Mucilage Layer Research Articles

Test of Level of Likes on Fermentation Length and Physical Quality Characteristics of Robusta Coffee After Storage

One of the stages in the wet processing of coffee that can affect its flavor is fermentation. Fermentation is the process of removing the mucilage layer that still adheres to the coffee beans, which results in a distinctive coffee aroma and reduces caffeine content, especially in robusta coffee. After the fermentation process, packaging and storage are carried out. Storage is the stage where raw coffee beans are kept to maintain their quality until the next process. This study aims to determine the effect of fermentation duration on panelists' preference levels and the impact of storage duration on the physical quality characteristics of robusta coffee. This research employs both quantitative and qualitative descriptive methods. The results show that storage duration does not affect the physical quality characteristics of robusta coffee beans. The moisture content parameter after storage was found to be 12.2%, the density parameter averaged 0.691 gr/ml, and the defect value parameter fell into grade 3. The study also shows that different fermentation durations do not significantly affect the panelists' preference levels, with an average score of 3-4,1.

The transcription factors ZAT5 and BLH2/4 regulate homogalacturonan demethylesterification in Arabidopsis seed coat mucilage.

The level of methylesterification alters the functional properties of pectin, which is believed to influence plant growth and development. However, the mechanisms that regulate demethylesterification remain largely unexplored. Pectin with a high degree of methylesterification is produced in the Golgi apparatus and then transferred to the primary cell wall where it is partially demethylesterified by pectin methylesterases (PMEs). Here, we show that in Arabidopsis (Arabidopsis thaliana) seed mucilage, pectin demethylesterification is negatively regulated by the transcription factor ZINC FINGER FAMILY PROTEIN5 (ZAT5). Plants carrying null mutations in ZAT5 had increased PME activity, decreased pectin methylesterification, and produced seeds with a thinner mucilage layer. We provide evidence that ZAT5 binds to a TGATCA motif and thereby negatively regulates methylesterification by reducing the expression of PME5, HIGHLY METHYL ESTERIFIED SEEDS (HMS)/PME6, PME12, and PME16. We also demonstrate that ZAT5 physically interacts with BEL1-LIKE HOMEODOMAIN2 (BLH2) and BLH4 transcription factors. BLH2 and BLH4 are known to modulate pectin demethylesterification by directly regulating PME58 expression. The ZAT5-BLH2/4 interaction provides a mechanism to control the degree of pectin methylesterification in seed coat mucilage by modifying each transcription factor's ability to regulate the expression of target genes encoding PMEs. Taken together, these findings reveal a transcriptional regulatory module comprising ZAT5, BLH2, and BLH4, that functions in modulating the demethylesterification of homogalacturonan in seed coat mucilage.

Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR‐mediated disruption of Transparent Testa 8

SummaryCamelina (Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well‐organized seed coat mucilage layer. Transcriptomic analysis of CsTT8‐edited seeds revealed significantly increased expression of the lipid‐related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis‐related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced‐oil germplasm in camelina. The resulting lines may directly contribute to future net‐zero carbon energy production or be combined with other traits to produce desired lipid‐derived bioproducts at high yields.

Genome wide association study of Arabidopsis seed mucilage layers at a regional scale

The myxospermous species Arabidopsis thaliana extrudes a polysaccharidic mucilage from the seed coat epidermis during imbibition. The whole seed mucilage can be divided into a seed-adherent layer and a fully soluble layer, both layers presenting natural genetic variations. The adherent mucilage is variable in size and composition, while the soluble mucilage is variable in composition and physical properties. Studies reporting both the genetic architecture and the putative selective agents acting on this natural genetic variation are scarce. In this study, we set up a Genome Wide Association study (GWAS) based on 424 natural accessions collected from 166 natural populations of A. thaliana located south-west of France and previously characterized for a very important number of abiotic and biotic factors. We identified an extensive genetic variation for both mucilage layers. The adherent mucilage was mainly related to precipitation and temperature whereas the non-adherent mucilage was unrelated to any environmental factors. By combining a hierarchical Bayesian model with a local score approach, we identified 55 and 28 candidate genes, corresponding to 26 and 10 QTLs for the adherent and non-adherent mucilages, respectively. Putative or characterized function and expression data available in the literature were used to filter the candidate genes. Only one gene among our set of candidate genes was already described as a seed mucilage actor, leaving a large set of new candidates putatively implicated inseed mucilage synthesis or release. The present study lay out foundation to understand the influence of regional ecological factors acting on seed mucilage in A. thaliana.

Influence of nanoscale spatial architecture of seed and root mucilage on its wettability

Mucilage secreted by root-tips may alter hydraulic conditions at the root-soil interface and seed coat mucilage increases water availability for seeds. Both significantly affect the water content dynamics of the rhizosphere. Therefore, we examined the nanostructure of different types of dried mucilage (root or seed mucilage and mucilage from different species) and how it affects their wettability. We hypothesized that with increasing roughness wettability of dried mucilage decreases based on the Cassie−Baxter relation due to entrapped air in holes formed in the mucilage layer during drying. In order to visualize the nanostructure of different mucilage species, we dried root mucilage from aeroponically grown seedlings (maize, cress, flax, wheat, barley) and seed mucilage (cress and flax) as thin layers on flat mica surfaces. Spatial structures of the samples were visualized by atomic force microscopy. Variogram analysis was used to analyze the spatially resolved topography data, and the coverage of mucilage was quantified using GeoDict® software. Moreover, by the pore size distribution, the effect of hole-size dispersity on mucilage's wettability was investigated. Our results demonstrate that mucilage of different species have different nanostructures. In contrast to expectations, the wettability of dried mucilage does not follow the Cassie−Baxter relation and does not decrease with increasing roughness. We conclude that wettability of surfaces decreases with increasing mucilage coverage. The lower the coverage the more hydrophilic area of the underlying mineral surface contributes to the overall wettability.

A Consumer Assessment of Fermented Green Coffee Beans with Common Beer/Wine Yeast Strains for Novel Flavor Properties

Fermentation is a critical step in the production of coffee when following standard wet processing, one of the most common methods used to remove the mucilage layer from coffee cherries. During this step, the de-pulped coffee cherries undergo fermentation with native yeast that modifies the flavor profile of the resultant coffee. This study aimed to ferment green coffee beans using commercial yeast strains from beer and wine prized for their ability to produce specific flavors, and subsequently evaluate the aroma and flavor of the coffee using coffee consumers. Four Saccharomyces cerevisiae strains were used: Belgian Ale, Sourvisiae, 71 B, and Tropical IPA, along with one non-Saccharomyces, Toluraspora delbrueckii (Biodiva), and a non-inoculated control sample. The green coffee beans underwent a controlled wet fermentation for 72 h, followed by roasting, grinding, and brewing. Results showed that flavor profiles varied broadly by yeast strain, suggesting that producing novel flavors in coffee through fermentation is feasible and that these flavors survive the roasting process; however, higher liking scores were still reported for the control sample compared to the fermented samples. Biodiva, a strain used in wine to produce esters and fruity flavors, resulted in coffee with highly fruity notes, and all strains were rated more floral than the control, while the sample fermented with Sourvisiae yeast used in the brewing of sour ales resulted in coffee that was both perceived as more sour and had the lowest pH, likely due to the degree of lactic acid this strain is engineered to produce. Further, there were significant color differences between the samples. In conclusion, fermenting green coffee beans with brewing and winemaking yeast strains strongly impacted the flavor and aroma of the resultant coffee; however, evaluating larger panels of strains or optimizing strain performance may yield flavor profiles more suitable for coffee.

Extracting high-molecular weight DNA from cyanobacteria using Promega's WizardⓇ HMW DNA extraction kit with a modified protocol, METIS

Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's WizardⓇ HMW DNA Extraction Kit to acquire high molecular weight DNA from two cyanobacterial species. The protocol used in the kit is the “3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria” protocol. During a key step in the protocol, we propose that the lingering remnants of the cellular debris such as the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes from cyanobacteria (Synechococcus sp. and Microcystis aeruginosa) and one from a gram-negative bacterium, Lacibacter. It also allows for HMW DNA to be rapidly extracted without the use of toxic chemicals such as phenol and without extra reagents to be purchased.•Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling•METIS reduces leftover remnants of the mucilage layer in the extraction•High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration

Yeasts are essential for mucilage degradation of coffee beans during wet fermentation.

During wet fermentation, mucilage layers in coffee cherries must be removed completely. To explain mucilage degradation, several controversial hypotheses have been proposed. The aim of this work was to improve our understanding of the kinetics of mucilage breakdown. Pulped coffee beans were wet fermented with seven different treatments for 36 h. Endogenous bacteria and yeasts are selectively suppressed, and pectinases or lactic acid are added. They also involve maintaining the beans at pH 7 throughout fermentation and using spontaneous fermentation without additives as a control. During spontaneous fermentation, yeast and lactic acid bacteria were detected and significantly increased to 5.5 log colony-forming units(CFU)/mL and 5.2 log CFU/mL, respectively. In the first 12 h of fermentation, there was a significant degree of endogenous pectinolytic activity, which resulted in partly destroyed beans in the absence of microorganisms. By adding pectinase and lactic acid to the fermentation mass, the breakdown process was accelerated in less than 8 h. When yeast was present throughout the fermentation, complete degradation was achieved. Bacteria played no critical role in the degradation. Klebsiella pneumoniae and Erwinia soli were found in a lower population and showed weaker pectinolytic activities compared to Hanseniaspora uvarum and Pichia kudriavzevii. During wet fermentation, mucilage degradation appears to be mediated by endogenous enzymes at the early stage, whereas microbial contributions, mainly yeasts, occur subsequently. H. uvarum and P. kudriavzevii may be promising candidates to be tested in future studies as coffee starter cultures to better control the mucilage degradation process.

Role of mucilage in improving seedling emergence of Ocimum basilicum L. exposed to salinity stress

The seeds of many desert plants have an extraneous mucilage layer that can raise water holding capability of seeds under stressful environments. The purpose of this study was to investigate the role of mucilage in improving the salinity resistance of Ocimum basilicum L. The treatments include salinity (0, 5, 10, 15 and 20 dS/m) and seed type (with and without mucilage) with three replications. Results showed that all studied traits were significantly (p < 0.01) effected by salinity stress. Seed type had a significant effect (p < 0.01) on the germination rate and root and shoot length, whereas this factor did not significantly affect other traits. Except for seed germination, other variables were significantly affected by salinity and seed type interaction. With increasing salinity stress, germination and seedling growth of both seed types were significantly decreased; however, seeds with mucilage were less affected by salinity stress than those without mucilage.

The competitive strategies of poisonous weeds Elsholtzia densa Benth. on the Qinghai Tibet Plateau: Allelopathy and improving soil environment.

The competitive strategies of plants play a crucial role in their growth. Allelopathy is one of the weapons that plants use to improve their competitive advantage. In order to explore the competitive strategy of a poisonous weed Elsholtzia densa Benth. (E. densa) on the Qinghai-Tibet Plateau (QTP), the effects of decomposing substances of E. densa on growth, root border cells (RBCs) characteristics of highland crop highland barley (Hordeum vulgare L.), and soil environment were determined. The decomposing allelopathic effect of E. densa on the germination and seedling growth of highland barley mainly occurred in the early stage of decomposing. The allelopathic effects were mainly on seed germination and root growth of highland barley. After treatment with its decomposing solution, the RBC's mucilage layer of highland barley thickened, and the RBC's activity decreased or even apoptosis compared with the control. However, only the above-ground part of the treatment group showed a significant difference. The effects of E. densa decomposed substances on the soil environment were evaluated from soil physicochemical properties and bacterial community. The results showed that soil bacteria varied greatly in the early stage of decomposion under different concentrations of E. densa. In addition, E. densa decomposing substances increased the soil nutrient content, extracellular enzyme activities, and bacterial community diversity. In the process of decomposition, the bacterial community structure changed constantly, but Actinobacteriota was always the dominant phylum. These results indicated that E. densa might adopt the following two strategies to help it gain an advantage in the competition: 1. Release allelochemicals that interfere with the defense function of surrounding plants and directly inhibit the growth and development of surrounding plants. 2. By changing the physical and chemical properties of soil and extracellular enzyme activity, residual plant decomposition can stimulate soil microbial activity, improve soil nutrition status, and create a more suitable soil environment for growth.

A fine-tuned defense at the pea root caps: Involvement of border cells and arabinogalactan proteins against soilborne diseases.

Plants have to cope with a myriad of soilborne pathogens that affect crop production and food security. The complex interactions between the root system and microorganisms are determinant for the whole plant health. However, the knowledge regarding root defense responses is limited as compared to the aerial parts of the plant. Immune responses in roots appear to be tissue-specific suggesting a compartmentalization of defense mechanisms in these organs. The root cap releases cells termed root "associated cap-derived cells" (AC-DCs) or "border cells" embedded in a thick mucilage layer forming the root extracellular trap (RET) dedicated to root protection against soilborne pathogens. Pea (Pisum sativum) is the plant model used to characterize the composition of the RET and to unravel its function in root defense. The objective of this paper is to review modes of action of the RET from pea against diverse pathogens with a special focus on root rot disease caused by Aphanomyces euteiches, one of the most widely occurring and large-scale pea crop diseases. The RET, at the interface between the soil and the root, is enriched in antimicrobial compounds including defense-related proteins, secondary metabolites, and glycan-containing molecules. More especially arabinogalactan proteins (AGPs), a family of plant extracellular proteoglycans belonging to the hydroxyproline-rich glycoproteins were found to be particularly present in pea border cells and mucilage. Herein, we discuss the role of RET and AGPs in the interaction between roots and microorganisms and future potential developments for pea crop protection.

Structure and formation of the perforated theca defining the Pelagophyceae (Heterokonta), and three new genera that substantiate the diverse nature of the class.

The pelagophytes, a morphologically diverse class of marine heterokont algae, have been historically united only by DNA sequences. Recently we described a novel perforated theca (PT) encasing cells from the Pelagophyceae and hypothesized it may be the first morphological feature to define the class. Here we consolidate that observation, describing a PT for the first time in an additional seven pelagophyte genera, including three genera new to science. We established clonal cultures of pelagophytes collected from intertidal pools located around Australia, and established phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA and psbC gene sequences that led to the discovery of three new species: Wyeophycus julieharrissiae and Chromopallida australis form a distinct lineage along with Ankylochrysis lutea within the Pelagomonadales, while Pituiglomerulus capricornicus is sister genus to Chrysocystis fragilis in the Chrysocystaceae (Sarcinochrysidales). Using fixation by high-pressure freezing for electron microscope observations, a distinctive PT was observed in the three new genera described in this paper, as well as four genera not previously investigated: Chrysoreinhardia, Sargassococcus, Sungminbooa and Andersenia. The mechanism of PT formation is novel, being fabricated from rafts in Golgi-derived vesicles before being inserted into an established PT. Extracellular wall and/or mucilage layers assemble exterior to the PT in most pelagophytes, the materials likewise secreted by Golgi-derived vesicles, though the mechanism of secretion is novel. Secretory vesicles never fuse with the plasma membrane as in classic secretion and deposition, but rather relocate extracellularly beneath the PT and disintegrate, the contents having to pass through the PT prior to wall and/or mucilage synthesis. This study substantiates the diverse nature of pelagophytes, and provides further evidence that the PT is a sound morphological feature to define the Pelagophyceae, with all 14 of the 20 known genera studied to date by TEM possessing a PT.

Sensory Properties and Volatile Compound Profile of Anaerobic Fermented Gayo Arabica Coffee In Non-Washed Processing

Coffee is a popular beverage that is consumed worldwide. Flavor is an important aspect of excellent coffee. Fermentation was applied in the wash coffee processing to degrade the mucilage layer and to improve the flavor quality. Fermentation in non-washed gayo arabica coffee processing has not been widely reported. This study aims to analyze the effect of anaerobic fermentation on non-washed Gayo arabica coffee processing to obtain coffee with the highest sensorial score. Coffee fruit were processed and fermented for 0-48 hours. Sensorial score, volatile, and non-volatil compounds were observed at roasted bean. A significant increase in sensory test scores was obtained in the Pulped Natural (PN) process which was fermented with a score of 85.58 ± 1.01. Concentrations of citric, malic, succinic, lactic, and acetic acids are varied between the treatments. Volatile analysis using SPME-GCMS produced 20 compounds with an Odor Active Value OAV&gt; 1 consisting of aldehydes, furans, ketones, phenols, pyrazines, and terpenes. β-Damascenone was a compound that has the highest OAV values with honey-like, fruity, apple, and rose aroma characteristics that dominate PN and Black Honey (BH). Fermentation for 48 h in the PN process is suitable for use in Gayo with humid conditions and high altitudes.&#x0D; &#x0D; Keywords: Coffea arabica, SPME-GCMS, Volatile compounds, HPLC, Non-volatile components, Coffee processing.

Pharmacognostic approach to evaluate the micromorphological, phytochemical and biological features of Citrus lumia seeds

This study evaluated the micro-morphology as well as the chemical and biological features of Citrus lumia seeds. The cream-colored pyriform seed showed a woody coat covered by a thick layer of mucilage and an embryo with two large cotyledons rich in oil bodies. Hydroxycinnamic acid glycosides and flavonoids are the most abundant compounds in methanol and ethyl acetate extracts (ME and EAE), respectively. Conversely, fatty acids and α-tocopherol represent the main bioactive compounds in the hexane extract (HE). ME showed the most promising antioxidant and anti-inflammatory activities already in cell-free assays. These results were confirmed by experiments carried out on human primary cells. Indeed, ME showed the best inhibitory activity against heat-induced haemolysis and ROS formation in erythrocytes. Moreover, the same order of potency (ME > EAE > HE) was observed also on peripheral blood mononuclear cells, in which the seed extracts were able to decrease TNF-α and IL-6 release after LPS-induced inflammation.

Morphological and Ultrastructural Features of Formation of the Skin of Wheat (Triticum aestivum L.) Kernel.

The integumentary tissues of plant seeds protect the embryo (new sporophyte) forming in them from unfavorable external conditions; therefore, comprehensive knowledge about the structural and functional specificity of seed covers in various plants may be of both theoretical and practical interest. As a result of our study, additional data were obtained on the morphological and ultrastructural features of the formation of a multilayer skin of wheat (Triticum aestivum L.) kernel (caryopsis). The ultrastructure research analysis showed that differentiation of the pericarp and inner integument of the ovule leads to the formation of functionally different layers of the skin of mature wheat grain. Thus, the differentiation of exocarp and endocarp cells is accompanied by a significant thickening of the cell walls, which reliably protect the ovule from adverse external conditions. The cells of the two-layer inner integument of the ovule differentiate into cuticular and phenolic layers, which are critical for protecting daughter tissues from various pathogens. The epidermis of the nucellus turns into a layer of mucilage, which apparently helps to maintain the water balance of the seed. Morphological and ultrastructural data showed that the formation of the kernel’s skin occurs in coordination with the development of the embryo and endosperm up to the full maturity of the kernel. This is evidenced by the structure of the cytoplasm and nucleus, characteristic of metabolically active protoplasts of cells, which is observed in most integumentary layers at the late stages of maturation. This activity can also be confirmed by a significant increase in the thickness of the cell walls in the cells of two layers of the exocarp and in cross cells in comparison with the earlier stages. Based on these results, we came to the conclusion that the cells of a majority in the covering tissues of the wheat kernel during its ontogenesis are transformed into specialized layers of the skin by terminal differentiation.

Sterol Glucosyltransferases Tailor Polysaccharide Accumulation in Arabidopsis Seed Coat Epidermal Cells.

The conjugation of sterols with a Glc moiety is catalyzed by sterol glucosyltransferases (SGTs). A portion of the resulting steryl glucosides (SG) are then esterified with a long-chain fatty acid to form acyl-SG (ASG). SG and ASG are prevalent components of plant cellular membranes and influence their organization and functional properties. Mutant analysis had previously inferred that two Arabidopsis SGTs, UGT80A2 and UGT80B1/TT15, could have specialized roles in the production of SG in seeds, despite an overlap in their enzymatic activity. Here, we establish new roles for both enzymes in the accumulation of polysaccharides in seed coat epidermal cells (SCEs). The rhamnogalacturonan-I (RG-I) content of the inner layer of seed mucilage was higher in ugt80A2, whereas RG-I accumulation was lower in mutants of UGT80B1, with double mutant phenotypes indicating that UGT80A2 acts independently from UGT80B1. In contrast, an additive phenotype was observed in double mutants for increased galactoglucomannan (GGM) content. Double mutants also exhibited increased polymer density within the inner mucilage layer. In contrast, cell wall defects were only observed in mutants defective for UGT80B1, while more mucilage cellulose was only observed when UGT80A2 was mutated. The generation of a range of phenotypic effects, simultaneously within a single cell type, demonstrates that the adjustment of the SG and ASG composition of cellular membranes by UGT80A2 and UGT80B1 tailors polysaccharide accumulation in Arabidopsis seeds.

The ecological roles of seed mucilage on germination of Lepidium perfoliatum, a desert herb with typical myxospermy in Xinjiang

Myxospermy is considered as an adaptation to unideal habitats, which is characterized as a gelatinous layer of mucilage around the seed upon contact with water. Studies on the ecological roles of seed mucilage should provide valuable information on seed germination strategy. In the present study, the germination properties of Lepidium perfoliatum L., an early spring ephemeral plant with typical myxospermy in desert of northwestern China, were investigated with the intact seeds and demucilaged seeds under different conditions. Results revealed that, firstly, the seed mucilage was essential for intact seeds to germinate quickly and efficiently under abundant or excess water condition, while had no effect on relief of seed germination at low water potential or other stressful conditions; secondly, the presence of mucilage layer stimulated intact seed germination under relatively higher temperature, appropriate light, permeable soil matrix, which helps seed germinate efficiently under favorable conditions; thirdly, the existence of mucilage may limit air diffusion into seed and subsequently reduce germination, which might be a strategy to ensure a proportion of seeds staying in soil seed bank to wait for next favourable conditions. Our results suggest that the seed mucilage of L. perfoliatum applies multiple ecological roles in protection of seed germination to avoid unfavourable conditions, which should be an adaptation strategy for population survival and succession in a harsh desert environment.

Datasets of seed mucilage traits for Arabidopsis thaliana natural accessions with atypical outer mucilage

The seeds of Arabidopsis thaliana become encapsulated by a layer of mucilage when imbibed. This polysaccharide-rich hydrogel is constituted of two layers, an outer layer that can be easily extracted with water and an inner layer that must be examined in situ in order to study its properties and structure in a non-destructive manner or disintegrated through hydrolysis or physical means in order to analyze its constituents. Mucilage production is an adaptive trait and we have exploited 19 natural accessions previously found to have atypical and varied outer mucilage characteristics. A detailed study using biochemical, histological and Time-Domain NMR analyses has been used to generate three related datasets covering 33 traits measured in four biological replicates. This data will be a rich resource for genetic, biochemical, structural and functional analyses investigating mucilage constituent polysaccharides or their role as adaptive traits.

Salt stress of two rice varieties: root border cell response and multi-logistic quantification.

How to capture the rice varieties salt stress sensitivity? Here, we measure responses of root border cells (1 day, ± 60 mM NaCl) and apply multi-logistic quantification of growth variables (21 days, ± 60 mM NaCl) to two rice varieties, salt-sensitive IR29 and tolerant Pokkali. Thus, logistic models determine the maximum response velocities (Vmax) and times of half-maximum (T0) for root border cell (RBC) and growth parameters. Thereof, seven variables show logistic models (0.58 < R ≤ 1) and monotonous responses in both Pokkali and IR29: root to shoot ratio by water content, primary root length, shoot water, adventitious root number, shoot dry and fresh weight, and root dry weight. Moreover, the regression to lognormal distribution (R = 0.99) of these seven Vmax fractionated by T0 represents the rice variety's comprehensive response. Its quotient IR29/Pokkali is peaking at 98-fold higher velocity of IR29, thus capturing the variety's sensitivity. Consequently, our finding of 66-fold higher Vmax of primary root length response of IR29 indicates an essential salt sensor in the root, including RBC. Finally, the effects of salt stress on RBC confirm multi-logistic quantification, showing 36% decrease of RBC mucilage layer in IR29, without change in Pokkali. Inversely, RBC number of Pokkali increases 43% without change in IR29. Briefly, this suggests both RBC and multi-logistic quantification for the screening for salt tolerance in two thousand rice varieties.

Characterization of Two Zygnema Strains (Zygnema circumcarinatum SAG 698-1a and SAG 698-1b) and a Rapid Method to Estimate Nuclear Genome Size of Zygnematophycean Green Algae.

Zygnematophyceae green algae (ZGA) have been shown to be the closest relatives of land plants. Three nuclear genomes (Spirogloea muscicola, Mesotaenium endlicherianum, and Penium margaritaceum) of ZGA have been recently published, and more genomes are underway. Here we analyzed two Zygnema circumcarinatum strains SAG 698-1a (mating +) and SAG 698-1b (mating −) and found distinct cell sizes and other morphological differences. The molecular identities of the two strains were further investigated by sequencing their 18S rRNA, psaA and rbcL genes. These marker genes of SAG 698-1a were surprisingly much more similar to Z. cylindricum (SAG 698-2) than to SAG 698-1b. Phylogenies of these marker genes also showed that SAG 698-1a and SAG 698-1b were well separated into two different Zygnema clades, where SAG 698-1a was clustered with Z. cylindricum, while SAG 698-1b was clustered with Z. tunetanum. Additionally, physiological parameters like ETRmax values differed between SAG 698-1a and SAG 698-1b after 2 months of cultivation. The de-epoxidation state (DEPS) of the xanthophyll cycle pigments also showed significant differences. Surprisingly, the two strains could not conjugate, and significantly differed in the thickness of the mucilage layer. Additionally, ZGA cell walls are highly enriched with sticky and acidic polysaccharides, and therefore the widely used plant nuclear extraction protocols do not work well in ZGA. Here, we also report a fast and simple method, by mechanical chopping, for efficient nuclear extraction in the two SAG strains. More importantly, the extracted nuclei were further used for nuclear genome size estimation of the two SAG strains by flow cytometry (FC). To confirm the FC result, we have also used other experimental methods for nuclear genome size estimation of the two strains. Interestingly, the two strains were found to have very distinct nuclear genome sizes (313.2 ± 2.0 Mb in SAG 698-1a vs. 63.5 ± 0.5 Mb in SAG 698-1b). Our multiple lines of evidence strongly indicate that SAG 698-1a possibly had been confused with SAG 698-2 prior to 2005, and most likely represents Z. cylindricum or a closely related species.