藻类学
植物生物化学 蛋白质 RNA 植物育种 植物细胞生物学 植物发育生物学 植物免疫 植物新陈代谢 植物分子生物学 植物生理学 植物转化Quantification of Protochlorophyllide (Pchlide) Content in Arabidopsis Seedlings Using a High-Performance Liquid Chromatography (HPLC) System
利用高效液相色谱法(HPLC)对拟南芥幼苗中原叶绿素酸酯(Pchlide)的含量进行定量分析
The protochlorophyllide (Pchlide) level is a crucial indicator of plant fitness. Precise quantification of Pchlide content is necessary not only in studies of flu-related mutants that over-accumulate Pchlide in the dark but also for research on plants suffering from environmental stresses. Due to its low content and interference of chlorophylls, quantitative determination of Pchlide content is a challenge. Here, we describe an optimized protocol for Pchlide extraction from Arabidopsis thaliana seedlings and subsequent analysis using high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Divinyl-Protochlorophyllide (DV-Pchlide, the major form of Pchlide in plants) quantification is achieved by interpolating fluorescence peak areas against an experimentally derived standard curve. This protocol provides a reliable workflow for Pchlide quantification, facilitating the deciphering of the underlying mechanism of plant environmental resilience.
High-Performance Liquid Chromatography Quantification of Glyphosate, Aminomethylphosphonic Acid, and Ascorbate in Culture Medium and Microalgal Cells
高效液相色谱法检测培养基及微藻细胞中草甘膦、氨基甲基膦酸和抗坏血酸的含量
Glyphosate (GLY) is a widely used herbicide that can induce oxidative stress in microalgae and other non-target organisms. The quantification of GLY in surface water is a difficult task, especially in trace-level concentrations, due to its high polarity and susceptibility to biotic and abiotic degradation. Several analytical methods have been developed for GLY quantification. Most of them use high-performance liquid chromatography (HPLC) coupled with detection by mass spectrometry (MS) and include a derivatization step to decrease the polarity of the herbicide to improve detection. This protocol describes an adaptation of an existing protocol for the quantification of GLY and its metabolite aminomethylphosphonic acid (AMPA) in a water-based microalgae culture medium using ultra-high-pressure liquid chromatography (UHPLC) coupled with fluorescence detection (FLR). The principal advantage of this protocol compared with other analytical methods that employ HPLC–MS is its low cost and accessibility since it does not require an MS detector nor radioactively labeled analytical standards. Ascorbic acid (AH-) is one of the most important hydrosoluble non-enzymatic antioxidants in eukaryotic cells and plays a key role in many metabolic pathways of critical importance in plants and algae. In this protocol, we also describe an adaptation of a previously published protocol to quantify AH- in blood samples to be used in microalgal cells exposed to GLY and GLY-based herbicides. The sample preparation procedure for this last protocol is fast, easy, and does not require expensive equipment. It uses an HPLC system coupled with an electrochemical detector (EC) for AH- quantification but may be adapted to be used with a UV-Vis detector.
Measurement of Total Phosphorus and Polyphosphate in Chlamydomonas reinhardtii
莱茵衣藻中总磷和多磷酸盐的测定
Phosphorus is an essential nutrient for plants. Green algae usually store excess P as polyphosphate (polyP) in the vacuoles. PolyP, a linear chain of three to hundreds of phosphate residues linked by phosphoanhydride bonds, is important for cell growth. Based on the previous method of polyP purification with silica gel columns (Werner et al., 2005; Canadell et al., 2016) in yeast cells, we developed a protocol to purify and determine the total P and polyP in Chlamydomonas reinhardtii by a quick, simplified, and quantitative method. We use hydrochloric acid or nitric acid to digest polyP or total P in dried cells and analyze P content using the malachite green colorimetric method. This method may be applied to other microalgae.
Isolation of Intact Vacuoles from Arabidopsis Root Protoplasts and Elemental Analysis
拟南芥根原生质体完整液泡的分离及元素分析
The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins.
Graphical abstract
A Quick Method to Quantify Iron in Arabidopsis Seedlings
一种快速定量拟南芥幼苗中铁的方法
Iron (Fe) is an indispensable micronutrient for plant growth and development. Since both deficiency, as well as a surplus of Fe, can be detrimental to plant health, plants need to constantly tune uptake rates to maintain an optimum level of Fe. Quantification of Fe serves as an important parameter for analyzing the fitness of plants from different accessions, or mutants and transgenic lines with altered expression of specific genes. To quantify metals in plant samples, methods based on inductively coupled plasma-optical emission spectrometry (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS) have been widely employed. Although these methods are highly accurate, these methodologies rely on sophisticated equipment which is not always available. Moreover, ICP-OES and ICP-MS allow for surveying several metals in the same sample, which may not be necessary if only the Fe status is to be determined. Here, we outline a simple and cost-efficient protocol to quantify Fe concentrations in roots and shoots of Arabidopsis seedlings, by using a spectroscopy-based assay to quantify Fe2+-BPDS3 complexes against a set of standards. This protocol provides a fast and reproducible method to determine Fe levels in plant samples with high precision and low costs, which does not depend on expensive equipment and expertise to operate such equipment.
MAMP-triggered Medium Alkalinization of Plant Cell Cultures
MAMP诱导植物细胞培养基质碱化
An Adjustable Protocol to Analyze Chemical Profiles of Non-sterile Rhizosphere Soil
一种可调整的用于分析研究非无菌根际土壤化学特性的方法
An Inexpensive and Comprehensive Method to Examine and Quantify Field Insect Community Influenced by Host Plant Olfactory Cues
一种分析量化寄主植物嗅觉引诱性物质对田间昆虫群落影响的低成本、综合方法
Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803
分析集胞藻属PCC6803全细胞、类囊体和光合蛋白复合体中的金属
In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts
拟南芥粗提物中硝酸还原酶体外活性测定
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