Genome-Wide Analysis of the Late Embryogenesis Abundant (LEA) and Abscisic Acid-, Stress-, and Ripening-Induced (ASR) Gene Superfamily from Canavalia rosea and Their Roles in Salinity/Alkaline and Drought Tolerance.

Canavalia rosea (bay bean), distributing in coastal areas or islands in tropical and subtropical regions, is an extremophile halophyte with good adaptability to seawater and drought. Late embryogenesis abundant (LEA) proteins typically accumulate in response to various abiotic stresses, including dehydration, salinity, high temperature, and cold, or during the late stage of seed development. Abscisic acid-, stress-, and ripening-induced (ASR) genes are stress and developmentally regulated plant-specific genes. In this study, we reported the first comprehensive survey of the LEA and ASR gene superfamily in C. rosea. A total of 84 CrLEAs and three CrASRs were identified in C. rosea and classified into nine groups. All CrLEAs and CrASRs harbored the conserved motif for their family proteins. Our results revealed that the CrLEA genes were widely distributed in different chromosomes, and all of the CrLEA/CrASR genes showed wide expression features in different tissues in C. rosea plants. Additionally, we introduced 10 genes from different groups into yeast to assess the functions of the CrLEAs/CrASRs. These results contribute to our understanding of LEA/ASR genes from halophytes and provide robust candidate genes for functional investigations in plant species adapted to extreme environments.

Ectopic Expression of CrPIP2;3, a Plasma Membrane Intrinsic Protein Gene from the Halophyte Canavalia rosea, Enhances Drought and Salt-Alkali Stress Tolerance in Arabidopsis.

Aquaporins are channel proteins that facilitate the transmembrane transport of water and other small neutral molecules, thereby playing vital roles in maintaining water and nutrition homeostasis in the life activities of all organisms. Canavalia rosea, a seashore and mangrove-accompanied halophyte with strong adaptability to adversity in tropical and subtropical regions, is a good model for studying the molecular mechanisms underlying extreme saline-alkaline and drought stress tolerance in leguminous plants. In this study, a PIP2 gene (CrPIP2;3) was cloned from C. rosea, and its expression patterns and physiological roles in yeast and Arabidopsis thaliana heterologous expression systems under high salt-alkali and high osmotic stress conditions were examined. The expression of CrPIP2;3 at the transcriptional level in C. rosea was affected by high salinity and alkali, high osmotic stress, and abscisic acid treatment. In yeast, the expression of CrPIP2;3 enhanced salt/osmotic and oxidative sensitivity under high salt/osmotic and H2O2 stress. The overexpression of CrPIP2;3 in A. thaliana could enhance the survival and recovery of transgenic plants under drought stress, and the seed germination and seedling growth of the CrPIP2;3 OX (over-expression) lines showed slightly stronger tolerance to high salt/alkali than the wild-type. The transgenic plants also showed a higher response level to high-salinity and dehydration than the wild-type, mostly based on the up-regulated expression of salt/dehydration marker genes in A. thaliana plants. The reactive oxygen species (ROS) staining results indicated that the transgenic lines did not possess stronger ROS scavenging ability and stress tolerance than the wild-type under multiple stresses. The results confirmed that CrPIP2;3 is involved in the response of C. rosea to salt and drought, and primarily acts by mediating water homeostasis rather than by acting as an ROS transporter, thereby influencing physiological processes under various abiotic stresses in plants.

Preliminary study of the production of metabolites from in vitro cultures of C. ensiformis.

BACKGROUND: Canavalia ensiformis is a legume native to Central and South America that has historically been a source of protein. Its main proteins, urease, and lectin have been extensively studied and are examples of bioactive compounds. In this work, the effect of pH and light effects on the growth of C. ensiformis were analyzed. Also, the bioactive compounds such as phenols, carotenoids, chlorophyll a/b, and the growth of callus biomass of C. ensiformis from the effect of different types of light treatments (red, blue and mixture) were evaluated. Likewise, the antioxidative activity of C. ensiformis extracts were studied and related to the production of bioactive compounds. For this, a culture of calluses obtained from seeds were carried out. For the light experiments, polypropylene boxes with red, blue, combination (1/3, 3/1 and 1/1 R-B, respectively) lights and white LED were used as control. In each treatment, three glass containers with 25 ml of MS salts containing 0.25 g of fresh callus were seeded. RESULTS: The results have shown that the pH of the culture medium notably affects the increase in callogenic biomass. It shows that the pH of 5.5 shows better results in the callogenic growth of C. ensiformis with an average increase of 1.3051 g (198.04%), regarding the initial weight. It was found that the pH 5.5 and the 1/3 R-B LED combination had higher production of bioactive compounds and better antioxidant activity. At the same time, the red-light treatment was least effective. CONCLUSIONS: It was possible to find the ideal conditions of important growth under conditions of pH and light of C. ensiformis. Likewise, it is evaluated whether the production of compounds of interest, such as phenolic compounds and carotenoids, occurs under these conditions. The highest production of calluses occurs in the 1/3 R-B LED combined light treatment, which showed a significant increase in biomass, followed by B. From this study, it could be demonstrated that C. ensiformis produces compounds such as phenols and carotenoids in vitro culture that are essential for the antioxidant activity of the plant.

Does Canavalia ensiformis inoculation with Bradyrhizobium sp. enhance phytoremediation of sulfentrazone-contaminated soil?

Symbiosis among herbicide-metabolising microorganisms and phytoremediation plants may be an efficient alternative to remediate sulfentrazone-contaminated soils. This work evaluated the bioremediation of sulfentrazone-contaminated soils by symbiosis between bacteria (Bradyrhizobium sp.) and jack bean (Canavalia ensiformis L.). The experiment was carried out in a greenhouse between March and May of 2018, in the Universidade Federal do Espírito Santo (UFES). Four doses of sulfentrazone (0, 400, 800, and 1200 g ha-1 a. i.) were tested with and without inoculation with Bradyrhizobium sp. BR 2003 (SEMIA 6156) After 80 days of cultivation, plants were cut and soil was collected for determination of the herbicide residual levels and millet bioassay. The sulfentrazone concentration was significantly reduced by plant inoculation with Bradyrhizobium sp.: on average, concentrations were 18.97%, 23.82%, and 22.10% lower than in the absence of inoculation at doses of 400, 800, and 1200 g ha-1, respectively. Symbiosis promoted a reduction of up to 65% in residual soil herbicides. Under the 1200 g ha-1 dose, inoculation promoted greater plant height than in the uninoculated plant. Regardless of the dose of sulfentrazone, the dry root mass was higher in the inoculated plants. The microbiological indicators showed satisfactory results mainly for the dose of 400 g ha-1. The results of this study highlight the potential of positive interactions between symbiotic microorganisms and leguminous species, aiming toward the phytoremediation of sulfentrazone herbicide.

Earthworms and mycorrhization increase copper phytoextraction by Canavalia ensiformis in sandy soil.

Phytoremediation is an alternative for remediating soil contamination by copper, and its efficiency has been shown to increase when arbuscular mycorrhizal fungi (AMF) and earthworms are separately inoculated into the soil. This study evaluated the isolated and combined effects of inoculating earthworms and arbuscular mycorrhizal fungi into a sandy soil on copper phytoremediation by Canavalia ensiformis. The plants were grown in a greenhouse in soil contaminated with 100 mg Cu kg-1 with and without being inoculated with the arbuscular mycorrhizal fungus Rhizoglomus clarum and the earthworm Eisenia andrei. The availabilities of solid-phase Cu and other nutrients in the soil solution and plant growth were evaluated along with Cu phytotoxicity based on photochemical efficiency and oxidative stress enzyme activity. Accumulation of Cu and other nutrients in the shoots and roots; mycorrhizal colonization, nodulation, and reproduction; and Cu accumulation in the earthworm tissues were also evaluated. The copper caused photosynthetic and biochemical damage that reduced the shoot dry weight by 44% and the root dry weight by 29%. However, the arbuscular mycorrhizal fungus alleviated the Cu toxicity to the plant and increased the shoot dry weight by 81% in the contaminated soil. The earthworms increased the Cu uptake and translocation to the shoot by 31%. The combined presence of the arbuscular mycorrhizal fungus and earthworms in the contaminated soil increased the growth and Cu content of the aerial plant tissues, yielding a 200% increase in Cu accumulation (metal content × biomass) in the C. ensiformis shoots. Combined inoculation with earthworms and arbuscular mycorrhizal fungi increased copper phytoextraction by Canavalia ensiformis in a sandy soil.

Vermicompost dose and mycorrhization determine the efficiency of copper phytoremediation by Canavalia ensiformis.

The phytoremediation of copper (Cu)-contaminated sandy soils can be influenced by the addition of vermicompost to the soil and the mycorrhization of plants. The objective of this study was to evaluate the effects of inoculation with the mycorrhizal fungus Rhizophagus clarus and the addition of different doses of bovine manure vermicompost on the phytoremediation of a sandy soil with a high Cu content using Canavalia ensiformis. Soil contaminated with 100 mg kg-1 Cu received five doses of vermicompost and was cultivated with C. ensiformis, with and without inoculation with mycorrhizal fungus, and the Cu and nutrients in the soil and soil solution were evaluated. The concentrations of Cu and other nutrients and the biomass and Cu phytotoxicity in the plants were quantified by gauging the photochemical efficiency, concentration of photosynthetic pigments and activity of oxidative stress enzymes. The vermicompost increased the soil pH and nutrient concentrations and reduced the Cu content of the solution. When the vermicompost was applied at a dose equivalent to 80 mg phosphorus (P) kg-1, the phytoextraction efficiency was higher, but the phytostabilization efficiency was higher for vermicompost doses of 10 and 20 mg P kg-1. The presence of mycorrhizal fungi increased Cu phytostabilization, especially at vermicompost doses of 10 and 20 mg P kg-1. The use of vermicompost at low doses and inoculation with mycorrhizal fungi increase the phytostabilization potential of C. ensiformis in sandy soil contaminated by Cu.

Development of Canavalia ensiformis in soil contaminated with diesel oil.

Hydrocarbons are the main components of diesel oil and are toxic for the majority of plants. A few plant species, known as phytoremediators, are tolerant of hydrocarbons and can survive the stressful conditions of soils contaminated with diesel oil. Canavalia ensiformis, a plant species that is well distributed throughout the tropics, possesses advantageous features for a potential resistance to soil contamination, such as fast growth and a deep root system. Thus, the aim of the present study was to evaluate the tolerance of C. ensiformis when it was exposed to soil contaminated with diesel oil. Seedlings were subjected to two treatments: contaminated soil (CS) (95 ml/kg of diesel oil) and non-contaminated soil (NCS) for a period of 30 days; its growth, morphology, anatomy, and physiology were analyzed. Despite the high level of toxicity, some individuals were able to survive in CS. These plants had root apical meristems with high levels of mitosis and were able to issue new roots with more developed aerenchyma tissue. Because the surviving plants presented no marks of cellular damage on the organs formed (root and leaves) during the experiment, the species capacity of growth on CS was confirmed. Although, long-term field experiments, applying different contaminant concentrations, should be considered to infer about the species resistance and use as phytoremediator.

Evaluation of mycorrhizal influence on the development and phytoremediation potential of Canavalia gladiata in Pb-contaminated soils.

Soil contamination by heavy metals is a serious problem to humans due to its high level of toxicity. The heavy metal lead (Pb) is commonly used in industries and if the disposal of residues that contain this element is not done properly may result in tragic consequences to the organisms. In this experiment we assessed the potential of a forrage leguminous, Canavalia gladiata, to phytoremediate lead-contaminated soil under mycorrhizal influence. The experimental design was composed of 4 Pb doses (0, 250, 500, and 1000 mg kg(-1) of soil) and the plants were inoculated or uninoculated with arbuscular mycorrhizal fungi (AMF). We observed that the nodulation was severely affected by the presence of Pb independently of the mycorrhizal status; most of the elements analyzed were affected independently of the mycorrhizal status with exception of P. The mycorrhizal colonization was able to restrict the entrance of Pb in plants under high concentrations of Pb but promoted it's accumulation in both organs under intermediate concentrations of this element. Besides the mycorrhization did not promote plant growth under Pb stress, the use of this plant may be considered to be used for phytostabilization purposes.

Global and targeted proteomics in developing jack bean (Canavalia ensiformis) seedlings: an investigation of urease isoforms mobilization in early stages of development.

Jack bean (Canavalia ensiformis) seeds are toxic for insects and the toxicity is due in part to an entomotoxic peptide enzymatically released from ureases in the midgut of susceptible insects. To characterize expression of urease isoforms in jack bean seed, particularly the more abundant urease isoform (JBU), quantitative proteomics was performed. Quiescent through 5-day germinating seeds were analyzed at 1-day intervals using a total proteomics approach (TPA) and also after co-immunoprecipitation (co-IP) with anti-JBU monoclonal antibodies. Jack bean proteins for TPA and co-IP were pre-fractionated by SDS-PAGE, segmented for in-gel trypsin digestion, and analyzed by liquid chromatography coupled to nanospray ionization tandem mass spectrometry (LC-MS/MS). Acquired MS(2) data were searched against a comprehensive plant database and the MEROPS peptidase database, in the absence of a jack bean EST database. Proteins detected in TPA were quantified by label-free spectral counting. A total of 234 and 106 non-redundant proteins were detected in TPA and co-IP, respectively. Mobilization of JBU was observed beginning 3-days after imbibition indicating that the entomotoxic peptide was not formed before this stage. A predicted urease isoform, JBURE-IIb, was detected in the co-IP study. Additionally, 46 plastid proteins, including RuBisCO and plastid ATPase were pulled down with JBU antibodies. These data shed new light on the behavior of urease isoforms during the early stages of plant development.

Zn uptake, physiological response and stress attenuation in mycorrhizal jack bean growing in soil with increasing Zn concentrations.

The influence of arbuscular mycorrhizal fungi (AMF) inoculation on Canavalia ensiformis growth, nutrient and Zn uptake, and on some physiological parameters in response to increasing soil Zn concentrations was studied. Treatments were applied in seven replicates in a 2 x 4 factorial design, consisting of the inoculation or not with the AMF Glomus etunicatum, and the addition of Zn to soil at the concentrations of 0, 100, 300 and 900 mg kg(-1). AMF inoculation enhanced the accumulation of Zn in tissues and promoted biomass yields and root nodulation. Mycorrhizal plants exhibited relative tolerance to Zn up to 300 mg kg(-1) without exhibiting visual symptoms of toxicity, in contrast to non-mycorrhizal plants which exhibited a significant growth reduction at the same soil Zn concentration. The highest concentration of Zn added to soil was highly toxic to the plants. Leaves of plants grown in high Zn concentration exhibited a Zn-induced proline accumulation and also an increase in soluble amino acid contents; however proline contents were lower in mycorrhizal jack beans. Plants in association or not with the AMF exhibited marked differences in the foliar soluble amino acid profile and composition in response to Zn addition to soil. In general, Zn induced oxidative stress which could be verified by increased lipid peroxidation rates and changes in catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase activities. In summary, G. etunicatum was able to maintain an efficient symbiosis with jack bean plants in moderately contaminated Zn-soils, improving plant performance under those conditions, which is likely to be due to a combination of physiological and nutritional changes caused by the intimate relation between fungus and plant. The enhanced Zn uptake by AMF inoculated jack bean plants might be of interest for phytoremediation purposes.

A comparative study of the expression of serine proteinases in quiescent seeds and in developing Canavalia ensiformis plants.

An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.