Aleš Soukup

Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin

• Polarized deposition of cell wall pectins is a key process in Arabidopsis thaliana myxospermous seed coat development. The exocyst, an octameric secretory vesicle tethering complex, has recently been shown to be involved in the regulation of cell polarity in plants. Here, we used the Arabidopsis seed coat to study the participation of the exocyst complex in polarized pectin delivery. • We characterized the amount of pectinaceous mucilage and seed coat structure in sec8 and exo70A1 exocyst mutants. Using a yeast two-hybrid screen, we identified a new interactor of the exocyst subunit Exo70A1, termed Roh1, a member of the DUF793 protein family. • T-DNA insertions in SEC8, EXO70A1 caused considerable deviations from normal seed coat development, in particular reduced pectin deposition and defects in the formation of the central columella of seed epidermal cells. A gain-of-function mutation of ROH1 also caused reduced pectin deposition. Interestingly, we observed a systematic difference in seed coat development between primary and secondary inflorescences in wild-type plants: siliques from secondary branches produced seeds with thicker seed coats. • The participation of exocyst subunits in mucilage deposition provides direct evidence for the role of the exocyst in polarized cell wall morphogenesis.

The lateral root initiation index: an integrative measure of primordium formation

BACKGROUND AND AIMS Lateral root initiation is an essential and continuous process in the formation of root systems; therefore, its quantitative analysis is indispensable. In this study a new measure of lateral root initiation is proposed and analysed, namely the lateral root initiation index (I(LRI)), which defines how many lateral roots and/or primordia are formed along a parent-root portion corresponding to 100 cortical cells in a file. METHODS For data collection, a commonly used root clearing procedure was employed, and a new simple root clearing procedure is also proposed. The I(LRI) was determined as 100dl, where d is the density of lateral root initiation events (number mm(-1)) and l is the average fully elongated cortical cell length (mm). KEY RESULTS Analyses of different Arabidopsis thaliana genotypes and of a crop plant, tomato (Solanum lycopersicum), showed that I(LRI) is a more precise parameter than others commonly used as it normalizes root growth for variations in cell length. Lateral root primordium density varied in the A. thaliana accessions Col, Ler, Ws, and C24; however, in all accessions except Ws, I(LRI) was similar under the same growth conditions. The nitrogen/carbon ratio in the growth medium did not change the lateral root primordium density but did affect I(LRI). The I(LRI) was also modified in a number of auxin-related mutants, revealing new root branching phenotypes in some of these mutants. The rate of lateral root initiation increased with Arabidopsis seedling age; however, I(LRI) was not changed in plants between 8 and 14 d post-germination. CONCLUSIONS The I(LRI) allows for a more precise comparison of lateral root initiation under different growth conditions, treatments, genotypes and plant species than other comparable methods.

The role of the testa during development and in establishment of dormancy of the legume seed

Timing of seed germination is one of the key steps in plant life cycles. It determines the beginning of plant growth in natural or agricultural ecosystems. In the wild, many seeds exhibit dormancy and will only germinate after exposure to certain environmental conditions. In contrast, crop seeds germinate as soon as they are imbibed usually at planting time. These domestication-triggered changes represent adaptations to cultivation and human harvesting. Germination is one of the common sets of traits recorded in different crops and termed the “domestication syndrome.” Moreover, legume seed imbibition has a crucial role in cooking properties. Different seed dormancy classes exist among plant species. Physical dormancy (often called hardseededness), as found in legumes, involves the development of a water-impermeable seed coat, caused by the presence of phenolics- and suberin-impregnated layers of palisade cells. The dormancy release mechanism primarily involves seed responses to temperature changes in the habitat, resulting in testa permeability to water. The underlying genetic controls in legumes have not been identified yet. However, positive correlation was shown between phenolics content (e.g., pigmentation), the requirement for oxidation and the activity of catechol oxidase in relation to pea seed dormancy, while epicatechin levels showed a significant positive correlation with soybean hardseededness. myeloblastosis family of transcription factors, WD40 proteins and enzymes of the anthocyanin biosynthesis pathway were involved in seed testa color in soybean, pea and Medicago, but were not tested directly in relation to seed dormancy. These phenolic compounds play important roles in defense against pathogens, as well as affecting the nutritional quality of products, and because of their health benefits, they are of industrial and medicinal interest. In this review, we discuss the role of the testa in mediating legume seed germination, with a focus on structural and chemical aspects.

Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy

Histochemical methods allow for identification and localization of various components within the tissue. Such information on the spatial heterogeneity is not available with biochemical methods. However, there is limitation of the specificity of such detection in context of complex tissue, which is important to consider, and interpretations of the results should regard suitable control treatments if possible. Hereby we present set of selected simple staining and histochemical methods with comments based on our laboratory experience.

Lateral root development in the maize (Zea mays) lateral rootless1 mutant.

BACKGROUND AND AIMS The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions. METHODS Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data. KEY RESULTS The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase. CONCLUSIONS The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no influences observed on the root apical meristem structure and maintenance; however, development of the epidermis and cortex are impaired.

Casparian bands and suberin lamellae in exodermis of lateral roots: an important trait of roots system response to abiotic stress factors

Background and Aims Root absorptive characteristics rely on the presence of apoplastic barriers. However, little is known about the establishment of these barriers within a complex root system, particularly in a major portion of them - the lateral roots. In Zea mays L., the exodermis differentiates under the influence of growth conditions. Therefore, the species presents a suitable model to elucidate the cross-talk among environmental conditions, branching pattern and the maturation of barriers within a complex root system involved in the definition of the plant-soil interface. The study describes the extent to which lateral roots differentiate apoplastic barriers in response to changeable environmental conditions. Methods The branching, permeability of the outer cell layers and differentiation of the endo- and exodermis were studied in primary roots and various laterals under different types of stress of agronomic importance (salinity, heavy metal toxicity, hypoxia, etc.). Histochemical methods, image analysis and apoplastic tracer assays were utilized. Key Results The results show that the impact of growth conditions on the differentiation of both the endodermis and exodermis is modulated according to the type/diameter of the root. Fine laterals clearly represent that portion of a complex root system with a less advanced state of barrier differentiation, but with substantial ability to modify exodermis differentiation in response to environmental conditions. In addition, some degree of autonomy in exodermal establishment of Casparian bands (CBs) vs. suberin lamellae (SLs) was observed, as the absence of lignified exodermal CBs did not always fit with the lack of SLs. Conclusions This study highlights the importance of lateral roots, and provides a first look into the developmental variations of apoplastic barriers within a complex root system. It emphasizes that branching and differentiation of barriers in fine laterals may substantially modulate the root system-rhizosphere interaction.

A Combined Comparative Transcriptomic, Metabolomic, and Anatomical Analyses of Two Key Domestication Traits: Pod Dehiscence and Seed Dormancy in Pea (Pisum sp.)

The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography–electrospray ionization/mass spectrometry and Laser desorption/ionization–mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in agreement in all four genotypes with Massive analysis of cDNA Ends (MACE) data. In case of pod dehiscence, the analysis of two candidate genes (SHATTERING and SHATTERPROOF) and three out of 20 MACE identified genes (MACE-P004, MACE-P013, MACE-P015) showed down-expression in dorsal and ventral pod suture of indehiscent genotypes. Moreover, MACE-P015, the homolog of peptidoglycan-binding domain or proline-rich extensin-like protein mapped correctly to predicted Dpo1 locus on PsLGIII. This integrated analysis of the seed coat in wild and cultivated pea provides new insight as well as raises new questions associated with domestication and seed dormancy and pod dehiscence.

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.

Tracing root permeability: comparison of tracer methods

Root epidermis and apoplastic barriers (endodermis and exodermis) are the critical root structures involved in setting up plant-soil interface by regulating free apoplastic movement of solutes within root tissues. Probing root apoplast permeability with “apoplastic tracers” presents one of scarce tools available for detection of “apoplastic leakage” sites and evaluation of their role in overall root uptake of water, nutrients, or pollutants. Although the tracers are used for many decades, there is still not an ideal apoplastic tracer and flawless procedure with straightforward interpretation. In this article, we present our experience with the most frequently used tracers representing various types of chemicals with different characteristics. We examine their behaviour, characteristics, and limitations. Here, we show that results gained with an apoplastic tracer assay technique are reliable but depend on many parameters–chemical properties of a selected tracer, plant species, cell wall properties, exposure time, or sample processing.

Towards Better Understanding of Pea Seed Dormancy Using Laser Desorption/Ionization Mass Spectrometry

Seed coats of six pea genotypes contrasting in dormancy were studied by laser desorption/ionization mass spectrometry (LDI-MS). Multivariate statistical analysis discriminated dormant and non-dormant seeds in mature dry state. Separation between dormant and non-dormant types was observed despite important markers of particular dormant genotypes differ from each other. Normalized signals of long-chain hydroxylated fatty acids (HLFA) in dormant JI64 genotype seed coats were significantly higher than in other genotypes. These compounds seem to be important markers likely influencing JI64 seed imbibition and germination. HLFA importance was supported by study of recombinant inbred lines (JI64xJI92) contrasting in dormancy but similar in other seed properties. Furthemore HLFA distribution in seed coat was studied by mass spectrometry imaging. HLFA contents in strophiole and hilum are significantly lower compared to other parts indicating their role in water uptake. Results from LDI-MS experiments are useful in understanding (physical) dormancy (first phases of germination) mechanism and properties related to food processing technologies (e.g., seed treatment by cooking).