Indole-3-acetic Acid Analysis Service

What is Indole-3-acetic Acid?

Indole-3-acetic acid (IAA) is the most well-known and widely distributed natural auxin, a class of plant hormones for plant growth and development. Structurally, IAA is a derivative of indole, an aromatic heterocyclic organic compound with a bicyclic structure, which includes a carboxymethyl group. The presence of this functional group makes IAA a highly bioactive molecule capable of influencing a broad range of physiological and developmental processes in plants.

Functions of IAA in Plants

Cell Elongation and Division: One of the primary functions of IAA is to promote cell elongation and division, particularly in the stem and root tissues. IAA activates proton pumps in the cell membrane, leading to cell wall loosening and expansion. This effect is vital for shoot elongation, root development, and overall plant size.

Apical Dominance: IAA regulates apical dominance, the phenomenon where the main central stem of the plant grows more robustly than the side shoots. The high concentration of IAA at the apex inhibits lateral bud growth, directing resources toward vertical growth.

Root Initiation and Growth: IAA promotes the formation of lateral and adventitious roots, crucial for the plant's ability to explore soil for water and nutrients. It enhances root hair development, which increases surface area for nutrient absorption.

Vascular Tissue Differentiation: IAA influences the differentiation of vascular tissues such as xylem and phloem, which are critical for transporting water, nutrients, and photosynthates throughout the plant. This function is essential for the establishment of healthy, functional vascular systems in plants.

Indole-3-acetic Acid Analysis Offered by Creative Proteomics

In Vitro IAA Quantification: Comprehensive measurement of IAA levels in plant and tissue cultures to monitor hormone concentrations during experiments.

In Vivo IAA Monitoring: Accurate detection and quantification of IAA levels within living plants, aiding in the study of real-time hormonal fluctuations and stress responses.

Environmental Microbiome IAA Analysis: Investigating IAA production by bacteria and fungi, with potential applications in sustainable agricultural practices and microbial ecology.

Custom Research Solutions: Tailored IAA analysis for unique experimental setups, offering flexibility to address specific research questions and requirements.

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Technology Platforms for Indole-3-acetic Acid Assay

At Creative Proteomics, our Indole-3-acetic acid (IAA) analysis is built on advanced instrumentation to ensure precision, sensitivity, and reproducibility across a wide range of sample types. Our primary technology platform is High-Performance Liquid Chromatography (HPLC), integrated with UV detection at 254 nm, which is ideally suited for the quantitative analysis of IAA in complex biological matrices.

High-Performance Liquid Chromatography (HPLC) with UV Detection

HPLC is a powerful separation technique, essential for analyzing non-volatile and thermally unstable compounds like IAA. At Creative Proteomics, we use Agilent 1260 Infinity II HPLC systems, renowned for their high resolution and throughput. Key features include:

• High Sensitivity and Selectivity: The HPLC system, combined with UV detection, allows precise identification of IAA, even in trace amounts, while effectively separating it from structurally similar compounds or impurities present in the sample.
• Quantitative Accuracy: The use of photodiode array detectors (PDA) ensures high-precision UV absorbance measurements at 254 nm, where IAA exhibits strong absorption, enabling accurate quantification of IAA levels.
• Versatile Sample Compatibility: Our HPLC system can handle various sample types such as plant tissues, extracts, and culture media, providing flexibility for different research applications.

Complementary Techniques: Mass Spectrometry (MS)

For enhanced sensitivity and structural confirmation, we offer HPLC-MS services using Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap MS. This high-resolution mass spectrometer provides:

• Unmatched Sensitivity: Ideal for detecting ultra-low concentrations of IAA in complex samples.
• Precise Molecular Characterization: Allows accurate determination of molecular weight and fragmentation patterns, confirming the identity of IAA and distinguishing it from similar molecules.

HPLC-MS is particularly useful for samples with low IAA concentrations or where precise molecular identification is critical.

Data Integrity and Automation

Our analysis platform is fully integrated with Empower 3 Chromatography Data System (CDS), which automates data acquisition, processing, and reporting. This ensures:

• Minimized Human Error: Automated workflows reduce manual handling, improving data reliability.
• High Throughput and Scalability: The system handles large datasets efficiently, ensuring rapid analysis without compromising accuracy.

Sample Requirements for Indole-3-acetic Acid Analysis

Plant Growth Promotion, Phytohormone Production and Genomics of the Rhizosphere-Associated Microalga, Micractinium rhizosphaerae sp. nov.

Journal: Plants

Published: 2023

Background

Microalgae are integral components of soil and plant microbiomes, contributing to soil health and promoting plant growth. This study focuses on the functional and genomic characterization of a newly identified microalga strain, NFX-FRZ, isolated from the rhizosphere of a wild Ficus tree in Portugal. The findings reveal that this strain not only binds to tomato plant tissues and promotes growth but also synthesizes various phytohormones and possesses multiple genes linked to phytohormone biosynthesis. This research underscores the potential of eukaryotic microalgae as agents for plant growth promotion, paving the way for further investigations into their roles in soil and plant interactions.

Materials & Methods

Strain Isolation, Identification, and Characterization

The NFX-FRZ strain was isolated from the roots of a wild Ficus tree in Portugal. Roots were washed with sterile water, and the solution was plated on algae culture agar, incubated under light and temperature conditions. Identification was confirmed through genomic data (18S-ITS1-5.8S-ITS2) and morphological observation via microscopy.

Phylogenetic Analysis

Sequences of Micractinium species were obtained from the NCBI database and aligned using MUSCLE. Phylogenetic relationships were analyzed with MEGA X, employing the maximum likelihood method and bootstrapping for robustness.

Growth Kinetics under Autotrophic Conditions

Axenic autotrophic growth experiments were conducted in 2 L Schott flasks with 1.5X algae culture broth. Cultivation occurred at 22 °C under specific light and aeration conditions, starting with an inoculum of approximately 1 × 10^6 cells/mL.

Tomato Plant Growth Promotion Assays

Tomato growth promotion was evaluated using various treatments on agar plates. NFX-FRZ exudates were tested by creating agar plates from microalgal exudates. Germinated seedlings were used to assess root and shoot elongation and fresh weight, with statistical analysis performed using ANOVA and Tukey's test.

Untargeted Metabolomic Analysis of NFX-FRZ Exudates

Supernatants from growth experiments were collected and subjected to metabolomic analysis. Samples were lyophilized, dissolved in methanol, and prepared for LC-MS. Metabolites were identified and quantified using Compound Discoverer software.

Genome Sequencing and Analysis

Total genomic DNA was extracted and sequenced, followed by assembly and annotation. The completeness and functional annotations of the nuclear genome, chloroplast, and mitochondrial genomes were assessed using bioinformatics tools, including GHOSTKOALA and BLASTp.

Results

Characterization of Micractinium rhizosphaerae sp. nov.

Strain NFX-FRZ is spherical, measuring ~3.5 × 3.2 μm, non-motile, reproducing via autospores. It contains a single cup-shaped chloroplast with a defined pyrenoid and distinct starch and lipid bodies. BLAST analysis confirmed a high similarity to Micractinium species, with phylogenetic analysis indicating NFX-FRZ as a novel species, Micractinium rhizosphaerae sp. nov.

Promotes Tomato Plant Growth and Binds to Roots

NFX-FRZ significantly enhanced tomato plant growth in exudate agar compared to controls, showing increased shoot elongation and fresh weight. In contrast, algae culture agar negatively affected growth, potentially due to nutrient imbalances. NFX-FRZ also attached to tomato roots, forming clusters, indicating its potential as a beneficial agent in agriculture.

Metabolomic Analysis Revealed Phytohormones and Growth Compounds

Untargeted metabolomic analysis identified 115 compounds from 5563 peaks in negative mode and 146 from 3497 in positive mode. Key compounds included organic acids and phytohormones, with indole-3-acetic acid (IAA) being predominant, suggesting it contributes to NFX-FRZ's plant growth-promoting effects.

Genomic Properties

The genome size of NFX-FRZ is 68.28 Mbp, with a GC content of 65.3%. It comprises 1497 contigs with functional annotations indicating similarities to Micractinium conductrix, though unique modules were identified in NFX-FRZ that could affect cell wall properties.

Insights into Phytohormone Production

The NFX-FRZ genome supports IAA synthesis through various pathways, and several genes related to transport and signaling were identified. It also contains genes for salicylic acid and jasmonic acid biosynthesis, though signaling mechanisms may differ from those in higher plants, indicating unique regulatory systems in Micractinium.

Table 1. Top 30 compounds identified in NFX-FRZ exudates (15 from each of negative and positive ionization modes).

Table 2. Phytohormones detected in M. rhizosphaerae NFX-FRZ exudates.

Reference

1. Quintas-Nunes, Francisco, et al. "Plant Growth Promotion, Phytohormone Production and Genomics of the Rhizosphere-Associated Microalga, Micractinium rhizosphaerae sp. nov." Plants 12.3 (2023): 651.