Monitoring Xylem Transport in Arabidopsis thaliana Seedlings Using Fluorescent Dyes.

Fluorescent dyes are often used to observe transport mechanisms in plant vascular tissues. However, it has been technically challenging to apply fluorescent dyes on roots to monitor xylem transport in vivo. Here, we present a fast, noninvasive, and high-throughput protocol to monitor xylem transport in seedlings. Using the fluorescent dyes 5(6)-carboxyfluorescein diacetate (CFDA) and Rhodamine WT, we were able to observe xylem transport on a cellular level in Arabidopsis thaliana roots. We describe how to apply these dyes on primary roots of young seedlings, how to monitor root-to-shoot xylem transport, and how to measure xylem transport velocity in roots. Moreover, we show that our protocol can also be applied to lateral roots and grafted seedlings to assess xylem (re)connection. Altogether, these techniques are useful for investigating xylem functionality in diverse experimental setups.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

Quantification of Tracheary Elements Types in Mature Hypocotyl of Arabidopsis thaliana.

Secondary growth is a highly relevant process for dicot and gymnosperm species development. The process relies on vascular tissue proliferation and culminates with the thickening of stems, roots, and hypocotyls. The formation of tracheary elements is a critical step during this process. Among such tracheary elements, four different cell types are distinguished depending on their secondary cell wall pattern, which is exclusive for each tracheary cell type. Here we describe a method to isolate, dye, and recognize each of these tracheary cell types. The method is optimized to be performed in the Arabidopsis thaliana hypocotyl. This is because, in this species, the hypocotyl is the organ undergoing the largest proportion of secondary growth. Results allow for determining the relative amounts of each of the tracheary cell types.

Inducible Pluripotent Suspension Cell Cultures (iPSCs) to Study Plant Cell Differentiation.

Inducing the differentiation of specific cell type(s) synchronously and on-demand is a great experimental system to understand the sequential progression of the cellular processes, their timing and their resulting properties for distinct isolated plant cells independently of their tissue context. The inducible differentiation in cell suspension cultures, moreover, enables to obtain large quantities of distinct cell types at specific development stage, which is not possible when using whole plants. The differentiation of tracheary elements (TEs) - the cell type responsible for the hydro-mineral sap conduction and skeletal support of plants in xylem tissues - has been the most studied using inducible cell suspension cultures. We herein describe how to establish and use inducible pluripotent suspension cell cultures (iPSCs) in Arabidopsis thaliana to trigger on-demand different cell types, such as TEs or mesophyll cells. We, moreover, describe the methods to establish, monitor, and modify the sequence, duration, and properties of differentiated cells using iPSCs.

Clearing of Vascular Tissue in Arabidopsis thaliana for Reporter Analysis of Gene Expression.

To study the gene regulatory mechanisms modulating development is essential to visualize gene expression patterns at cellular resolution. However, this kind of analysis has been limited as a consequence of the plant tissues' opacity. In the last years, ClearSee has been increasingly used to obtain high-quality imaging of plant tissue anatomy combined with the visualization of gene expression patterns. ClearSee is established as a major tissue clearing technique due to its simplicity and versatility.In this chapter, we outline an easy-to-follow ClearSee protocol to analyze gene expression of reporters using either ß-glucuronidase (GUS) or fluorescent protein (FP) tags, compatible with different dyes to stain cell walls. We detail materials, equipment, solutions, and procedures to easily implement ClearSee for the study of vascular development in Arabidopsis thaliana, but the protocol can be easily adapted to a variety of plant tissues in a wide range of plant species.

The apple lipoxygenase MdLOX3 positively regulates zinc tolerance.

Various abiotic stresses, especially heavy metals near factories around the world, limit plant growth and productivity worldwide. Zinc is a light gray transition metal, and excessive zinc will inactivate enzymes in the soil, weaken the biological function of microorganisms, and enter the food chain through enrichment, thus affecting human health. Lipoxygenase (LOX) can catalyze the production of fatty acid derivatives from phenolic triglycerides in plants and is an important pathway of fatty acid oxidation in plants, which usually begins under unfavorable conditions, especially under biotic and abiotic stresses. Lipoxygenase can be divided into 9-LOX and 13-LOX. MdLOX3 is a homolog of AtLOX3 and has been identified in apples (housefly apples). MdLOX3 has a typical conserved lipoxygenase domain, and promoter analysis shows that it contains multiple stress response elements. In addition, different abiotic stresses and hormonal treatments induced the MdLOX3 response. In order to explore the inherent anti-heavy metal mechanism of MdLOX3, this study verified the properties of MdLOX3 based on genetic analysis and overexpression experiments, including plant taproots length, plant fresh weight, chlorophyll, anthocyanins, MDA, relative electrical conductivity, hydrogen peroxide and superoxide anion, NBT\DAB staining, etc. In the experiment, overexpression of MdLOX3 in apple callus and Arabidopsis effectively enhanced the tolerance to zinc stress by improving the ability to clear ROS. Meanwhile, tomato materials with overexpression of ectopia grew better under excessive zinc ion stress. These results indicated that MdLOX3 had a good tolerance to heavy metal zinc. Homologous mutants are more sensitive to zinc, which proves that MdLOX3 plays an important positive role in zinc stressed apples, which broadens the range of action of LOX3 in different plants.

Begomovirus Inoculation in Arabidopsis and Cassava.

The use of infectious clones to inoculate plant viruses allows for controlled studies that lead to a better understanding of plant-virus interactions. The main methods used for laboratory inoculation of geminiviruses are agroinoculation and biolistics. We describe how to successfully inoculate geminiviruses, focusing on Arabidopsis as a model plant and cassava as a crop.

Replication Assay of Begomovirus in Arabidopsis Protoplasts.

Protoplasts are isolated plant cells from which the cell walls have been removed by treatment with fungal cellulase and macerozyme enzymes, which degrade the primary components of the cell wall. The protoplasts are totipotent, sensitive, and versatile; thereby, they have been extensively used to study signal transduction pathways, cell-autonomous responses, and replication of plant viruses. This system has several advantages over the use of whole plants for viral replication, including a high percentage of infected cells and uncoupling virus movement from replication assays. Here, we describe a simple and efficient protocol for begomovirus transfection and replication in Arabidopsis protoplasts. The method consists of four steps, (i) protoplast isolation, (ii) PEG-calcium transfection of begomovirus infectious clones, (iii) elimination of input plasmid DNA by DpnI digestion, and (iv) quantification of the viral newly synthesized DNA by qPCR. The protoplasts can be transformed efficiently with begomovirus infectious clones, and virus replication can be monitored by the accumulation of nascent viral DNA in the infected protoplasts.

Co-immunoprecipitation Assays to Detect Protein-Protein Interactions.

Proteins usually do not function as monomers but rather perform their functions by interacting with themselves or other proteins. Co-immunoprecipitation is an essential assay for detecting protein interactions in vivo. In this chapter, we describe how to use co-immunoprecipitation to detect protein interactions in Arabidopsis protoplasts, seedlings, and Nicotiana benthamiana leaves. When using co-immunoprecipitation assays to detect protein interactions, it is necessary to pay attention to the design of the experimental and control groups.

In Situ Protein Microarray for Identifying the Geminivirus-Arabidopsis Interactome.

The protein-protein interactions (PPI) by protein array technology complement other PPI assay technologies such as AP-MS and Y2H. The in situ protein array technology (NAPPA) enables low-cost, rapid, and comprehensive protein detection. It allows standardized and simultaneous assay of a wide range of proteins with a broad range of expression in cells. This technology facilitates the detection of protein-protein interactions within species and between heterologous species such as host-microbe. Here, we described the technique that identified a syntaxin-6 protein-mediated begomovirus infection using an array containing 4600 Arabidopsis genes. The protein microarray assay also identified several other viral protein-host protein interactions.

Virus-Induced Heritable Gene Editing in Plants.

Gene editing using clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease is an excellent tool for assessing gene function in plants. However, delivery of CRISPR/Cas-editing components into plant cells is still a major bottleneck and requires tissue culture-based approaches and regeneration of plants. To overcome this limitation, several plant viral vectors have recently been engineered to deliver single-guide RNA (sgRNA) targets into SpCas9-expressing plants. Here, we describe an optimized, step-by-step protocol based on the tobacco rattle virus (TRV)-based vector system to deliver sgRNAs fused to mobile tRNA sequences for efficient heritable editing in Nicotiana benthamiana and Arabidopsis thaliana model systems. The protocol described here could be adopted to study the function of any gene of interest.

Mitigating cadmium accumulation in dicotyledonous vegetables by iron fertilizer through inhibiting Fe transporter IRT1-mediated Cd uptake.

The solubility of cadmium (Cd) in soil and its transfer to plants are influenced by soil pH. While increasing soil pH reduces Cd solubility and accumulation in rice plants grown in acidic soils, its effect on Cd accumulation in vegetables remains inconclusive. Here, we investigated the impact of soil pH on Cd accumulation in dicotyledonous vegetables and elucidated the underlying molecular mechanisms. Soils collected from various locations were supplemented with varying quantities of lime to achieve soil pH values of around 5.0, 6.0, 7.0, and 8.0. Raising soil pH from around 5.0 to 8.0 markedly decreased extractable Cd. However, increasing soil pH tended to promote shoot Cd accumulation in dicotyledonous vegetable species including lettuce, pakchoi, and Chinese cabbage, and the model dicotyledonous plant Arabidopsis thaliana. Conversely, soil pH increase resulted in a monotonic decrease in rice Cd accumulation. In our hydroponic experiments, we discovered that iron (Fe) deficiency substantially increased Cd uptake and accumulation in dicotyledonous plants but not in rice. Increasing soil pH reduced soil Fe availability and induced the Fe transporter gene IRT1 expression in dicotyledonous vegetables roots, which led to an increase in IRT1-mediated Cd uptake and subsequently increased Cd accumulation as soil pH increases. A comprehensive model incorporating extractable Cd and root IRT1 expression better explained Cd accumulation in vegetable shoots. The application of 50 mg/kg of Fe fertilizer in neutral or alkaline soils resulted in a significant reduction in Cd accumulation by 34-58% in dicotyledonous vegetables. These findings reveal that increasing soil pH has two opposite effects, decreasing soil Cd availability while promoting Cd uptake through IRT1 upregulation, reconciling the inconsistency in its effect on Cd accumulation in dicotyledonous plants. Our findings provide important insights for understanding the factors affecting Cd uptake in plants and offer a practical solution to mitigate Cd contamination in vegetables.

Quantification of Plant Virus Seed Transmission Rate in Arabidopsis thaliana.

More than 25% of all known plant viruses are transmitted through seeds, which makes this mode of dispersal of great importance for plant virus epidemics. Virus detection in seed stocks remains the most frequent approach for seed health testing, but current methods are not always standardized and/or do not allow analyzing large numbers of seeds. Here, we describe a high-throughput method to quantify plant virus seed transmission rate based on classical grow-out tests, which can be applied to widely different viruses and host species.

The mechanism of KpMIPS gene significantly improves resistance of Koelreuteria paniculata to heavy metal cadmium in soil.

Cadmium (Cd) pollution in soil is an important factor endangering plant growth and harming human health through food chains. Koelreuteria paniculata is an important woody plant for ecological restoration of Cd-contaminated soils. In this study, KpMIPS gene of K. paniculata was cloned, and the expressed protein (60 kDa) had 1-phosphate synthase activity. The results showed that KpMIPS significantly promoted root development of K. paniculata and Arabidopsis thaliana, reduced damage to the roots of Arabidopsis thaliana caused by Cd, and decreased transfer of Cd to the aboveground parts of K. paniculata and Arabidopsis thaliana . In the K. paniculata plants overexpressing KpMIPS integrity of the root cells was maintained and the content of pectin and phytic acid was significantly increased. Overexpression of KpMIPS increased the Cd accumulation in the roots and decreased the Cd content in the stems and leaves. Clearly, KpMIPS could regulate the contents of pectin and phytic acid in K. paniculata, thereby passivating Cd2+ and enriching it in the root cell wall, reducing the transfer of free Cd2+ to other parts of K. paniculata, and providing a positive regulatory effect on the Cd resistance of K. paniculata. The results of the study provide a technical introduction for the selection and genetic improvement of target genes regulating heavy metal resistance of plants in phytoremediation technology.

Dark complexes of the Calvin-Benson cycle in a physiological perspective.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two enzymes of the Calvin Benson cycle that stand out for some peculiar properties they have in common: (i) they both use the products of light reactions for catalysis (NADPH for GAPDH, ATP for PRK), (ii) they are both light-regulated through thioredoxins and (iii) they are both involved in the formation of regulatory supramolecular complexes in the dark or low photosynthetic conditions, with or without the regulatory protein CP12. In the complexes, enzymes are transiently inactivated but ready to recover full activity after complex dissociation. Fully active GAPDH and PRK are in large excess for the functioning of the Calvin-Benson cycle, but they can limit the cycle upon complex formation. Complex dissociation contributes to photosynthetic induction. CP12 also controls PRK concentration in model photosynthetic organisms like Arabidopsis thaliana and Chlamydomonas reinhardtii. The review combines in vivo and in vitro data into an integrated physiological view of the role of GAPDH and PRK dark complexes in the regulation of photosynthesis.

Genome-wide identification and expression analysis of the GASA gene family in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

BACKGROUND: The Gibberellic Acid-Stimulated Arabidopsis (GASA) gene family is widely involved in the regulation of plant growth, development, and stress response. However, information on the GASA gene family has not been reported in Chinese cabbage (Brassica rapa L. ssp. pekinensis). RESULTS: Here, we conducted genome-wide identification and analysis of the GASA genes in Chinese cabbage. In total, 15 GASA genes were identified in the Chinese cabbage genome, and the physicochemical property, subcellular location, and tertiary structure of the corresponding GASA proteins were elucidated. Phylogenetic analysis, conserved motif, and gene structure showed that the GASA proteins were divided into three well-conserved subfamilies. Synteny analysis proposed that the expansion of the GASA genes was influenced mainly by whole-genome duplication (WGD) and transposed duplication (TRD) and that duplication gene pairs were under negative selection. Cis-acting elements of the GASA promoters were involved in plant development, hormonal and stress responses. Expression profile analysis showed that the GASA genes were widely expressed in different tissues of Chinese cabbage, but their expression patterns appeared to diverse. The qRT-PCR analysis of nine GASA genes confirmed that they responded to salt stress, heat stress, and hormonal triggers. CONCLUSIONS: Overall, this study provides a theoretical basis for further exploring the important role of the GASA gene family in the functional genome of Chinese cabbage.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Golgi ELMO1 binds QUA1, QUA2, GAUT9, and ELMO4 and is required for pectin accumulation in Arabidopsis.

Pectin and its modification influence the plasticity and strength of the plant cell wall controlling cell adhesion, size, shape, and pathogen resistance. The Golgi membrane anchored QUA1, QUA2, and GAUT9 Golgi enzymes synthesize and esterify pectin, which is then secreted and selectively de-esterified to potentiate structure influencing crosslinks in the cell wall. Mutations in members of the family of non-enzymatic ELMO Golgi membrane proteins lead to a reduction of pectin levels, cell adhesion, and hypocotyl tensile strength. Results from immunoprecipitation of Golgi protein complexes reveal that ELMO1-GFP is associated with pectin biosynthesis and modifying enzymes QUA1, QUA2, and GAUT9. In a yeast two and three hybrid assay, ELMO1 can bind directly to QUA1, GAUT9 or ELMO4, but QUA1, QUA2 or GAUT9 do not bind to each other. A yeast 3 hybrid assay provides evidence that ELMO1 can mediate the binding of QUA1 and QUA2. Taken together, these results indicate that the 20 kDa ELMO1 serves to facilitate some aspect of pectin synthesis and modification that leads to sufficient accumulation to allow cell adhesion, and we speculate that ELMOs help to scaffold key enzymes in this process.

Genome-wide identification and molecular expression profile analysis of FHY3/FAR1 gene family in walnut (Juglans sigillata L.) development.

BACKGROUND: Juglans sigillata L. (walnut) has a high economic value for nuts and wood and has been widely grown and eaten around the world. Light plays an important role in regulating the development of the walnut embryo and promoting nucleolus enlargement, which is one of the factors affecting the yield and quality of walnut. However, little is known about the effect of light on the growth and quality of walnuts. Studies have shown that far red prolonged hypocotyl 3 (FHY3) and far red damaged response (FAR1) play important roles in plant growth, light response, and resistance. Therefore, FHY3/FAR1 genes were identified in walnuts on a genome-wide basis during their growth and development to reveal the potential regulation mechanisms involved in walnut kernel growth and development. RESULTS: In the present study, a total of 61 FHY3/FAR1 gene family members in walnuts have been identified, ranging in length from 117 aa to 895 aa. These gene family members have FHY3 or FAR1 conserved domains, which are unevenly distributed on the 15 chromosomes (Chr) of the walnut (except for the Chr16). All 61 FHY3/FAR1 genes were divided into five subclasses (I, II, III, IV, and V) by phylogenetic tree analysis. The results indicated that FHY3/FAR1 genes in the same subclasses with similar structures might be involved in regulating the growth and development of walnut. The gene expression profiles were analyzed in different walnut kernel varieties (Q, T, and F). The result showed that some FHY3/FAR1 genes might be involved in the regulation of walnut kernel ripening and seed coat color formation. Seven genes (OF07056-RA, OF09665-RA, OF24282-RA, OF26012-RA, OF28029-RA, OF28030-RA, and OF08124-RA) were predicted to be associated with flavonoid biosynthetic gene regulation cis-acting elements in promoter sequences. RT-PCR was used to verify the expression levels of candidate genes during the development and color change of walnut kernels. In addition, light responsiveness and MeJA responsiveness are important promoter regulatory elements in the FHY3/FAR1 gene family, which are potentially involved in the light response, growth, and development of walnut plants. CONCLUSION: The results of this study provide a valuable reference for supplementing the genomic sequencing results of walnut, and pave the way for further research on the FHY3/FAR1 gene function of walnut.

EASTR: Identifying and eliminating systematic alignment errors in multi-exon genes.

Accurate alignment of transcribed RNA to reference genomes is a critical step in the analysis of gene expression, which in turn has broad applications in biomedical research and in the basic sciences. We reveal that widely used splice-aware aligners, such as STAR and HISAT2, can introduce erroneous spliced alignments between repeated sequences, leading to the inclusion of falsely spliced transcripts in RNA-seq experiments. In some cases, the 'phantom' introns resulting from these errors make their way into widely-used genome annotation databases. To address this issue, we present EASTR (Emending Alignments of Spliced Transcript Reads), a software tool that detects and removes falsely spliced alignments or transcripts from alignment and annotation files. EASTR improves the accuracy of spliced alignments across diverse species, including human, maize, and Arabidopsis thaliana, by detecting sequence similarity between intron-flanking regions. We demonstrate that applying EASTR before transcript assembly substantially reduces false positive introns, exons, and transcripts, improving the overall accuracy of assembled transcripts. Additionally, we show that EASTR's application to reference annotation databases can detect and correct likely cases of mis-annotated transcripts.