of Aquilaria cumingiana. The highest percentage of rooting was detected in cuttings with two leaves (83.62%) and lowest in cuttings with the greatest (3) number of leaves (50%). Comparison of the means revealed a significant increase in percent rooting, number of roots, and length of the longest root on cuttings with two leaves. Correlation analysis among root traits revealed significant (P < 0.0001) low to high positive relationships (r2 = 0.26–0.91). The strongest association was recorded between the average root length and the length of the longest root (r2 = 0.91, P < 0.0001). All relationships between the rooting trait and the cutting size displayed weak and non-significant (r2 = 0.04–0.17, P > 0.05) – suggesting that aside from amount, variation in distribution and transport of naturally occurring endogenous auxin might probably affect the rooting. This study generated an economical and practical protocol to vegetatively mass- produce this premium species. Further studies are recommended, particularly with regards to auxin quantification to identify and quantify the specific naturally occurring endogenous auxin that is affecting the rooting of A. cumingiana.

Coumarin Interferes with Polar Auxin Transport Altering Microtubule Cortical Array Organization in Arabidopsis thaliana (L.) Heynh. Root Apical Meristem.

Coumarin is a phytotoxic natural compound able to affect plant growth and development. Previous studies have demonstrated that this molecule at low concentrations (100 µM) can reduce primary root growth and stimulate lateral root formation, suggesting an auxin-like activity. In the present study, we evaluated coumarin’s effects (used at lateral root-stimulating concentrations) on the root apical meristem and polar auxin transport to identify its potential mode of action through a confocal microscopy approach. To achieve this goal, we used several Arabidopsis thaliana GFP transgenic lines (for polar auxin transport evaluation), immunolabeling techniques (for imaging cortical microtubules), and GC-MS analysis (for auxin quantification). The results highlighted that coumarin induced cyclin B accumulation, which altered the microtubule cortical array organization and, consequently, the root apical meristem architecture. Such alterations reduced the basipetal transport of auxin to the apical root apical meristem, inducing its accumulation in the maturation zone and stimulating lateral root formation.

The unique bryophyte-specific repeat-containing protein SHORT-LEAF regulates gametophore development in moss.

Convergent evolution of shoot development across plant lineages has prompted numerous comparative genetic studies. Though functional conservation of gene networks governing flowering plant shoot development has been explored in bryophyte gametophore development, the role of bryophyte-specific genes remains unknown. Previously, we have reported Tnt1 insertional mutants of moss defective in gametophore development. Here, we report a mutant (short-leaf; shlf) having two-fold shorter leaves, reduced apical dominance, and low plasmodesmata frequency. UHPLC-MS/MS-based auxin quantification and analysis of soybean (Glycine max) auxin-responsive promoter (GH3:GUS) lines exhibited a striking differential auxin distribution pattern in the mutant gametophore. Whole-genome sequencing and functional characterization of candidate genes revealed that a novel bryophyte-specific gene (SHORT-LEAF; SHLF) is responsible for the shlf phenotype. SHLF represents a unique family of near-perfect tandem direct repeat (TDR)-containing proteins conserved only among mosses and liverworts, as evident from our phylogenetic analysis. Cross-complementation with a Marchantia homolog partially recovered the shlf phenotype, indicating possible functional specialization. The distinctive structure (longest known TDRs), absence of any known conserved domain, localization in the endoplasmic reticulum, and proteolytic cleavage pattern of SHLF imply its function in bryophyte-specific cellular mechanisms. This makes SHLF a potential candidate to study gametophore development and evolutionary adaptations of early land plants.

Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants.

The plant hormone auxin, indole-3-acetic acid, regulates many aspects of plant growth and development. Auxin quantification should offer broad insights into its mechanistic action in plants. However, limited auxin content in plant tissues hampers the establishment of quantification methods without the highest graded instruments or deeply specialized experimental techniques. In this study, we detailed optimized conditions for high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (LC/MS). We compared LC/MS conditions, such as columns, mobile phases, parameters of acquisition methods (selective or multiple ion monitoring), dwell times (DTs), and channel numbers, using differentially mixed authentic auxin and its related compounds. We further investigated pretreatment methods through the optimization of auxin recovery and irrelative compound removal from plant tissues prior to the LC/MS analysis. Our LC/MS analysis demonstrated the particular importance of the column, DTs, and channel numbers on detection sensitivity. Our comparative analysis developed optimal pretreatment methods, including the pulverization of plants, concentration of extract through centrifugal evaporation, and removal of irrelative metabolites using liquid-liquid extraction and a spin filter. We injected plant samples into our LC/MS system, quantified auxin and eight related compounds in a single measurement, and determined the auxin increase in an auxin over-producing mutant. Our practical optimization of LC/MS conditions and pretreatment methods provides detailed experimental processes toward the sensitive quantification of auxin from 10 mg of plant tissue. These processes have not always been clearly documented; therefore, our protocol could broadly contribute to technical advances in plant growth and development research.

Lipo-chitooligosaccharides promote lateral root formation and modify auxin homeostasis in Brachypodium distachyon.

Lipo-chitooligosaccharides (LCOs) are microbial symbiotic signals that also influence root growth. In Medicago truncatula, LCOs stimulate lateral root formation (LRF) synergistically with auxin. However, the molecular mechanisms of this phenomenon and whether it is restricted to legume plants are not known. We have addressed the capacity of the model monocot Brachypodium distachyon (Brachypodium) to respond to LCOs and auxin for LRF. For this, we used a combination of root phenotyping assays, live-imaging and auxin quantification, and analysed the regulation of auxin homeostasis genes. We show that LCOs and a low dose of the auxin precursor indole-3-butyric acid (IBA) stimulated LRF in Brachypodium, while a combination of LCOs and IBA led to different regulations. Both LCO and IBA treatments locally increased endogenous indole-3-acetic acid (IAA) content, whereas the combination of LCO and IBA locally increased the endogenous concentration of a conjugated form of IAA (IAA-Ala). LCOs, IBA and the combination differentially controlled expression of auxin homeostasis genes. These results demonstrate that LCOs are active on Brachypodium roots and stimulate LRF probably through regulation of auxin homeostasis. The interaction between LCO and auxin treatments observed in Brachypodium on root architecture opens interesting avenues regarding their possible combined effects during the arbuscular mycorrhizal symbiosis.

The Use of Auxin Quantification for Understanding Clonal Tree Propagation

Qualitative and quantitative hormone analyses have been essential for understanding the metabolic, physiological, and morphological processes that are influenced by plant hormones. Auxins are key hormones in the control of many aspects of plant growth and development and their endogenous levels are considered critical in the process of adventitious root induction. Exogenous auxins are used extensively in the clonal propagation of tree species by cuttings or tissue culture. Understanding of auxin effects has advanced with the development of increasingly accurate methods for auxin quantification. However, auxin analysis has been challenging because auxins typically occur at low concentrations, while compounds that interfere with their detection often occur at high concentrations, in plant tissues. Interference from other compounds has been addressed by extensive purification of plant extracts prior to auxin analysis, although this means that quantification methods have been limited by their expense. This review explores the extraction, purification, and quantification of auxins and the application of these techniques in developing improved methods for the clonal propagation of forestry trees.

Fast Detection of Auxins by Microplate Technique

Plant growth promotion indole-3-acetic acid (IAA) is the most abundant natural auxin that plays diverse roles in plant growth, development and plant immunity. Perturbing auxin homeostasis appears to be a common virulence mechanism, as many pathogens can synthesize auxin-like molecules. In other hand, the addition of plant growth promotion rhizobacteria (PGPR) that are able to produce auxins promotes plant growth and provides protection against pathogens. Techniques as high performance liquid chromatography (HPLC) and gas chromatography (GC) are used to quantify auxins produced by microorganism and plants at high precision and sensitivity, even though those techniques are expensive and require a big number of solvents. For these reasons, the aim of the present study was to develop a fast microplate technique for auxin detection, in Bacillus subtilis strains using salkowski reagent. For auxin quantification, calibration curves were done with alcohol, landy medium and water and the R2 were calculated. The microplate techniques were able to quantify auxin production by B. subtillis stains.

Auxin-producing Bacillus sp.: Auxin quantification and effect on the growth of Solanum tuberosum

Plant-associated bacteria are known to improve plant growth and play a major role in the development of plants. The present study is concerned with the isolation of two auxin-producing plant growth-promoting bacteria (PGPB). On the basis of 16S rRNA sequencing, both of the strains are identified as Bacillus sp. Maximum auxin production was observed at 37 °C after 48 h of incubation. Increase in tryptophan concentration stimulated auxin production by the isolates. High-performance liquid chromatography analysis showed that the bacterial auxin exhibited similar retention time as the standard indole-3-acetic acid (IAA). Sprouts of Solanum tuberosum var. Desiree were inoculated with the isolates. Comparison of various growth parameters of inoculated plants with non-inoculated plants revealed the improvement of plant growth by bacterial inoculation. Almost 40 and 35 % increase in shoot length with P4 and S6 inoculation, respectively, was observed. Considerable improvement in root growth was observed with an increase in the number and length of roots. On the basis of the above findings, it is concluded that the plant growth-promoting Bacillus strains affect S. tuberosum beneficially, resulting in improved plant growth.

Auxin producing Bacillus sp.: Auxin quantification and effect on the growth of Solanum tuberosum

Plant-associated bacteria are known to improve plant growth and play a major role in the development of plants. The present study is concerned with the isolation of two auxin- producing plant growth-promoting bacteria (PGPB). On the basis of 16S rRNA sequencing, both of the strains are identified as Bacillus sp. Maximum auxin production was observed at 37 °C after 48 h of incubation. Increase in tryptophan concentration stimulated auxin pro- duction by the isolates. High-performance liquid chromatography analysis showed that the bacterial auxin exhibited similar retention time as the standard indole-3-acetic acid (IAA). Sprouts of Solanum tuberosum var. Desiree were inoculated with the isolates. Comparison of various growth parameters of inoculated plants with non-inoculated plants revealed the im- provement of plant growth by bacterial inoculation. Almost 40 and 35 % increase in shoot length with P4 and S6 inoculation, respectively, was observed. Considerable improvement in root growth was observed with an increase in the number and length of roots. On the basis of the above findings, it is concluded that the plant growth-promoting Bacillus strains affect S. tuberosum beneficially, resulting in improved plant growth.

Auxin producing Bacillus Sp.: Auxin quantification and effect on the growth of Solanum tuberosum

Auxin producing Bacillus Sp.: Auxin quantification and effect on the growth of Solanum tuberosum

Auxin quantification based on histochemical staining of GUS under the control of auxin-responsive promoter

To visualize phytohormone localization in plant tissues, transgenic plants comprising the GUS reporter gene are often used. However, until now only qualitative assessment of the hormone presence was available. In this work, we suggested the method for IAA quantification in transgenic DR5::GUS Arabidopsis thaliana L. plants by the analysis of digital images. An empirical quadratic dependence was established between the IAA concentration in medium and the level of GUS-dependent staining. Using this method, we demonstrated that, after A. thaliana root gravistimulation for 90 min, auxin lateral redistribution occurred. It resulted in the increase in the IAA concentration in the lower root part (in the elongation zone and apical meristem) by 200% on the average.

Immunophilin-like TWISTED DWARF1 Modulates Auxin Efflux Activities of Arabidopsis P-glycoproteins

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.