The Chief Function Of Root Hairs Is To

November 12, 2022 0 Comments

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Transcriptional networks are tightly controlled in plant development and stress response. Alternative polyadenylation (APA) has been shown to regulate gene expression under abiotic stress by increasing heterogeneity at mRNA 3′ ends. Heavy metals such as cadmium pollute water and land due to mining and industrial applications. Understanding how plants cope with heavy metal stress is still an interesting question. Arabidopsis root hairs were selected as a single-cell model to investigate the functional role of APA in cadmium stress response. Inhibition of primary root growth and damaged root hair morphotypes were observed. Poly(A)-tag (PAT) libraries from single cell types, ie. root hair cells, non-hair epidermal cells and whole root tips under cadmium stress were prepared and sorted. Interestingly, root hair cell type-specific gene expression was detected during short-term cadmium exposure, but was not related to long-term treatment. Differential poly(A) sites were identified that significantly contributed to altered gene expression and were enriched in the pentose and glucuronate interconversion pathways as well as the phenylpropanoid biosynthesis pathway. Many genes with poly(A) site changes were found, mainly for functions in cell wall modification, root epidermal differentiation and root hair tip growth. Our results suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment.

The Chief Function Of Root Hairs Is To

The Chief Function Of Root Hairs Is To

Heavy metals are natural components of the soil. However, the increase in industrial manufacturing over the past two centuries has led to heavy metal pollution in the environment. Plants grown under high heavy metal conditions show stunted growth and reduced yield (Chibuike and Obiora, 2014). One of these metals, cadmium (Cd), is a non-essential element in plant growth and is highly toxic to plants (Tran and Popova, 2013). Cd is released into the soil through the use of fertilizers as well as various industrial processes, such as additives in plastic stabilizers, NiCd batteries, pigments and electroplating (Sanitá di Toppi and Gabbrielli, 1999). In addition to the effects of Cd on plant growth and development, it has been reported that Cd can accumulate in plant products such as rice grains, leading to production losses and contamination of the food chain (Chunhabundit, 2016). How plants can cope with Cd-contaminated soil environments is not yet clear.

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The majority of plant species (excluding plants) have evolved strategies to limit the translocation of heavy metals and maintain low levels of metal contaminants in their aerial tissues (Baker, 1981). For example, roots in Arabidopsis are the major organs for metal accumulation. The cell wall, which contains polysaccharides, is the first barrier against heavy metal accumulation by preventing heavy metals from entering the cytosol (Parrotta et al., 2015). Large amounts of heavy metals are immobilized by phytochelatins (PC) and sequestered in vacuoles (Hall, 2002). The negative effect of Cd has been observed through a reduction in the rate of photosynthesis and root elongation, and has been proposed as a general stress-induced morphogenic response (SIMR) similar to that detected with other heavy metals or UV-B radiation (Parrotta et al… , 2015). A SIMR signature, e.g. root hairs clustered near the tip, shown in canola seeds under Cd stress (Sun and Guo, 2013). However, no molecular mechanism has been proposed to explain this phenomenon.

One aspect of post-transcriptional regulation, alternative splicing, has been shown to alter the expression of specific genes through intron retention in fungi under Cd stress (Georg et al., 2009). In addition, several alternative splicing events, including exon skipping, intron retention, and alternative use of 5′ and 3′ splice sites, were identified in rice under Cd stress (He et al., 2015). Another aspect of post-transcriptional regulation, alternative polyadenylation (APA), has been documented to regulate gene expression in response to abiotic stress (Zhang et al., 2008). Together with alternative splicing, APA has a functional role in drought stress in sorghum (Abdel-Ghany et al., 2016). In addition, APA was found to be a common phenomenon in eukaryotes for other biological processes, including embryonic development and cell differentiation (Tian and Manley, 2017). Previous studies have found the use of multiple polyadenylation sites in tissue-specific and developmental APAs in zebrafish (Ulitsky et al., 2012), worms (Mangone et al., 2010), and rice (Fu et al., 2016). Recently, a study on plant hypoxia stress investigated the regulatory function of APA through the production of non-canonical mRNA isoforms (De Lorenzo et al., 2017).

Root hair has a function in absorption and perception, but it is also an interesting cell model in molecular and physiological studies due to the nature of the single cell as a simple and tubular structure of the root epidermis that forms the hair (Qiao and Libault, 2013). An RNA-Seq study of Arabidopsis root hair cells revealed many alternative splicing events enriched in biological processes associated with Cd stress ( Lan et al., 2013 ). However, the underlying mechanisms of Cd-induced root hair morphotypes are still unknown.

A cell type-specific alternative splicing map was revealed in Arabidopsis roots by combined fluorescence-activated cell sorting (FACS) and next-generation sequencing ( Li et al., 2016 ). Applying the same methodology using FACS and microarray identified core regulators of abiotic stress responses (Dinneny et al., 2008). Construction of two cell-type markers (root hairs and non-hairy epidermal cells) in Arabidopsis with isolation of labeled nuclei in a cell type-specific system (INTACT) enabled transcriptome profiling of Arabidopsis root hairs (Deal and Henikoff, 2011). Here, we used FACS and Illumina sequencing to explore the regulatory function of APA in plant root hair development under Cd stress.

Trait Variations And Expression Profiling Of Ospht1 Gene Family At The Early Growth Stages Under Phosphorus Limited Conditions

Arabidopsis thaliana (L.) Heynh. ecotype Col-0 (CS60000) was used in this study. Two GFP reporter lines, ADF8p:NTF/ACT2p:BirA and GL2p:NTF/ACT2p:BirA] provided by Drs. Roger Deal, now at Emory University, (Deal and Henikoff, 2010)], used for cell sorting. In the ADF8p:NTF/ACT2p:BirA cell line, GFP was fused to the ADF8 cell type-specific promoter, which showed green fluorescence in root hair cells. In the second cell line GL2p:NTF/ACT2p:BirA, the GL2 cell type-specific promoter drives GFP expression only in non-hairy root epidermal cells. Seedlings were sterilized with 50% bleach and 0.05% Tween 20 for 8 min. After being rinsed 5 times with sterile distilled water, seeds were plated at 4 °C for 2 days and grown on top of 100 μm nylon mesh (Genesee Scientific) in ½ Murashige and Skoog (MS) (Sigma-Aldrich) medium with 1% sucrose, 1% agar at a temperature of 22°C vertically with a dark cycle of 16 hours/8 hours. Five-day-old seedlings were transferred to ½ MS medium (control) or MS medium containing 100 μM CdCl.

(stress) for 24, 48 and 72 hours. Seeds were plated at a density of ∼20/cm in two rows on top of 100 μm nylon mesh for sorting experiments in three biological replicates.

Wild-type Col-0 plants were grown on ½ MS agar plates for 5 days and transferred to ½ MS medium (control) or MS medium containing 100 μM CdCl.

The Chief Function Of Root Hairs Is To

(stress) for 24, 48 and 72 h for phenotypic analysis. Seedlings were pressed and root length measured using Figure J (National Institutes of Health; http://rsb.info.nih.gov/ij). A total of 35–40 plants were used in each experiment and the experiment was repeated four times. Means and standard errors were analyzed by Repeated-Measurement ANOVA using SAS (SAS Institute Inc., NC, USA).

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Specific GFP expression in each marker line was confirmed by confocal microscopy. Briefly, buds were stained with propidium iodide 200 μg/ml for 2 min, rinsed with water, and imaged with a Carl Zeiss LSM710 confocal microscope. Protoplastization was performed as previously described (Bargmann and Birnbaum, 2010) with slight modifications. Briefly, 1.25% (w/v) cellulase (Calbiochem, #Cat219466) and 0.3% (w/v) macerozyme R-10 (Research Product International, Cat.# M22010) were used to protoplastize root cells. Enzymes were dissolved in wash solution [0.6 M Mannitol, 1 mg/ml BSA, 10 mM MgCl

O, 10 mM KCl, 0.39 mg/ml MES hydrate, pH 5.5 adjusted with 1 M Tris]. Chopped root tips were incubated at room temperature for 1 h with 85 rpm agitation. The protoplast suspension was centrifuged at 1200 rpm for 6 min (RT). The protoplast pellet was then washed and filtered through 70 μm and 40 μm cell strainers (Fisher Scientific).

A Moflo XDP fluorescence-activated cell sorter (Beckman Coulter, Inc) with a 100-μm nozzle ( Lan et al., 2013 ) was used to collect GFP-positive cells at a rate of 2,000 to 5,000 events per second. others at a fluid pressure of 25 psi (Research Flow Cytometry Core at Cincinnati Children’s Hospital Medical Center). The red tip of Col-0 was used as a negative control to collect GFP-labeled cells, and the expected number of sorting events was approximately 5,000 to 20,000 cells.

The collected cells were sorted in RA1 lysis buffer (in the kit for RNA isolation) supplemented with the reducing agent tris-2-carboxyethylphosphine (TCEP) at room temperature. Total RNA was extracted immediately after cell sorting (NucleoSpin RNA XS kit, Macherey Nagel) by on-column treatment with RNase-free DNase I (supplied with the kit) according to the manufacturer’s recommendations. The quality and total quantity of extracted RNA was assessed by RNA pico-chip using an Agilent Bioanalyzer 2100 (Agilent Biotechnologies). Samples with an RNA integrity number (RIN) above 6.9 were retained

Soil Nutrient Cycling

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