A Walk on the Wild Side: Genome Editing of Tuber-Bearing Solanum bulbocastanum.

Solanum bulbocastanum is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of S. bulbocastanum protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of Solanum tuberosum can be optimized to be applied to a wild Solanum species.

The Hypoxic Proteome and Metabolome of Barley (Hordeum vulgare L.) with and without Phytoglobin Priming.

Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O2 in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley (Hordeum vulgare L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca2+-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants.

Expression of starch-binding factor CBM20 in barley plastids controls the number of starch granules and the level of CO2 fixation.

The biosynthesis of starch granules in plant plastids is coordinated by the orchestrated action of transferases, hydrolases, and dikinases. These enzymes either contain starch-binding domain(s) themselves, or are dependent on direct interactions with co-factors containing starch-binding domains. As a means to competitively interfere with existing starch-protein interactions, we expressed the protein module Carbohydrate-Binding Motif 20 (CBM20), which has a very high affinity for starch, ectopically in barley plastids. This interference resulted in an increase in the number of starch granules in chloroplasts and in formation of compound starch granules in grain amyloplasts, which is unusual for barley. More importantly, we observed a photosystem-independent inhibition of CO2 fixation, with a subsequent reduced growth rate and lower accumulation of carbohydrates with effects throughout the metabolome, including lower accumulation of transient leaf starch. Our results demonstrate the importance of endogenous starch-protein interactions for controlling starch granule morphology and number, and plant growth, as substantiated by a metabolic link between starch-protein interactions and control of CO2 fixation in chloroplasts.

Destabilization of ROS metabolism in tomato roots as a phytotoxic effect of meta-tyrosine.

meta-Tyrosine (m-Tyr) is a non-protein amino acid produced in both plants and animals. Primary mode of action of this phenylalanine analog is its incorporation into protein structure leading to formation of aberrant molecules. An increased level of m-Tyr in animal cells is detected under oxidative stress and during age-related processes characterized by overproduction of reactive oxygen species (ROS). The aim of this study was to link m-Tyr physiological action to disturbances in ROS metabolism in tomato (Solanum lycopersicum L.) seedlings roots. Treatment of tomato seedlings with m-Tyr (50 or 250 µM) for 24-72 h led to inhibition of root growth without a lethal effect. Toxicity of m-Tyr after 72 h was connected with an increase in hydrogen peroxide concentration in roots and ROS leakage into the surrounding medium. On the contrary, membrane permeability and lipid peroxidation in roots were the same as for the control. This was accompanied by a decrease in total antioxidant activity and an increased accumulation of phenolic compounds. Catalase (CAT) activity declined in roots exposed to 50 µM m-Tyr after 24 h while after 72 h activity of this enzyme was inhibited in both treated and non-treated samples. Activities of different superoxide dismutase (SOD) isoforms were similar in m-Tyr stressed roots and in the control. Prolonged culture resulted in decrease of transcript level of genes coding CAT and SOD with the exception of FeSOD. Moreover, m-Tyr increased the level of protein carbonyl groups indicating induction of oxidative stress as a non-direct mode of action.

Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.

Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known as near-waxy or waxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Most waxy varieties are a result of either low or no expression of a GBSS gene. However, there are some waxy cultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one such waxy barley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both the non-waxy type and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa from non-waxy cultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa from waxy CDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 µm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.

meta-Tyrosine induces modification of reactive nitrogen species level, protein nitration and nitrosoglutathione reductase in tomato roots.

A non-protein amino acid (NPAA) - meta-Tyrosine (m-Tyr), is a harmful compound produced by fescue roots. Young (3-4 days old) tomato (Solanum lycopersicum L.) seedlings were supplemented for 24-72 h with m-Tyr (50 or 250 µM) inhibiting root growth by 50 or 100%, without lethal effect. Fluorescence of DAF-FM and APF derivatives was determined to show reactive nitrogen species (RNS) localization and level in roots of tomato plants. m-Tyr-induced restriction of root elongation growth was related to formation of nitrated proteins described as content of 3-nitrotyrosine. Supplementation with m-Tyr enhanced superoxide radicals generation in extracts of tomato roots and stimulated protein nitration. It correlated well to increase of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives corresponding respectively to NO and ONOO- formation. Alterations in RNS formation induced by m-Tyr were linked to metabolism of nitrosoglutathione (GSNO). Activity of nitrosoglutatione reductase (GSNOR), catalyzing degradation of GSNO was enhanced by long term plant supplementation with m-Tyr, similarly as protein abundance, while transcripts level were only slightly altered by tested NPAA. We conclude, that although in animal cells m-Tyr is considered as a marker of oxidative stress, its secondary mode of action in tomato plants involves perturbation in RNS formation, alteration in GSNO metabolism and modification of protein nitration level.

Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings.

Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins' structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24-72 h with CAN (10 or 50 µM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant's secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 µM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the formation of carbonyl groups than to nitration, and are detected particularly after 24 h. Thus, oxidative stress and oxidative modifications of proteins seems to be of significant importance in the rapid response of plants to CAN.

Toxicity of canavanine in tomato (Solanum lycopersicum L.) roots is due to alterations in RNS, ROS and auxin levels.

Canavanine (CAN) is non-proteinogenic aminoacid and a structural analog of arginine (Arg). Naturally, CAN occurs in legumes e.g. jack bean and is considered as a strong allelochemical. As a selective inhibitor of inducible nitric oxide synthase in mammalians, it could act as a modifier of nitric oxide (NO) concentration in plants. Modifications in the content of endogenous reactive nitrogen species (RNS) and reactive oxygen species (ROS) influence root structure and architecture, being also under hormonal control. The aim of the work was to investigate regulation of root growth in tomato (Solanum lycopersicum L. cv. Malinowy Ozarowski) seedling by application of CAN at concentration (10 and 50 µM) leading to 50% or 100% restriction of root elongation. CAN at higher concentration led to slight DNA fragmentation, increased total RNA and protein level. Decline in total respiration rate after CAN supplementation was not associated with enhanced membrane permeability. Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and ONOO(-) localization; determined mainly in peridermal cells and some border cells. Although, CAN resulted in low RNS production, addition of exogenous NO by usage of NO donors did not reverse its negative effect, nor recovery effect was detected after roots imbibition in Arg. To build up a comprehensive view on mode of action of CAN as root growth inhibitor, it was shown an elevated level of auxin. To summarize, we demonstrated several secondary mode of action of CAN, indicating its toxicity in plants linked to restriction in RNS formation accompanied by simultaneous overaccumulation of ROS.

Variations of morphology of activated sludge flocs studied at full-scale wastewater treatment plants.

Digital image analysis has been intensively developed over the last two decades including its application to describe morphology of activated sludge flocs. However, only few studies concerned the variation of flocs morphology with respect to the operational conditions, particularly oxido-reductive conditions, in a full-scale wastewater treatment plant (WWTP). In this work, morphology of activated sludge flocs was monitored over one year in two different full-scale WWTPs. The main aim of this study was to find the relationship between the operational parameters and morphology of sludge flocs. Simultaneously, the variations in floc size along activated sludge chamber were studied with respect to the oxido-reductive conditions. It was found that the sludge loading rate was one of the most important operational parameters influencing floc size. It was estimated that its values higher than 0.1 kg BOD5 kg TS(-1) d(-1) contributed to the decrease in floc size. Also, the oxido-reductive conditions influenced the floc size. It was statistically proved that flocs from the anaerobic zone were usually smaller than flocs from the anoxic or aerobic zones. Distribution of floc size in a full-scale WWTP usually could be described by a log-normal model.

Validation of a new image analysis procedure for quantifying filamentous bacteria in activated sludge.

Quantification of filamentous bacteria in activated sludge systems can be made by manual counting under a microscope or by the application of various automated image analysis procedures. The latter has been significantly developed in the last two decades. In this work a new method based upon automated image analysis techniques was elaborated and presented. It consisted of three stages: (a) Neisser staining, (b) grabbing of microscopic images, and (c) digital image processing and analysis. This automated image analysis procedure possessed the features of novelty. It simultaneously delivered data about aggregates and filaments in an individual calculation routine, which is seldom met in the procedures described in the literature so far. What is more important, the macroprogram performing image processing and calculation of morphological parameters was written in the same software which was used for grabbing of images. Previously published procedures required using two different types of software, one for image grabbing and another one for image processing and analysis. Application of this new procedure for the quantification of filamentous bacteria in the full-scale as well as laboratory activated sludge systems proved that it was simple, fast and delivered reliable results.