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.

Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis.

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.

A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements.

Plant cells can change their identity based on positional information, a mechanism that confers developmental plasticity to plants. This ability, common to distinct multicellular organisms, is particularly relevant for plant phloem cells. Protophloem sieve elements (PSEs), one type of phloem conductive cells, act as the main organizers of the phloem pole, which comprises four distinct cell files organized in a conserved pattern. Here, we report how Arabidopsis roots generate a reservoir of meristematic phloem cells competent to swap their cell identities. Although PSE misspecification induces cell identity hybridism, the activity of RECEPTOR LIKE PROTEIN KINASE 2 (RPK2) by perceiving CLE45 peptide contributes to restrict PSE identity to the PSE position. By maintaining a spatiotemporal window when PSE and PSE-adjacent cells' identities are interchangeable, CLE45 signaling endows phloem cells with the competence to re-pattern a functional phloem pole when protophloem fails to form.

Methods of Phloem Visualization: A Clear Future in Sight?

There have been exciting new results in phloem research in recent years, at least in part made possible by the rapid advancement of microscopic techniques. Several methods for visualizing phloem cells are available. The suitability of each method depends on the organ and species being studied, and on the scientific question being addressed. This review will briefly explain the specific challenges associated with phloem cell visualization. It will then focus on common methods currently being used for studying phloem anatomy, development, and function. Emphasis will be placed on the most recent improvements in imaging techniques which had, or most certainly will have, an impact on phloem research.

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch.

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.

OCTOPUS-LIKE 2, a novel player in Arabidopsis root and vascular development, reveals a key role for OCTOPUS family genes in root metaphloem sieve tube differentiation.

Protophloem and metaphloem sieve tubes are essential for transporting carbohydrates and signalling molecules towards sink tissues. OCTOPUS (OPS) was previously identified as an important regulator of protophloem differentiation in Arabidopsis roots. Here, we investigated the role of OCTOPUS-LIKE 2 (OPL2), a gene homologous to OPS. OPL2 expression patterns were analysed, and functional equivalence of OPS and OPL2 was tested. Mutant and double mutant phenotypes were investigated. OPS and OPL2 displayed overlapping expression patterns and a high degree of functional overlap. A mutation in OPL2 revealed redundant functions of OPS and OPL2 in developmental processes in which OPS was known to play a role, notably cotyledon vascular patterning and protophloem development. Moreover, we also uncovered redundant roles for OPS and OPL2 in leaf vascular patterning and, most interestingly, metaphloem sieve tube differentiation. Our results reveal a novel OPS-like protein that, together with OPS, is an important regulator of vascular patterning, root growth and phloem development. OPS and OPL2 are the first genes identified that play a role in metaphloem sieve tube differentiation.

Plant Physiology: Unveiling the Dark Side of Phloem Translocation.

Sugars and other macromolecules arrive in heterotrophic plant tissues through the phloem, a long-distance transport system. Owing to a recent study, we now have a better understanding of how these molecules exit the phloem at their final destinations.

Xylem Characterization Using Improved Pseudo-Schiff Propidium Iodide Staining of Whole Mount Samples and Confocal Laser-Scanning Microscopy.

An improved pseudo-Schiff propidium iodide staining technique well suited for, but not limited to, the visualization of xylem cell walls in whole mount samples is presented. The pseudo-Schiff reaction results in covalent binding of the fluorescent dye propidium iodide to cell walls. This stable linkage permits the use of clearing agents after staining, which is itself improved following pretreatment of the plant tissue. A subsequent acid alcohol washing step eliminates unbound propidium iodide to reduce background fluorescence. The method can be used for characterizing xylem cell structure in different organs and species without the need for tissue sectioning.

Amino Acid Export in Developing Arabidopsis Seeds Depends on UmamiT Facilitators.

Essential amino acids cannot be synthesized by humans and animals. They often are limiting in plant-derived foods and determine the nutritional value of a given diet. Seeds and fruits often represent the harvestable portion of plants. In order to improve the amino acid composition of these tissues, it is indispensable to understand how these substrates are transported within the plant. Amino acids result from nitrogen assimilation, which often occurs in leaves, the source tissue. They are transported via the vasculature, the xylem, and the phloem into the seeds, the so-called sink tissue, where they are stored or consumed. In seeds, several tissues are symplasmically isolated, i.e., not connected by plasmodesmata, channels in the cell walls that enable a cytoplasmic continuum in plants. Consequently, amino acids must be exported from cells into the apoplast and re-imported many times to support seed development. Several amino acid importers are known, but exporters remained elusive. Here, we characterize four members of the plant-specific UmamiT transporter family from Arabidopsis, related to the amino acid facilitator SIAR1 and the vacuolar auxin transporter WAT1. We show that the proteins transport amino acids along their (electro)chemical potential across the plasma membrane. In seeds, they are found in tissues from which amino acids are exported. Loss-of-function mutants accumulate high levels of free amino acids in fruits and produce smaller seeds. Our results strongly suggest a crucial role for the UmamiTs in amino acid export and possibly a means to improve yield quality.

ZLL/AGO10 maintains shoot meristem stem cells during Arabidopsis embryogenesis by down-regulating ARF2-mediated auxin response.

BACKGROUND: The shoot meristem gives rise to new organs throughout a plant's life by the activity of pluripotent stem cells in the meristem center. Organ initiation at the periphery of the shoot meristem is triggered by the accumulation of the phytohormone auxin at the initiation site. Loss-of-function mutants of the ZWILLE/ARGONAUTE10/PINHEAD (ZLL/AGO10/PNH) gene terminate shoot meristem stem cells late in embryogenesis and can form a leaf or a leaf-like structure instead, indicating that AGO10 activity is required to maintain shoot meristem stem cells undifferentiated. RESULTS: Here, we addressed whether stem cell maintenance by AGO10 involves regulation of auxin. We found that in zll-1 mutants, auxin accumulation and expression of the response reporter DR5:GFP are elevated, and transcription of the Auxin Response Factor 2 (ARF2) gene is upregulated. Downregulation of ARF2 significantly restores stem cells in zll-1 mutants, whereas increased expression of ARF2 enhances differentiation of stem cells in zll-1 mutants. We further found that upregulation of the AGO10 effector gene REVOLUTA restores ARF2 expression and stem cell maintenance in zll-1 embryos. CONCLUSIONS: Our results indicate that maintenance of shoot meristem stem cells by AGO10 involves negative regulation of auxin signaling and, via REV-mediated downregulation of ARF2 expression, auxin response.

Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.

In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis.

Phloem imaging.

The phloem is the long-distance solute-conducting tissue of plants. The observation of phloem cells is particularly challenging for several reasons and many recent advances in microscopy are, therefore, especially beneficial for the study of phloem anatomy and physiology. This review will give an overview of the imaging techniques that have been used for studying different aspects of phloem biology. It will also highlight some new imaging techniques that have emerged in recent years that will certainly advance our knowledge about phloem function.

Accession-specific modifiers act with ZWILLE/ARGONAUTE10 to maintain shoot meristem stem cells during embryogenesis in Arabidopsis.

BACKGROUND: Stem cells located in the centre of the shoot apical meristem are required for the repetitive formation of new organs such as leaves, branches and flowers. In Arabidopsis thaliana, the ZWILLE/PINHEAD/AGO10 (ZLL) gene encodes a member of the ARGONAUTE (AGO) protein family and is required to maintain shoot meristem stem cells during embryogenesis. In the Landsberg erecta (Ler) acession, ZLL is essential for stem cell maintenance, whereas in the Columbia (Col) accession its requirement appears masked by genetic modifiers. The genetic basis for this variation has remained elusive. RESULTS: To understand the impact of natural variation on shoot stem cell maintenance, we analysed 28 wild-type Arabidopsis accessions from around the world and show that ZLL function is essential for stem cell maintenance in accessions mainly originating from Germany, but is dispensable for accessions from other regions. Quantitative Trait Loci (QTL) mapping using Ler/Col recombinant inbred lines indicated that at least five genomic regions, referred to as FLETSCHE (FHE) 1-5, modify ZLL function in stem cell maintenance. Characterisation of Col zll near isogenic lines confirmed that the major QTL, FHE2, is preferentially maintained as a Ler allele in seedlings lacking stem cells, suggesting that this region harbours an important modifier of ZLL function. Comparison of torpedo-stage embryo expression profiles to QTL map data revealed candidate FHE genes, including the Arabidopsis Cyclophilin-40 homologue SQUINT (SQN), and functional studies revealed a previously uncharacterised role for SQN in stem cell regulation. CONCLUSIONS: Multiple genetic modifiers from different Arabidopsis accessions influence the role of ZLL in embryonic stem cell maintenance. Of the five FHE loci modifying stem cell maintenance in Ler-0 and Col-0, FHE2 was the most prominent and was tightly linked to the SQN gene, which encodes a cofactor that supports AGO1 activity. SQN shows variable embryonic expression levels between accessions and altered ZLL-dependency in transgenic assays, confirming a key role in stem cell maintenance. Reduced SQN expression levels in Col-0 correlate with transposon insertions adjoining the transcriptional start site, which may contribute to stem cell maintenance in other ZLL-independent accessions.

OCTOPUS, a polarly localised membrane-associated protein, regulates phloem differentiation entry in Arabidopsis thaliana.

Vascular development is embedded into the developmental context of plant organ differentiation and can be divided into the consecutive phases of vascular patterning and differentiation of specific vascular cell types (phloem and xylem). To date, only very few genetic determinants of phloem development are known. Here, we identify OCTOPUS (OPS) as a potentiator of phloem differentiation. OPS is a polarly localised membrane-associated protein that is initially expressed in provascular cells, and upon vascular cell type specification becomes restricted to the phloem cell lineage. OPS mutants display a reduction of cotyledon vascular pattern complexity and discontinuous phloem differentiation, whereas OPS overexpressers show accelerated progress of cotyledon vascular patterning and phloem differentiation. We propose that OPS participates in vascular differentiation by interpreting longitudinal signals that lead to the transformation of vascular initials into differentiating protophloem cells.

Looking deeper: whole-mount confocal imaging of plant tissue for the accurate study of inner tissue layers.

Building on previous work, we further developed a staining procedure that fluorescently labels plant cell walls and, when combined with confocal microscopy, allows visualization of plant cellular organisation in whole mounts to depths exceeding 200 microm. This technique can be combined with beta-glucuronidase histochemical activity assays, allowing the simultaneous study of gene expression. Images taken from stained samples can be used for three-dimensional tissue reconstruction. We used the technique to study Arabidopsis protophloem development. The phloem is one of the innermost tissues of a plant and therefore especially difficult to visualise. Furthermore, in general the technique will improve significantly the speed and accuracy with which any kind of plant tissue organisation can be studied, and it is not limited to the study of Arabidopsis tissues.

High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of Phloem development and structure in Arabidopsis.

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.

A simple way to identify non-viable cells within living plant tissue using confocal microscopy.

BACKGROUND: Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. RESULTS: Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. CONCLUSION: The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.

Arabidopsis thaliana outer ovule integument morphogenesis: ectopic expression of KNAT1 reveals a compensation mechanism.

BACKGROUND: The Arabidopsis outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated. RESULTS: To better characterise outer ovule integument morphogenesis, we have isolated some marker lines that show GFP expression in this organ. We have used those lines to identify distinct cell types in the outer integument and to demonstrate similarities between leaves and the outer integument. Using confocal microscopy, we showed that cell sizes and shapes differ between the two cell layers of the outer integument. Expression of KNAT1 in the integuments leads to extra cell divisions specifically in the outer layer of the outer integument. This is being compensated for by a decrease of cell volume in this layer, thus showing that mechanisms exist to control proper ovule integument morphogenesis. CONCLUSION: The Arabidopsis outer ovule integument can be used as a good model system to study the basic principles of plant organ morphogenesis. This work provides new insights into its development and opens new possibilities for the identification of factors involved in the regulation of cell division and elongation during plant organ growth.

Immunogenic tagging of chloroplasts allows their isolation from defined cell types.

Chloroplast structure varies depending on cell type. Currently it is difficult to obtain insight into how differences in chloroplast structure relate to function, as it is often not possible to isolate chloroplasts from specific cells. To address this, we have developed an approach that involves labelling chloroplasts from individual cell types by placing the foreign yellow fluorescent protein (YFP) on their outer surface, and then isolating those labelled chloroplasts immunogenically. Cell specificity is achieved through the use of enhancer trap lines. When whole leaves are homogenized, a mixture of labelled and unlabelled chloroplasts is released, but magnetic beads coated with antibodies to the green fluorescent protein (GFP) allow the labelled chloroplasts to be isolated. Chloroplasts from spongy mesophyll, vascular and epidermal cells of Arabidopsis thaliana were obtained in this way, and semi-quantitative RT-PCR showed that the abundance of various chloroplast transcripts differed between these three cell types. The approach is based on genetic logic, and so could be applied to the isolation of various organelles or subcellular compartments from transformable organisms other than A. thaliana.