Autophagic degradation of the chloroplastic 2-phosphoglycolate phosphatase TaPGLP1 in wheat.

KEY MESSAGE: TaPGLP1, a chloroplast stromal 2-phosphoglycolate phosphatase of wheat, is an ATG8-interacting protein and undergoes autophagic degradation in starvation-treated wheat mesophyll protoplasts. Selective autophagy in plants has been shown to target diverse cellular cargoes including whole chloroplasts (Chlorophagy) and several chloroplast components (Piecemeal chlorophagy). Most cargoes of selective autophagy are captured by the autophagic machinery through their direct or indirect interactions with the autophagy-essential factor ATG8. Here, we reported a new ATG8-interacting cargo of piecemeal chlorophagy, the wheat photorespiratory 2-phosphoglycolate phosphatase TaPGLP1. The TaPGLP1-mCherry fusions expressed in wheat protoplasts located in the chloroplast stroma. Strikingly, these fusions are translocated into newly formed chloroplast surface protrusions after a long time incubation of protoplasts in a nutrition-free solution. Visualization of co-expressed TaPGLP1-mCherry and the autophagy marker GFP-TaATG8a revealed physical associations of TaPGLP1-mCherry-accumulating chloroplast protrusions with autophagic structures, implying the delivery of TaPGLP1-mCherry fusions from chloroplasts to the autophagic machinery. TaPGLP1-mCherry fusions were also detected in the GFP-TaATG8a-labelled autophagic bodies undergoing degradation in the vacuoles, which suggested the autophagic degradation of TaPGLP1. This autophagic degradation of TaPGLP1 was further demonstrated by the enhanced stability of TaPGLP1-mCherry in protoplasts with impaired autophagy. Expression of TaPGLP1-mCherry in protoplasts stimulated an enhanced autophagy level probably adopted by cells to degrade the over-produced TaPGLP1-mCherry fusions. Results from gene silencing assays showed the requirement of ATG2s and ATG7s in the autophagic degradation of TaPGLP1. Additionally, TaPGLP1 was shown to interact with ATG8 family members. Collectively, our data suggest that autophagy mediates the degradation of the chloroplast stromal protein TaPGLP1 in starvation-treated mesophyll protoplasts.

Cellular and transcriptomic analyses reveal two-staged chloroplast biogenesis underpinning photosynthesis build-up in the wheat leaf.

BACKGROUND: The developmental gradient in monocot leaves has been exploited to uncover leaf developmental gene expression programs and chloroplast biogenesis processes. However, the relationship between the two is barely understood, which limits the value of transcriptome data to understand the process of chloroplast development. RESULTS: Taking advantage of the developmental gradient in the bread wheat leaf, we provide a simultaneous quantitative analysis for the development of mesophyll cells and of chloroplasts as a cellular compartment. This allows us to generate the first biologically-informed gene expression map of this leaf, with the entire developmental gradient from meristematic to fully differentiated cells captured. We show that the first phase of plastid development begins with organelle proliferation, which extends well beyond cell proliferation, and continues with the establishment and then the build-up of the plastid genetic machinery. The second phase is marked by the development of photosynthetic chloroplasts which occupy the available cellular space. Using a network reconstruction algorithm, we predict that known chloroplast gene expression regulators are differentially involved across those developmental stages. CONCLUSIONS: Our analysis generates both the first wheat leaf transcriptional map and one of the most comprehensive descriptions to date of the developmental history of chloroplasts in higher plants. It reveals functionally distinct plastid and chloroplast development stages, identifies processes occurring in each of them, and highlights our very limited knowledge of the earliest drivers of plastid biogenesis, while providing a basis for their future identification.

Characterization of histological changes at the tillering stage (Z21) in resistant and susceptible wheat plants infected by Tilletia controversa Kühn.

BACKGROUND: Dwarf bunt, which is caused by Tilletia controversa Kühn, is a soilborne and seedborne disease that occurs worldwide and can lead to 70% or even total losses of wheat crops. However, very little information is available about the histological changes that occur in dwarf bunt-resistant and dwarf bunt-susceptible wheat plants at the tillering stage (Z21). In this study, we used scanning electron microscopy and transmission electron microscopy to characterize the histological changes at this stage in resistant and susceptible wheat cultivars infected by T. controversa. RESULTS: Using scanning electron microscopy, the root, stem, and leaf structures of resistant and susceptible cultivars were examined after T. controversa infection. The root epidermal and vascular bundles were more severely damaged in the susceptible T. controversa-infected plants than in the resistant plants. The stem cell and longitudinal sections were much more extensively affected in susceptible plants than in resistant plants after pathogen infection. However, slightly deformed mesophyll cells were observed in the leaves of susceptible plants. With transmission electron microscopy, we found that the cortical bundle cells and the cell contents and nuclei in the roots were more severely affected in the susceptible plants than in the resistant plants; in the stems and leaves, the nuclei, chloroplasts, and mesophyll cells changed significantly in the susceptible plants after fungal infection. Moreover, we found that infected susceptible and resistant plants were affected much more severely at the tillering stage (Z21) than at the seedling growth stage (Z13). CONCLUSION: Histological changes in the wheat roots, stems and leaves were much more severe in T. controversa-infected susceptible plants than in infected resistant plants at the tillering stage (Z21).

Comparison of Light Condition-Dependent Differences in the Accumulation and Subcellular Localization of Glutathione in Arabidopsis and Wheat.

This study aimed to clarify whether the light condition-dependent changes in the redox state and subcellular distribution of glutathione were similar in the dicotyledonous model plant Arabidopsis (wild-type, ascorbate- and glutathione-deficient mutants) and the monocotyledonous crop species wheat (Chinese Spring variety). With increasing light intensity, the amount of its reduced (GSH) and oxidized (GSSG) form and the GSSG/GSH ratio increased in the leaf extracts of both species including all genotypes, while far-red light increased these parameters only in wheat except for GSH in the GSH-deficient Arabidopsis mutant. Based on the expression changes of the glutathione metabolism-related genes, light intensity influences the size and redox state of the glutathione pool at the transcriptional level in wheat but not in Arabidopsis. In line with the results in leaf extracts, a similar inducing effect of both light intensity and far-red light was found on the total glutathione content at the subcellular level in wheat. In contrast to the leaf extracts, the inducing influence of light intensity on glutathione level was only found in the cell compartments of the GSH-deficient Arabidopsis mutant, and far-red light increased it in both mutants. The observed general and genotype-specific, light-dependent changes in the accumulation and subcellular distribution of glutathione participate in adjusting the redox-dependent metabolism to the actual environmental conditions.

Plant Cell Wall Changes in Common Wheat Roots as a Result of Their Interaction with Beneficial Fungi of Trichoderma.

Plant cell walls play an important role in shaping the defense strategies of plants. This research demonstrates the influence of two differentiators: the lifestyle and properties of the Trichoderma species on cell wall changes in common wheat seedlings. The methodologies used in this investigation include microscopy observations and immunodetection. In this study was shown that the plant cell wall was altered due to its interaction with Trichoderma. The accumulation of lignins and reorganization of pectin were observed. The immunocytochemistry indicated that low methyl-esterified pectins appeared in intercellular spaces. Moreover, it was found that the arabinogalactan protein epitope JIM14 can play a role in the interaction of wheat roots with both the tested Trichoderma strains. Nevertheless, we postulate that modifications, such as the appearance of lignins, rearrangement of low methyl-esterified pectins, and arabinogalactan proteins due to the interaction with Trichoderma show that tested strains can be potentially used in wheat seedlings protection to pathogens.

Subcellular distribution governing accumulation and translocation of pesticides in wheat (Triticum aestivum L.).

Root uptake, translocation, and subcellular distribution of six pesticides (dinotefuran, thiamethoxam, imidacloprid, imazethapyr, propiconazole, and chlorpyrifos) with Kow ranging from -0.549 to 4.7 were investigated in wheat to study transportation and accumulation of pesticides. The root bioconcentration factor (RCF) of pesticides decreased with water solubility (R2 = 0.6121) and increased with hydrophobicity (when the pH-adjusted log Kow > 2, R2 = 0.925), respectively. The translocation of neutral pesticides from roots to shoots increased positively with water solubility (R2 > 0.6484) but decreased with hydrophobicity (R2 > 0.8039). The subcellular fraction concentration factor (SFCF) increased linearly with hydrophobicity of the tested pesticides (R2 > 0.958). The log RCF was positively correlated with log SFCF in root cell walls (R2 = 0.9894) and organelles (R2 = 0.9786). Transportation of the pesticides from roots to stems and stems to leaves was adversely affected by the log SFCF of cell walls and organelles of roots (R2 > 0.7997) and stems (R2 > 0.6666), respectively. Hydrophobicity-dependent SFCF is a factor governing accumulation of pesticides in roots after uptake and their subsequent upward translocation.

Comparative transcriptome analysis indicates that a core transcriptional network mediates isonuclear alloplasmic male sterility in wheat (Triticum aestivum L.).

BACKGROUND: Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of heterosis and various types of CMS often have different abortion mechanisms. Therefore, it is important to understand the molecular mechanisms related to anther abortion in wheat, which remain unclear at present. RESULTS: In this study, five isonuclear alloplasmic male sterile lines (IAMSLs) and their maintainer were investigated. Cytological analysis indicated that the abortion type was identical in IAMSLs, typical and stainable abortion, and the key abortive period was in the binucleate stage. Most of the 1,281 core shared differentially expressed genes identified by transcriptome sequencing compared with the maintainer in the vital abortive stage were involved in the metabolism of sugars, oxidative phosphorylation, phenylpropane biosynthesis, and phosphatidylinositol signaling, and they were downregulated in the IAMSLs. Key candidate genes encoding chalcone--flavonone isomerase, pectinesterase, and UDP-glucose pyrophosphorylase were screened and identified. Moreover, further verification elucidated that due to the impact of downregulated genes in these pathways, the male sterile anthers were deficient in sugar and energy, with excessive accumulations of ROS, blocked sporopollenin synthesis, and abnormal tapetum degradation. CONCLUSIONS: Through comparative transcriptome analysis, an intriguing core transcriptome-mediated male-sterility network was proposed and constructed for wheat and inferred that the downregulation of genes in important pathways may ultimately stunt the formation of the pollen outer wall in IAMSLs. These findings provide insights for predicting the functions of the candidate genes, and the comprehensive analysis of our results was helpful for studying the abortive interaction mechanism in CMS wheat.

Dissection and cytological mapping of chromosome arm 4VS by the development of wheat-Haynaldia villosa structural aberration library.

KEY MESSAGE: A cytological map of Haynaldia villosa chromosome arm 4VS was constructed to facilitate the identification and utilization of beneficial genes on 4VS. Induction of wheat-alien chromosomal structure aberrations not only provides new germplasm for wheat improvement, but also allows assignment of favorable genes to define physical regions. Especially, the translocation or introgression lines carrying alien chromosomal fragments with different sizes are useful for breeding and alien gene mapping. Chromosome arm 4VS of Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum (L.) P. Candargy) confers resistances to eyespot and wheat yellow mosaic virus (WYMV). In this research, we used both irradiation and the pairing homoeologous gene (Ph) mutant to induce chromosomal aberrations or translocations. By using the two approaches, a structural aberration library of chromosome arm 4VS was constructed. In this library, there are 57 homozygous structural aberrations, in which, 39 were induced by the Triticum aestivum cv. Chinese Spring (CS) ph1b mutant (CS ph1b) and 18 were induced by irradiation. The aberrations included four types, i.e., terminal translocation, interstitial translocation, deletion and complex structural aberration. The 4VS cytological map was constructed by amplification in the developed homozygous aberrations using 199 4VS-specific markers, which could be allocated into 39 bins on 4VS. These bins were further assigned to their corresponding physical regions of chromosome arm 4DS based on BLASTn search of the marker sequences against the reference sequence of Aegilops tauschii Cosson. The developed genetic stocks and cytological map provide genetic stocks for wheat breeding as well as alien gene tagging.

A stripe rust effector Pst18363 targets and stabilises TaNUDX23 that promotes stripe rust disease.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a tremendous threat to the production of wheat worldwide. The molecular mechanisms of Pst effectors that regulate wheat immunity are poorly understood. In this study, we identified an effector Pst18363 from Pst that suppresses plant cell death in Nicotiana benthamiana and in wheat. Knocking down Pst18363 expression by virus-mediated host-induced gene silencing significantly decreased the number of rust pustules, indicating that Pst18363 functions as an important pathogenicity factor in Pst. Pst18363 was proven to interact with wheat Nudix hydrolase 23 TaNUDX23. In wheat, silencing of TaNUDX23 by virus-induced gene silencing increased reactive oxygen species (ROS) accumulation induced by the avirulent Pst race CYR23, whereas overexpression of TaNUDX23 suppressed ROS accumulation induced by flg22 in Arabidopsis. In addition, TaNUDX23 suppressed Pst candidate effector Pst322-trigged cell death by decreasing ROS accumulation in N. benthamiana. Knocking down of TaNUDX23 expression attenuated Pst infection, indicating that TaNUDX23 is a negative regulator of defence. In N. benthamiana, Pst18363 stabilises TaNUDX23. Overall, our data suggest that Pst18363 stabilises TaNUDX23, which suppresses ROS accumulation to facilitate Pst infection.

Characterization of a wheat-tetraploid Thinopyrum elongatum 1E(1D) substitution line K17-841-1 by cytological and phenotypic analysis and developed molecular markers.

BACKGROUND: Tetraploid Thinopyrum elongatum (2n = 4x = 28) is a promising source of useful genes, including those related to adaptability and resistance to diverse biotic (Fusarium head blight, rust, powdery mildew, and yellow dwarf virus) and abiotic (cold, drought, and salt) stresses. However, gene transfer rates are low for this species and relatively few species-specific molecular markers are available. RESULTS: The wheat-tetraploid Th. elongatum line K17-841-1 derived from a cross between a hexaploid Trititrigia and Sichuan wheat cultivars was characterized based on sequential genomic and fluorescence in situ hybridizations and simple sequence repeat markers. We revealed that K17-841-1 is a 1E (1D) chromosomal substitution line that is highly resistant to stripe rust pathogen strains prevalent in China. By comparing the sequences generated during genotyping-by-sequencing (GBS), we obtained 597 specific fragments on the 1E chromosome of tetraploid Th. elongatum. A total of 235 primers were designed and 165 new Th. elongatum-specific markers were developed, with an efficiency of up to 70%. Marker validation analyses indicated that 25 specific markers can discriminate between the tetraploid Th. elongatum chromosomes and the chromosomes of other wheat-related species. An evaluation of the utility of these markers in a F2 breeding population suggested these markers are linked to the stripe rust resistance gene on chromosome 1E. Furthermore, 28 markers are unique to diploid Th. elongatum, tetraploid Th. elongatum, or decaploid Thinopyrum ponticum, which carry the E genome. Finally, 48 and 74 markers revealed polymorphisms between Thinopyrum E-genome- containing species and Thinopyrum bessarabicum (Eb) and Pseudoroegneria libanotica (St), respectively. CONCLUSIONS: This new substitution line provide appropriate bridge-breeding-materials for alien gene introgression to improve wheat stripe rust resistance. The markers developed using GBS technology in this study may be useful for the high-throughput and accurate detection of tetraploid Th. elongatum DNA in diverse materials. They may also be relevant for investigating the genetic differences and phylogenetic relationships among E, Eb, St, and other closely-related genomes and for further characterizing these complex species.

YR36/WKS1-Mediated Phosphorylation of PsbO, an Extrinsic Member of Photosystem II, Inhibits Photosynthesis and Confers Stripe Rust Resistance in Wheat.

Wheat stripe rust, due to infection by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease that causes significant global grain yield losses. Yr36, which encodes Wheat Kinase START1 (WKS1), is an effective high-temperature adult-plant resistance gene and confers resistance to a broad spectrum of Pst races. We previously showed that WKS1 phosphorylates the thylakoid ascorbate peroxidase protein and reduces its ability to detoxify peroxides, which may contribute to the accumulation of reactive oxygen species (ROS). WKS1-mediated Pst resistance is accompanied by leaf chlorosis in Pst-infected regions, but the underlying mechanisms remain elusive. Here, we show that WKS1 interacts with and phosphorylates PsbO, an extrinsic member of photosystem II (PSII), to reduce photosynthesis, regulate leaf chlorosis, and confer Pst resistance. A point mutation in PsbO-A1 or reduction in its transcript levels by RNA interference resulted in chlorosis and reduced Pst sporulation. Biochemical analyses revealed that WKS1 phosphorylates PsbO at two conserved amino acids involved in physical interactions with PSII and reduces the binding affinity of PsbO with PSII. Presumably, phosphorylated PsbO proteins dissociate from the PSII complex and then undergo rapid degradation by cysteine and aspartic proteases. Taken together, these results demonstrate that perturbations of wheat PsbO by point mutation or phosphorylation by WKS1 reduce the rate of photosynthesis and delay the growth of Pst pathogen before the induction of ROS.

Stripe Rust Effector PstGSRE1 Disrupts Nuclear Localization of ROS-Promoting Transcription Factor TaLOL2 to Defeat ROS-Induced Defense in Wheat.

Puccinia striiformis f. sp. tritici (Pst), a biotrophic plant pathogen, secretes numerous effectors to modulate host defense systems. Understanding the molecular mechanisms by which Pst effectors regulate wheat immunity is of great importance for the development of novel strategies for durable control of stripe rust. In this study, we identified a glycine-serine-rich effector gene, PstGSRE1, which is highly induced during early infection. Transgenic expression of PstGSRE1 RNAi constructs in wheat significantly reduced virulence of Pst and increased H2O2 accumulation in wheat. PstGSRE1 was shown to target the reactive oxygen species (ROS)-associated transcription factor TaLOL2, a positive regulator of wheat immunity. PstGSRE1 disrupted nuclear localization of TaLOL2 and suppressed ROS-mediated cell death induced by TaLOL2, thus compromising host immunity. This work reveals a previously unrecognized strategy whereby rust fungi exploit the PstGSRE1 effector to defeat ROS-associated plant defense by modulating the subcellular compartment of a host immune regulator and facilitate pathogen infection.

Effect of ZnO, TiO2, Al2O3 and ZrO2 nanoparticles on wheat callus cells.

Effect of metal oxide nanoparticles on calli of two wheat varieties: Parabola (stress tolerant) and Raweta (sensitive) was studied. ZnO induced 10% larger membrane damage in Raweta calli. TiO2, Al2O3, and ZrO2 caused nearly 30% greater lactate dehydrogenase leakage for Raweta compared to Parabola. UV-irradiation of samples containing ZnO particles intensified this effect. Membrane lipid peroxidation in ZnO treated Raweta calli was twice as high as in Parabola and further increased after UV-irradiation. TiO2, Al2O3, and ZrO2 nanoparticles caused a 4-fold increase in malondialdehyde concentration in Raweta calli in comparison to Parabola calli. The nanoparticles studied damaged the cellular defense system by inactivating the antioxidative enzymes.

Centromere Satellite Repeats Have Undergone Rapid Changes in Polyploid Wheat Subgenomes.

Centromeres mediate the pairing of homologous chromosomes during meiosis; this pairing is particularly challenging for polyploid plants such as hexaploid bread wheat (Triticum aestivum), as their meiotic machinery must differentiate homologs from similar homoeologs. However, the sequence compositions (especially functional centromeric satellites) and evolutionary history of wheat centromeres are largely unknown. Here, we mapped T. aestivum centromeres by chromatin immunoprecipitation sequencing using antibodies to the centromeric-specific histone H3 variant (CENH3); this identified two types of functional centromeric satellites that are abundant in two of the three subgenomes. These centromeric satellites had unit sizes greater than 500 bp and contained specific sites with highly phased binding to CENH3 nucleosomes. Phylogenetic analysis revealed that the satellites have diverged in the three T. aestivum subgenomes, and the more homogeneous satellite arrays are associated with CENH3. Satellite signals decreased and the degree of satellites variation increased from diploid to hexaploid wheat. Moreover, several T. aestivum centromeres lack satellite repeats. Rearrangements, including local expansion and satellite variations, inversions, and changes in gene expression, occurred during the evolution from diploid to tetraploid and hexaploid wheat. These results reveal the asymmetry in centromere organization among the wheat subgenomes, which may play a role in proper homolog pairing during meiosis.

Wheat egg in vitro fusion with wheat and green bristlegrass sperm.

SummaryIsolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30-35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300-500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

Multi-excitation hyperspectral autofluorescence imaging for the exploration of biological samples.

Many plant tissues can be observed thanks to autofluorescence of their cell wall components. Hyperspectral autofluorescence imaging using confocal microscopy is a fast and efficient way of mapping fluorescent compounds in samples with a high spatial resolution. However a huge spectral overlap is observed between molecular species. As a consequence, a new data analysis approach is needed in order to fully exploit the potential of this spectroscopic technique and extract unbiased chemical information about complex biological samples. The objective of this work is to evaluate multi-excitation hyperspectral autofluorescence imaging to identify biological components in wheat grains during their development through their spectral profiles and corresponding contribution maps using Multivariate Curve Resolution - Alternating Least-Squares (MCR-ALS), a signal unmixing algorithm under proper constraints. For this purpose two different scenarios are used: 1) analyzing the total spectral domain of data sets using MCR-ALS under non negativity constraint in both spectral and spatial modes; 2) analyzing a reduced spectral domain of data sets using MCR-ALS under non negativity in both modes and trilinearity constraint in spectral mode. Considering the original instrumental setup and our data analysis approach, we will demonstrate that extracted contribution maps and spectral profiles of constituents can provide complementary information used to identify molecules in complex biological samples.

The highly divergent Jekyll genes, required for sexual reproduction, are lineage specific for the related grass tribes Triticeae and Bromeae.

Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.

LSM-W2: laser scanning microscopy worker for wheat leaf surface morphology.

BACKGROUND: Microscopic images are widely used in plant biology as an essential source of information on morphometric characteristics of the cells and the topological characteristics of cellular tissue pattern due to modern computer vision algorithms. High-resolution 3D confocal images allow extracting quantitative characteristics describing the cell structure of leaf epidermis. For some issues in the study of cereal leaves development, it is required to apply the staining techniques with fluorescent dyes and to scan rather large fragments consisting of several frames. We aimed to develop a tool for processing multi-frame multi-channel 3D images obtained from confocal laser scanning microscopy and taking into account the peculiarities of the cereal leaves staining. RESULTS: We elaborated an ImageJ-plugin LSM-W2 that allows extracting data on Leaf Surface Morphology from Laser Scanning Microscopy images. The plugin is a crucial link in a workflow for obtaining data on structural properties of leaf epidermis and morphological properties of epidermal cells. It allows converting large lsm-files (laser scanning microscopy) into segmented 2D/3D images or tables with data on cells and/or nuclei sizes. In the article, we also represent some case studies showing the plugin application for solving biological tasks. Namely the plugin is applied in the following cases: defining parameters of jigsaw-puzzle pattern for maize leaf epidermal cells, analysis of the pavement cells morphological parameters for the mature wheat leaf grown under control and water deficit conditions, initiation of cell longitudinal rows, and detection of guard mother cells emergence at the initial stages of the stomatal morphogenesis in the growth zone of a wheat leaf. CONCLUSION: The proposed plugin is efficient for high-throughput analysis of cellular architecture for cereal leaf epidermis. The workflow implies using inexpensive and rapid sample preparation and does not require the applying of transgenesis and reporter genetic structures expanding the range of species and varieties to study. Obtained characteristics of the cell structure and patterns further could act as a basis for the development and verification for spatial models of plant tissues formation mechanisms accounting for structural features of cereal leaves. AVAILABILITY: The implementation of this workflow is available as an ImageJ plugin distributed as a part of the Fiji project (FijiisjustImageJ: https://fiji.sc/ ). The plugin is freely available at https://imagej.net/LSM_Worker , https://github.com/JmanJ/LSM_Worker and http://pixie.bionet.nsc.ru/LSM_WORKER/ .

A reductionist approach to dissecting grain weight and yield in wheat.

Grain yield is a highly polygenic trait that is influenced by the environment and integrates events throughout the life cycle of a plant. In wheat, the major grain yield components often present compensatory effects among them, which alongside the polyploid nature of wheat, makes their genetic and physiological study challenging. We propose a reductionist and systematic approach as an initial step to understand the gene networks regulating each individual yield component. Here, we focus on grain weight and discuss the importance of examining individual sub-components, not only to help in their genetic dissection, but also to inform our mechanistic understanding of how they interrelate. This knowledge should allow the development of novel combinations, across homoeologs and between complementary modes of action, thereby advancing towards a more integrated strategy for yield improvement. We argue that this will break barriers in terms of phenotypic variation, enhance our understanding of the physiology of yield, and potentially deliver improved on-farm yield.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.