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The cation exchanger family members CAX1 and CAX3 form homomeric and heteromeric complexes that are likely to modulate calcium transport during signaling events., Arabidopsis thaliana cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca2+/H+) antiporters that contribute to cellular Ca2+ homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remained elusive. Here, we demonstrate that expression of CAX1 and CAX3 occurs in guard cells. Additionally, CAX1 and CAX3 are co-expressed in mesophyll tissue in response to wounding or flg22 treatment, due to the induction of CAX3 expression. Having shown that the transporters can be co-expressed in the same cells, we demonstrate that CAX1 and CAX3 can form homomeric and heteromeric complexes in plants. Consistent with the formation of a functional CAX1-CAX3 complex, CAX1 and CAX3 integrated into the yeast genome suppressed a Ca2+-hypersensitive phenotype of mutants defective in vacuolar Ca2+ transport, and demonstrated enzyme kinetics different from those of either CAX protein expressed by itself. We demonstrate that the interactions between CAX proteins contribute to the functioning of stomata, because stomata were more closed in cax1-1, cax3-1, and cax1-1/cax3-1 loss-of-function mutants due to an inability to buffer Ca2+ effectively. We hypothesize that the formation of CAX1-CAX3 complexes may occur in the mesophyll to affect intracellular Ca2+ signaling during defense responses.

Reverse transcription of RNA is fallible, introducing biases and confounding the quantification of transcript abundance. We demonstrate that circular RNAs (circRNAs) are more subjective to overestimation of transcript abundance than cognate linear RNAs due to their covalently closed, circular form, producing multiple concatameric products from a single priming of reverse transcriptase. We developed SplintQuant, where custom DNA oligonucleotides are ligated by PBCV-1 DNA ligase only when bound to their target RNA. These circRNA-specific DNA oligonucleotides are terminally tagged with universal primers, allowing SplintQuant to accurately quantify even lowly abundant circRNAs through highly specific quantitative PCR (qPCR) in the absence of reverse transcription. SplintQuant is sensitive, specific, highly reproducible, and applicable to the quantification of canonical and noncanonical RNA transcripts including alternative splice variants, gene fusions, and offers a gold-standard approach for accurately quantifying circRNAs. Refereed/Peer-reviewed

Quantification of gene expression is a crucial research tool in the life sciences, which makes it important to identify any factors that could compromise its accuracy. One of these factors are non-polyadenylated (poly(A) -) transcripts, including circular RNAs (circRNAs) that can skew quantification of gene expression as they resemble messenger RNAs (mRNAs). Here, we highlight the impact circRNAs and other poly(A) - transcripts have on gene expression profiling and the biological conclusions drawn from such experiments. We also propose easily adoptable strategies to increase the accuracy of gene expression quantification. Refereed/Peer-reviewed

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200cand another epithelial-enriched miRNA, miR-375, exert wide spread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing. Refereed/Peer-reviewed

Magnesium accumulates at high concentrations in dicotyledonous leaves but it is not known in which leaf cell types it accumulates, by what mechanism this occurs and the role it plays when stored in the vacuoles of these cell types. Cell-specific vacuolar elemental profiles from Arabidopsis thaliana (Arabidopsis) leaves were analysed by X-ray microanalysis under standard and serpentine hydroponic growth conditions and correlated with the cell-specific complement of magnesium transporters identified through microarray analysis and quantitative polymerase chain reaction (qPCR). Mesophyll cells accumulate the highest vacuolar concentration of magnesium in Arabidopsis leaves and are enriched for members of the MGT/MRS2 family of magnesium transporters. Specifically, AtMGT2/AtMRS2-1 and AtMGT3/AtMRS2-5 were shown to be targeted to the tonoplast and corresponding T-DNA insertion lines had perturbed mesophyll-specific vacuolar magnesium accumulation under serpentine conditions. Furthermore, transcript abundance of these genes was correlated with the accumulation of magnesium under serpentine conditions, in a low calcium-accumulating mutant and across 23 Arabidopsis ecotypes varying in their leaf magnesium concentrations. We implicate magnesium as a key osmoticum required to maintain growth in low calcium concentrations in Arabidopsis. Furthermore, two tonoplast-targeted members of the MGT/MRS2 family are shown to contribute to this mechanism under serpentine conditions. Refereed/Peer-reviewed

Circular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT. Furthermore, by modulating QKI levels, we show the effect on circRNA abundance is dependent on intronic QKI binding motifs. Critically, the addition of QKI motifs is sufficient to induce de novo circRNA formation from transcripts that are normally linearly spliced. These findings demonstrate circRNAs are both purposefully synthesized and regulated by cell-type specific mechanisms, suggesting they play specific biological roles in EMT. Refereed/Peer-reviewed

Background: An important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment. In situ PCR is a relatively simple and rapid method that involves the amplification of specific mRNA directly within plant tissue whilst incorporating labelled nucleotides that are subsequently detected by immunohistochemistry. Another notable advantage of this technique is that it can be used on plants that are not easily genetically transformed Conclusions: The in situ PCR method presented here is highly sensitive and specific. It reliably identifies the cellular expression pattern of even highly homologous and low abundance transcripts within target tissues, and can be completed within two days of harvesting tissue. As such, it has considerable advantages over other methods, especially in terms of time and cost. We recommend its adoption as the standard laboratory technique of choice for demonstrating the cellular expression pattern of a gene of interest Results: An optimised workflow for in-tube and on-slide in situ PCR is presented that has been evaluated using multiple plant species and tissue types. The protocol includes optimised methods for: (i) fixing, embedding,and sectioning of plant tissue; (ii) DNase treatment; (iii) in situ RT-PCR with the incorporation of DIG-labellednucleotides; (iv) signal detection using colourimetric alkaline phosphatase substrates; and (v) mounting and microscopy. We also provide advice on troubleshooting and the limitations of using fluorescence as an alternative detection method. Using our protocol, reliable results can be obtained within two days from harvesting plant material.This method requires limited specialized equipment and can be adopted by any laboratory with a vibratome (vibratingblade microtome), a standard thermocycler, and a microscope. We show that the technique can be used to localise gene expression with cell-specific resolution Refereed/Peer-reviewed

Background Hydroponic growth systems are a convenient platform for studying whole plant physiology. However, we found through trialling systems as they are described in the literature that our experiments were frequently confounded by factors that affected plant growth, including algal contamination and hypoxia. We also found the way in which the plants were grown made them poorly amenable to a number of common physiological assays. Results The drivers for the development of this hydroponic system were: 1) the exclusion of light from the growth solution; 2) to simplify the handling of individual plants, and 3) the growth of the plant to allow easy implementation of multiple assays. These aims were all met by the use of pierced lids of black microcentrifuge tubes. Seed was germinated on a lid filled with an agar-containing germination media immersed in the same solution. Following germination, the liquid growth media was exchanged with the experimental solution, and after 14-21 days seedlings were transferred to larger tanks with aerated solution where they remained until experimentation. We provide details of the protocol including composition of the basal growth solution, and separate solutions with altered calcium, magnesium, potassium or sodium supply whilst maintaining the activity of the majority of other ions. We demonstrate the adaptability of this system for: gas exchange measurement on single leaves and whole plants; qRT-PCR to probe the transcriptional response of roots or shoots to altered nutrient composition in the growth solution (we demonstrate this using high and low calcium supply); producing highly competent mesophyll protoplasts; and, accelerating the screening of Arabidopsis transformants. This system is also ideal for manipulating plants for micropipette techniques such as electrophysiology or SiCSA. Conclusions We present an optimised plant hydroponic culture system that can be quickly and cheaply constructed, and produces plants with similar growth kinetics to soil-grown plants, but with the advantage of being a versatile platform for a myriad of physiological and molecular biological measurements on all plant tissues at all developmental stages. We present ‘tips and tricks’ for the easy adoption of this hydroponic culture system.

The plant ionome varies both inter- and intraspecifically despite the highly conserved roles for particular elements across the plant kingdom. Element storage requires transport across the plasma membrane and commonly deposition within the central vacuole. Therefore, tonoplast transport characteristics can be highly influential in controlling the plant ionome. As a result, individual cell types of the same plant, each with unique transcriptomes and vacuolar proteomes, can display very different elemental profiles. Here we address the use of natural variation in Arabidopsis thaliana for identifying genes involved in elemental accumulation. We present a conceptual framework, exploiting publicly available leaf ionomic and transcriptomic data across 31 Arabidopsis accessions, that promises to accelerate conventional forward genetics approaches for candidate gene discovery. Utilizing this framework, we identify numerous genes with documented roles in accumulation of calcium, magnesium and zinc and implicate additional candidate genes. Where appropriate, we discuss their role in cell-specific elemental accumulation. Currently, this framework could represent an alternate approach for identifying genes suitable for element biofortification of plants. Integration of additional cell-specific and whole-plant 'omics' datasets across Arabidopsis accessions under diverse environmental conditions should enable this concept to be developed into a scalable and robust tool for linking genotype and phenotype. Refereed/Peer-reviewed

The ability of wheat to maintain a low sodium concentration ([Na +]) in leaves correlates with improved growth under saline conditions. This trait, termed Na + exclusion, contributes to the greater salt tolerance of bread wheat relative to durum wheat. To improve the salt tolerance of durum wheat, we explored natural diversity in shoot Na + exclusion within ancestral wheat germplasm. Previously, we showed that crossing of Nax2, a gene locus in the wheat relative Triticum monococcum into a commercial durum wheat (Triticum turgidum ssp. durum var. Tamaroi) reduced its leaf [Na +] (ref. 5). Here we show that a gene in the Nax2 locus, TmHKT1;5-A, encodes a Na +-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, which is therefore ideally localized to withdraw Na + from the xylem and reduce transport of Na + to leaves. Field trials on saline soils demonstrate that the presence of TmHKT1;5-A significantly reduces leaf [Na +] and increases durum wheat grain yield by 25% compared to near-isogenic lines without the Nax2 locus. Refereed/Peer-reviewed