Polysaccharide assemblies in fungal and plant cell walls explored by solid-state NMR.

Structural analysis of macromolecular complexes within their natural cellular environment presents a significant challenge. Recent applications of solid-state NMR (ssNMR) techniques on living fungal cells and intact plant tissues have greatly enhanced our understanding of the structure of extracellular matrices. Here, we selectively highlight the most recent progress in this field. Specifically, we discuss how ssNMR can provide detailed insights into the chemical composition and conformational structure of pectin, and the consequential impact on polysaccharide interactions and cell wall organization. We elaborate on the use of ssNMR data to uncover the arrangement of the lignin-polysaccharide interface and the macrofibrillar structure in native plant stems or during degradation processes. We also comprehend the dynamic structure of fungal cell walls under various morphotypes and stress conditions. Finally, we assess how the combination of NMR with other techniques can enhance our capacity to address unresolved structural questions concerning these complex macromolecular assemblies.

Signals and Their Perception for Remodelling, Adjustment and Repair of the Plant Cell Wall.

The integrity of the cell wall is important for plant cells. Mechanical or chemical distortions, tension, pH changes in the apoplast, disturbance of the ion homeostasis, leakage of cell compounds into the apoplastic space or breakdown of cell wall polysaccharides activate cellular responses which often occur via plasma membrane-localized receptors. Breakdown products of the cell wall polysaccharides function as damage-associated molecular patterns and derive from cellulose (cello-oligomers), hemicelluloses (mainly xyloglucans and mixed-linkage glucans as well as glucuronoarabinoglucans in Poaceae) and pectins (oligogalacturonides). In addition, several types of channels participate in mechanosensing and convert physical into chemical signals. To establish a proper response, the cell has to integrate information about apoplastic alterations and disturbance of its wall with cell-internal programs which require modifications in the wall architecture due to growth, differentiation or cell division. We summarize recent progress in pattern recognition receptors for plant-derived oligosaccharides, with a focus on malectin domain-containing receptor kinases and their crosstalk with other perception systems and intracellular signaling events.

Live Fluorescence Visualization of Cellulose and Pectin in Plant Cell Walls.

The plant cell wall comprises various types of macromolecules whose abundance and spatial distribution change dynamically and are crucial for plant architecture. High-resolution live cell imaging of plant cell wall components is, therefore, a powerful tool for plant cell biology and plant developmental biology. To acquire suitable data, the experimental setup for staining and imaging of non-fixed samples must be straightforward and avoid creating stress-induced artifacts. We present a detailed sample preparation and live image acquisition protocol for fluorescence visualization of cell wall components using commercially available probes and stains.

Metabolic and Transcriptional Stress Memory in Sorbus pohuashanensis Suspension Cells Induced by Yeast Extract.

Plant stress memory can provide the benefits of enhanced protection against additional stress exposure. Here, we aimed to explore the responses of recurrent and non-recurrent yeast extract (YE) stresses in Sorbus pohuashanensis suspension cells (SPSCs) at metabolomics and transcriptional levels. Biochemical analyses showed that the cell wall integrity and antioxidation capacity of SPSCs in the pretreated group were evidently improved. Metabolic analysis showed that there were 39 significantly altered metabolites in the pretreated group compared to the non-pretreated group. Based on the transcriptome analysis, 219 differentially expressed genes were obtained, which were highly enriched in plant-pathogen interaction, circadian rhythm-plant, oxidative phosphorylation, and phenylpropanoid biosynthesis. Furthermore, the correlation analysis of the transcriptome and metabolome data revealed that phenylpropanoid biosynthesis involved in the production of biphenyl phytoalexins may play a critical role in the memory response of SPSC to YE, and the key memory genes were also identified, including PAL1, BIS1, and BIS3. Collectively, the above results demonstrated that the memory responses of SPSC to YE were significant in almost all levels, which would be helpful for better understanding the adaptation mechanisms of medicinal plants in response to biotic stress, and laid a biotechnological foundation to accumulate favorable antimicrobial drug candidates from plant suspension cells.

HISTONE DEACETYLASE 9 transduces heat signal in plant cells.

Heat stress limits plant growth, development, and crop yield, but how plant cells precisely sense and transduce heat stress signals remains elusive. Here, we identified a conserved heat stress response mechanism to elucidate how heat stress signal is transmitted from the cytoplasm into the nucleus for epigenetic modifiers. We demonstrate that HISTONE DEACETYLASE 9 (HDA9) transduces heat signals from the cytoplasm to the nucleus to play a positive regulatory role in heat responses in Arabidopsis. Heat specifically induces HDA9 accumulation in the nucleus. Under heat stress, the phosphatase PP2AB'ß directly interacts with and dephosphorylates HDA9 to protect HDA9 from 26S proteasome-mediated degradation, leading to the translocation of nonphosphorylated HDA9 to the nucleus. This heat-induced enrichment of HDA9 in the nucleus depends on the nucleoporin HOS1. In the nucleus, HDA9 binds and deacetylates the target genes related to signaling transduction and plant development to repress gene expression in a transcription factor YIN YANG 1-dependent and -independent manner, resulting in rebalance of plant development and heat response. Therefore, we uncover an HDA9-mediated positive regulatory module in the heat shock signal transduction pathway. More important, this cytoplasm-to-nucleus translocation of HDA9 in response to heat stress is conserved in wheat and rice, which confers the mechanism significant implication potential for crop breeding to cope with global climate warming.

Building an Artificial Plant Cell Wall on a Lipid Bilayer by Assembling Polysaccharides and Engineered Proteins.

The cell wall constitutes a fundamental structural component of plant cells, providing them with mechanical resistance and flexibility. Mimicking this wall is a critical step in the conception of an experimental model of the plant cell. The assembly of cellulose/hemicellulose in the form of cellulose nanocrystals and xyloglucans as a representative model of the plant cell wall has already been mastered; however, these models lacked the pectin component. In this work, we used an engineered chimeric protein designed for bridging pectin to the cellulose/hemicellulose network, therefore achieving the assembly of complete cell wall mimics. We first engineered a carbohydrate-binding module from Ruminococcus flavefaciens able to bind oligogalacturonan, resulting in high-affinity polygalacturonan receptors with Kd in the micromolar range. A Janus protein, with cell wall gluing property, was then designed by assembling this carbohydrate-binding module with a Ralstonia solanacearum lectin specific for fucosylated xyloglucans. The resulting supramolecular architecture is able to bind fucose-containing xyloglucans and homogalacturonan, ensuring high affinity for both. A two-dimensional assembly of an artificial plant cell wall was then built first on synthetic polymer and then on the supported lipid bilayer. Such an artificial cell wall can serve as a basis for the development of plant cell mechanical models and thus deepen the understanding of the principles underlying various aspects of plant cells and tissues.

A comprehensive study on the interaction of Eu(III) and U(VI) with plant cells (Daucus carota) in suspension.

Daucus carota suspension cells showed a high affinity towards Eu(III) and U(VI) based on a single-step bioassociation process with an equilibrium after 48-72 h. Cells responded with an increased metabolic activity towards heavy metal stress. Luminescence spectroscopy pointed to multiple species for both f-block elements in the culture media, providing initial hints of their interaction with cells and released metabolites. Using nuclear magnetic resonance spectroscopy, we could prove that malate, as an released metabolite in the culture medium, was found to complex with U. Luminescence spectroscopy also showed that Eu(III)-EDTA species are interacting with the cells. Furthermore, Eu(III) and U(VI) coordination is dominated by phosphate groups provided by the cells. We found that Ca ion channels of D. carota cells were involved in the uptake of U(VI), which led to a bioprecipitation of U(VI) in the vacuole of the cells, most probably as uranyl(VI) phosphates along with an intracellular sorption of U(VI) on biomembranes by lipid structures. Eu(III) could be found locally concentrated in the cell wall and in the cytoplasm with a co-localization with phosphorous and oxygen.

Isolation of Specialized Plant Cells by Fluorescence-Activated Cell Sorting.

Plant organs are built of different cell types, characterized by specific transcription programs and metabolic profiles. The possibility of isolation of such cell types to perform differential transcriptomic, proteomic and metabolomic analyses is highly important to understand many aspects of plant physiology, namely, the structure and regulation of economically valuable specialized metabolic pathways. Here, we describe the isolation of idioblast leaf protoplasts of the medicinal plant Catharanthus roseus by fluorescence-activated cell sorting, taking advantage of the differential autofluorescence properties of those specialized cells.

Hairy Roots of Scutellaria spp. (Lamiaceae) as Promising Producers of Antiviral Flavones.

We measured and studied the growth parameters and the qualitative and quantitative composition of the flavones of hairy roots of the Scutellaria genus: S. lateriflora, S. przewalskii and S. pycnoclada. Hairy roots were obtained using wild-type Agrobacterium rhizogenes A4 by co-cultivation of explants (cotyledons) in a suspension of Agrobacterium. The presence of the rol-genes was confirmed by PCR analysis. The hairy roots of the most studied plant from the Scutellaria genus, S. baicalensis, were obtained earlier and used as a reference sample. HPLC-MS showed the predominance of four main flavones (baicalin, baicalein, wogonin and wogonoside) in the methanol extracts of the studied hairy roots. In addition to the four main flavones, the other substances which are typical to the aerial part of plants were found in all the extracts: apigenin, apigetrin, scutellarin and chrysin-7-O-ß-d-glucuronide. According to the total content of flavones, the hairy roots of the studied skullcaps form the following series: S. przewalskii (33 mg/g dry weight) > S. baicalensis (17.04 mg/g dry weight) > S. pycnoclada (12.9 mg/g dry weight) > S. lateriflora (4.57 mg/g dry weight). Therefore, the most promising producer of anti-coronavirus flavones is S. przewalskii.

Effects of Abiotic Elicitors on Expression and Accumulation of Three Candidate Benzophenanthridine Alkaloids in Cultured Greater Celandine Cells.

Efforts to develop the necessary biotechnologies in Greater Celandine (Chelidonium majus L.), a leading plant resource for the development of plant-derived medicines, have been hampered by the lack of knowledge about transcriptome and metabolome regulations of its medicinal components. Therefore, this study aimed to examine the effect of abiotic elicitors, methyl jasmonate (MJ) and salicylic acid (SA), at different time courses (12, 24, 48, and 72 h), on expression and metabolome of key benzophenanthridine alkaloids (BPAs) in an optimized in vitro culture. Gene expression analysis indicated the upregulation of CFS (cheilanthifoline synthase) to 2.62, 4.85, and 7.28 times higher than the control at 12, 24, and 48 h respectively, under MJ elicitation. Besides, MJ upregulated the expression of TNMT (tetrahydroprotoberberine N-methyltransferase) to 2.79, 4.75, and 7.21 times at 12, 24, and 48 h respectively, compared to the control. Investigation of BPAs revealed a significant enhancement in the chelidonine content (9.86 µg/mg) after 72 h of MJ elicitation. Additionally, sanguinarine content increased to its highest level (3.42 µg/mg) after 24 h of MJ elicitation; however, no significant enhancement was detected in its content in shorter elicitation time courses. Generally, higher gene expression and BPAs' level was observed through longer elicitation courses (48 and 72 h). Our findings take part in improving the understanding of transcription and metabolic regulation of BPAs in cultured Greater Celandine cells.

TOR coordinates nucleotide availability with ribosome biogenesis in plants.

TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic Ser/Thr protein kinase that coordinates growth and metabolism with nutrient availability. We conducted a medium-throughput functional genetic screen to discover essential genes that promote TOR activity in plants, and identified a critical regulatory enzyme, cytosolic phosphoribosyl pyrophosphate (PRPP) synthetase (PRS4). PRS4 synthesizes cytosolic PRPP, a key upstream metabolite in nucleotide synthesis and salvage pathways. We found that prs4 knockouts are embryo-lethal in Arabidopsis thaliana, and that silencing PRS4 expression in Nicotiana benthamiana causes pleiotropic developmental phenotypes, including dwarfism, aberrant leaf shape, and delayed flowering. Transcriptomic analysis revealed that ribosome biogenesis is among the most strongly repressed processes in prs4 knockdowns. Building on these results, we discovered that TOR activity is inhibited by chemical or genetic disruption of nucleotide biosynthesis, but that this effect can be reversed by supplying plants with nucleobases. Finally, we show that TOR transcriptionally promotes nucleotide biosynthesis to support the demands of ribosomal RNA synthesis. We propose that TOR coordinates ribosome biogenesis with nucleotide availability in plants to maintain metabolic homeostasis and support growth.

The genetic basis of cytoplasmic male sterility and fertility restoration in wheat.

Hybrid wheat varieties give higher yields than conventional lines but are difficult to produce due to a lack of effective control of male fertility in breeding lines. One promising system involves the Rf1 and Rf3 genes that restore fertility of wheat plants carrying Triticum timopheevii-type cytoplasmic male sterility (T-CMS). Here, by genetic mapping and comparative sequence analyses, we identify Rf1 and Rf3 candidates that can restore normal pollen production in transgenic wheat plants carrying T-CMS. We show that Rf1 and Rf3 bind to the mitochondrial orf279 transcript and induce cleavage, preventing expression of the CMS trait. The identification of restorer genes in wheat is an important step towards the development of hybrid wheat varieties based on a CMS-Rf system. The characterisation of their mode of action brings insights into the molecular basis of CMS and fertility restoration in plants.

UV-C mediated accumulation of pharmacologically significant phytochemicals under light regimes in in vitro culture of Fagonia indica (L.).

Fagonia indica (L.) is an important medicinal plant with multitude of therapeutic potentials. Such application has been attributed to the presence of various pharmacological important phytochemicals. However, the inadequate biosynthesis of such metabolites in intact plants has hampered scalable production. Thus, herein, we have established an in vitro based elicitation strategy to enhance such metabolites in callus culture of F. indica. Cultures were exposed to various doses of UV radiation (UV-C) and grown in different photoperiod regimes and their impact was evaluated on biomass accumulation, biosynthesis of phytochemicals along antioxidant expression. Cultures grown under photoperiod (16L/8D h) after exposure to UV-C (5.4 kJ/m2) accumulated optimal biomass (438.3 g/L FW; 16.4 g/L DW), phenolics contents (TPC: 11.8 µgGAE/mg) and flavonoids contents (TFC: 4.05 µgQE/mg). Similarly, HPLC quantification revealed that total production (6.967 µg/mg DW) of phytochemicals wherein kaempferol (1.377 µg/mg DW), apigenin (1.057 µg/mg DW), myricetin (1.022 µg/mg DW) and isorhamnetin (1.022 µg/mg DW) were recorded highly accumulated compounds in cultures at UV-C (5.4 kJ/m2) dose than other UV-C radiations and light regimes.. The antioxidants activities examined as DPPH (92.8%), FRAP (182.3 µM TEAC) and ABTS (489.1 µM TEAC) were also recorded highly expressed by cultures under photoperiod after treatment with UV-C dose 5.4 kJ/m2. Moreover, same cultures also expressed maximum % inhibition towards phospholipase A2 (sPLA2: 35.8%), lipoxygenase (15-LOX: 43.3%) and cyclooxygenases (COX-1: 55.3% and COX-2: 39.9%) with 1.0-, 1.3-, 1.3- and 2.8-fold increased levels as compared with control, respectively. Hence, findings suggest that light and UV can synergistically improve the metabolism of F. indica and could be used to produce such valuable metabolites on commercial scale.

Yeast extract improved biosynthesis of astragalosides in hairy root cultures of Astragalus membranaceus.

The dried root of Astragalus membranaceus is a well-known herbal medicine, and it is useful in treating chronic diseases and weakness, as well as for improving overall health and vitality. Astragalosides, which are root quality indicators of A. membranaceus, are natural triterpenoid saponins that are used in the treatment of diabetes and cardiovascular diseases. Currently, there is an urgent need to improve their production because of their low quantity in plants and the difficulty of chemical synthesis. In this study, yeast extract was added to facilitate elicitation in Agrobacterium-mediated hairy root cultures, thereby enhancing astragaloside production in A. membranaceus. Results showed that yeast extract could stimulate astragaloside content effectively in the hairy roots of A. membranaceus. Moreover, astragaloside accumulation was positively correlated with the upregulation of mevalonate biosynthetic gene expression in the presence of yeast extract. Our study demonstrated that pretreatment with yeast extract (3.65 mM) for 72 h serves as an effective strategy to enhance astragaloside levels in A. membranaceus hairy root cultures. Thus, these optimal conditions can provide valuable information for the improvement of astragaloside industrial production in A. membranaceus.

Investigation of Drug Efficacy by Screening Bioactive Chemical Effects on Plant Cell Subcellular Architecture.

New biologically active compounds are regularly discovered through screening procedures using microorganisms. This very cheap procedure is followed by drug discovery that is usually seen as a highly focused approach, testing new compounds on animals or cell lines. In vivo assays of candidate drugs in mammals are expensive and sometimes not affordable at the preliminary stages of drug development. Early screening approaches in transgenic plants would allow chemotherapeutic drug candidates further selection before their characterization in expensive biological models. The proposed screening approach is based on cell subcellular architecture observations in transgenic plants within a short time of treatment, which is better than observing the effects of compounds on growth.

Age-Dependent and Tissue-Specific Alterations in the rDNA Clusters of the Panax ginseng C. A. Meyer Cultivated Cell Lines.

Long-term cultivation of Panax ginseng cell lines leads to a decreasing synthesis of the biologically active substances used in traditional medicine. To gain insight into the cellular mechanisms which may influence this process, we analyzed variations within the rDNA cluster of the Oriental ginseng cell lines. The cell lines were cultivated for 6 and 24 years; the number of nucleoli and chromosomes was analyzed. The complete 18S rDNA sequences were cloned and sequenced. The nucleotide polymorphism and phylogenetic relations of the sequences were analyzed, and the secondary structures for separate 18S rRNA regions were modeled. The 18S rDNA accumulated mutations during cell cultivation that correlate well with an increase in the number of chromosomes and nucleoli. The patterns of nucleotide diversity are culture-specific and the increasing polymorphism associates with cytosine methylation sites. The secondary structures of some 18S rRNA regions and their interaction can alter during cultivation. The phylogenetic tree topologies are particular for each cell line.The observed alterations in rDNA clusters are associated with a somaclonal variation, leading to changes in the pattern of intracellular synthesis during cell cultivation. The identified divergent rRNAs could provide additional gene expression regulation in P. ginseng cells by forming heterogeneous ribosomes.

Production of ascorbic acid, total protein, callus and root in vitro of non-heading Chinese cabbage by tissue culture.

The objective of the present work was the selection of cultivar, suitable medium and explant type for callus, root production, ascorbic acid, total ascorbic acid, dehydroascorbic and total protein of non-heading Chinese cabbage in two cultivars 'Caixin' and 'Suzhouqing'. We compared 10 types of MS media supplemented with 0.0, 1.0, 2.0 and 3.0 mg/l TDZ; 0.0, 0.25, 0.50 and 1.0 mg/l NAA and 0.0, 5.0, 7.5 and 9.0 mg/l AgNO3 and 5 kinds of explants as embryo, leaf, root, cotyledon and hypocotyl. Maximum frequency of callus fresh weight was recorded with hypocotyl explant, which were cultured on MS + 2.0 mg/l TDZ + 1.0 mg/l NAA + 9.0 mg/l AgNO3 in 'Suzhouqing', optimum callus dry weight was obtained on the same media. The highest result for root fresh and dry weight recorded with 'Caixin' with MS + 3.0 mg/l TDZ + 1.0 mg/l NAA + 9.0 mg/l AgNO3 when we used embryo as explant. The highest ascorbic acid content was found with callus cultured on MS + 1.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3, when used leaf explant in 'Caixin' or root in 'Suzhouqing', and there were no significant difference between them. While the highest value of total AsA content was registered with callus cultured on MS + 2.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 extracted from cotyledon in 'Caixin'. The highest content of DHA was registered with MS + 2.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 with cotyledon in 'Caixin'. Also, in 'Caixin' MS + 3.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 recorded the highest value of total protein content with embryo explant.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Developmental Methylome of the Medicinal Plant Catharanthus roseus Unravels the Tissue-Specific Control of the Monoterpene Indole Alkaloid Pathway by DNA Methylation.

Catharanthus roseus produces a wide spectrum of monoterpene indole alkaloids (MIAs). MIA biosynthesis requires a tightly coordinated pathway involving more than 30 enzymatic steps that are spatio-temporally and environmentally regulated so that some MIAs specifically accumulate in restricted plant parts. The first regulatory layer involves a complex network of transcription factors from the basic Helix Loop Helix (bHLH) or AP2 families. In the present manuscript, we investigated whether an additional epigenetic layer could control the organ-, developmental- and environmental-specificity of MIA accumulation. We used Whole-Genome Bisulfite Sequencing (WGBS) together with RNA-seq to identify differentially methylated and expressed genes among nine samples reflecting different plant organs and experimental conditions. Tissue specific gene expression was associated with specific methylation signatures depending on cytosine contexts and gene parts. Some genes encoding key enzymatic steps from the MIA pathway were found to be simultaneously differentially expressed and methylated in agreement with the corresponding MIA accumulation. In addition, we found that transcription factors were strikingly concerned by DNA methylation variations. Altogether, our integrative analysis supports an epigenetic regulation of specialized metabolisms in plants and more likely targeting transcription factors which in turn may control the expression of enzyme-encoding genes.

Identification of MTP gene family in tea plant (Camellia sinensis L.) and characterization of CsMTP8.2 in manganese toxicity.

Cation diffusion facilitators (CDFs) play central roles in metal homeostasis and tolerance in plants, but the specific functions of Camellia sinensis CDF-encoding genes and the underlying mechanisms remain unknown. Previously, transcriptome sequencing results in our lab indicated that the expression of CsMTP8.2 in tea plant shoots was down-regulated exposed to excessive amount of Mn2+ conditions. To elucidate the possible mechanisms involved, we systematically identified 13 C. sinensis CsMTP genes from three subfamilies and characterized their phylogeny, structures, and the features of the encoded proteins. The transcription of CsMTP genes was differentially regulated in C. sinensis shoots and roots in responses to high concentrations of Mn, Zn, Fe, and Al. Differences in the cis-acting regulatory elements in the CsMTP8.1 and CsMTP8.2 promoters suggested the expression of these two genes may be differentially regulated. Transient expression analysis indicated that CsMTP8.2 was localized to the plasma membrane in tobacco and onion epidermal cells. Moreover, when heterologously expressed in yeast, CsMTP8.2 conferred tolerance to Ni and Mn but not to Zn. Additionally, heterologous expression of CsMTP8.2 in Arabidopsis thaliana revealed that CsMTP8.2 positively regulated the response to manganese toxicity by decreasing the accumulation of Mn in plants. However, there was no difference in the accumulation of other metals, including Cu, Fe, and Zn. These results suggest that CsMTP8.2 is a Mn-specific transporter that contributes to the efflux of excess Mn2+ from plant cells.