A search of Brassica SI-involved orthologs in buckwheat leads to novel buckwheat sequence identification: MLPK possibly involved in SI response

Kod biljaka cvetnica postoje genetički određeni sistemi self-inkompatibilnosti (SI), koji sprečavaju samooprašivanje i ukrštanje u srodstvu održavajući genetičku raznovrsnost vrsta. SI se javlja u dva oblika, kao gametofitna i sporofitna SI, koje se razlikuju u načinu određivanja SI fenotipa polena - kod GSI je SI fenotip polena određen polenovim sopstvenim haploidnim genomom, dok je kod SSI određen dipolidnim genotipom majke biljke. SSI se javlja kao homomorfna (jedan tip cveta u biljaka jedne vrste) i heteromorfna (dva ili tri tipa cveta u biljaka jedne vrste). Heteromorfna SSI je u poređenju sa homomorfnom SSI i GSI izuzetno malo proučena i za sada je upoznavanje na molekularnom nivou tek započelo. Kod heljde je prisutna distilna heteromorfna SSI, o kojoj je sakupljeno dosta podataka na fiziološkom nivou, ali o kojoj za sada nema molekularnih podataka. Na osnovu fiziološke sličnosti SI odgovora biljaka rodova Brassica i Prunus sa tram i pin morfom heljde, respektivno, zatim na osnovu toga što postoje dokazi da slični biohemijski mehanizmi leže u osnovi različitih SI odgovora i na osnovu toga što i evolutivno udaljene SI vrste mogu posedovati iste ili slične predačke SI gene, mi smo odlučili da ispitamo prisustvo ortologih gena uključenih u SI odgovore Brassica i Prunus u genomu heljde. Upotrebom izrođenih prajmera dizajniranih na osnovu evolutivno očuvanih regiona SRK, SLG, SP11 i MLPK sekvenci Brassica rapa, kao i S-RNaza i SFB gena roda Prunus, dostupnih u NCBI bazi podataka, ispitano je prisustvo ortologa ovih gena u genomu heljde. Takođe je prisustvo S-RNaza ispitano u proteinskim izolatima neoprašenih i kompatibilno i inkompatibilno oprašenih tučkova heljde oba morfa. Rezultati su pokazali da nema ortologa SRK, SLG, SP11, kao ni S-RNaza i SFB u genomu heljde, ali da postoji MLPK ortolog kod heljde. Izvedena aminokiselinska sekvenca pokazala je 80 % sličnosti sa MLPKf2 sekvencom Brassica rapa i APK1A Arabidopsis thaliana, potvrđujući da su u pitanju ortolozi koji bi mogli da imaju i sličnu ulogu. Naš sledeći korak je dobijanje cele nukleotidne sekvence MLPK heljde uz is- pitivanje postojanja alternativnih mesta iskrajanja i određivanje nivoa ekspresije po tkivima, kao i ispitivanje moguće uloge u SI odgovoru heljde. Ovi odgovori omogućiće bolje upoznavanje heteromorfnih SSI sistema koji su još uvek u svojoj najranijoj fazi istraživanja i obezbediće podatke nužne za uvid u evoluciju SI sistema biljaka cvetnica. Najzad, rasvetljavanjem SSI sistema heljde, koja se koristi u ishrani, biće moguće genetički kontrolisati ukrštanje heljde i dobijanje linija sa željenim hranljivim i/ili fiziološkim osobinama.Self-incompatibility (SI) systems, gamethophytic (GSI) and sporophytic (SSI), prevent self-pollination in angiosperms. Buckwheat displays heteromorphic SSI, with pollination allowed only between different flower morphs - thrum and pin. The physiology of thrum and pin morph SI responses are entirely different, resembling homomorphic Brassica SSI and Prunus GSI responses, respectively. Considering angiosperm species may share ancestral SI genes, we examined the presence of Brassica and Prunus SI-involved gene orthologs in the buckwheat genome. We did not find evidence of SRK, SLG and SP11 Brassica or S-RNase and SFB Prunus orthologs in the buckwheat genome, but we found a Brassica MLPK ortholog. We report the partial nucleotide sequence of the buckwheat MLPK and discuss the possible implications of this finding

Molecular tools for utilization of mitochondrial diversity in faba bean (vicia faba)

We performed in silico PCR analyses utilizing complete mitochondrial (mtDNA) genome sequences of faba bean (Vicia faba) and two related species, Vigna angularis and Vigna radiata, currently available in GenBank, to infer whether 15 published universal primer pairs for amplification of all 14 cis-spliced introns in genes of NADH subunits (nad genes) are suitable for V. faba and related species. Then, we tested via PCR reactions whether seven out of 15 primer pairs would generate PCR products suitable for further manipulation in 16 genotypes of V. faba representing all botanical varieties of this species (major, minor, equina and subsp. paucijuga) of various levels of improvement (traditional and improved cultivars) originating from Europe, Africa, Asia and south America. We provide new PCR primers for amplification of nad1 intron 2/3 in V. faba, and demonstrate intraspecific variability in primary nucleotide sequences at this locus. Based on outcomes of both in silico predictions and PCR amplification, we report a set of PCR primers for amplification of five introns in nad genes that are promising molecular tools for future phylogeographic and other studies in this species for which unambiguous data on wild ancestors, centre of origin and domestication are lacking

Analysis of some nutritional and antinutritional compounds in dry seeds of grass pea and field pea cultivars from Croatia

Field pea (Pisum sativum var. arvense) and grass pea (Lathyrus sativus) are important high protein crops used as feed. Legume seeds are also rich in many other nutrient compounds including starch, sugar, dietary fiber and micronutrients (vitamins and minerals.) They are also a rich source of many bioactive non-nutrient compounds including phenolic antioxidants. Besides their beneficial effects, some phenolic compounds, especially condensed tannins in feed, are considered as antinutritional factors for ruminants, owing to their capacity for binding to macromolecules such as fiber and proteins. Seed mass and diameter are valuable parameters used in breeding, as well as seed mineral composition, since significant quantitative trait loci were identified for Ca, Fe, Mn, K, Mg, Ni, P and Zn. Thus we conducted this study in order to determine some parameters, in three cultivars of previously non-tested field pea and five grass pea cultivars from Croatia, which are important for breeders. We determined seed mass, diameter and proximate mineral composition as well as content of soluble sugars and total phenolic content in dry seeds of these cultivars. Cultivars differed significantly in total phenollics and mineral composition

CRISPR/Cas9-Targeted Disruption of Two Highly Homologous Arabidopsis thaliana DSS1 Genes with Roles in Development and the Oxidative Stress Response

Global climate change has a detrimental effect on plant growth and health, causing serious losses in agriculture. Investigation of the molecular mechanisms of plant responses to various environmental pressures and the generation of plants tolerant to abiotic stress are imperative to modern plant science. In this paper, we focus on the application of the well-established technology CRISPR/Cas9 genome editing to better understand the functioning of the intrinsically disordered protein DSS1 in plant response to oxidative stress. The Arabidopsis genome contains two highly homologous DSS1 genes, AtDSS1(I) and AtDSS1(V). This study was designed to identify the functional differences between AtDSS1s, focusing on their potential roles in oxidative stress. We generated single dss1(I) and dss1(V) mutant lines of both Arabidopsis DSS1 genes using CRISPR/Cas9 technology. The homozygous mutant lines with large indels (dss1(I)del25 and dss1(V)ins18) were phenotypically characterized during plant development and their sensitivity to oxidative stress was analyzed. The characterization of mutant lines revealed differences in root and stem lengths, and rosette area size. Plants with a disrupted AtDSS1(V) gene exhibited lower survival rates and increased levels of oxidized proteins in comparison to WT plants exposed to oxidative stress induced by hydrogen peroxide. In this work, the dss1 double mutant was not obtained due to embryonic lethality. These results suggest that the DSS1(V) protein could be an important molecular component in plant abiotic stress respons

Simple cooking and in vitro gastrointestinal digestion improves antioxidant activity of legume seed flour extracts

Antioxidant activities and phenolic contents were studied in methanolic and water extracts of non-processed, cooked and in vitro enzymatically digested seed flour. Antioxidant activities of total protein hydrolysates and small peptide fractions (MW<3kDa and MW<3kDa) isolated from seed were also determined. Study showed that cooking and enzymatic digestion strongly enhanced release of phenolic compounds. Scavenging activity against 2,2-diphenyl-1-picryhydrazyl radical (DPPH•), hydroxyl radical and hydrogen peroxide were also increased. Protein hydrolysates especially MW<3kDa fraction possess significant antioxidative potential. Taken together our findings indicate that in methanolic and water extracts of cooked and digested seed flour small peptide fraction, beside phenolic compounds, can also significantly contribute to free radical and hydrogen peroxide scavenging activity

Salt stress response in three pea species (Pisum arvense, P. sativum and P. fulvum) in vitro cultures

Salinity is one of the major abiotic factors limiting plants productivity. Processes, such as seed germination, seedling growth and vigor, vegetative growth, flowering, and fruit set, are adversely affected by high saltconcentrations, ultimately causing diminished economic yield and also quality of products. Shoots of micropropagated Pisum fulvum, P. sativum and P. arvense were treated with 75, 120, 150 and 200 mMNaClin order to study pea tissues capacity to tolerate salt stress and their antioxidant capacity. We used the phenol content and DPPH antioxidant assay as oxidative damage indicators. Results showed that salt stress caused slower plant growth, leading to an increase in lipid peroxidation and a decrease in total chlorophyll in pea tissues. Further, results showed that P. fulvum is to be considered as potentially less sensitive to high NaCl concentrations in comparison to P. sativum and P. arvense and could represents a gene pool which could be used for improving the characteristics of field pea

Dehydration affected the expression of miR398 and miR408 in pea (Pisum sativum L.)

MicroRNAs (miRNAs), recently recognized as important regulator of gene expression at posttranscriptional level, have been found to be involved in plant stress responses. The observation that some miRNAs are up- or down regulated by stress implies that that could play vital roles in plant resistance to abiotic and biotic stress. We investigated the effect of water stress treatment during 10 days on expression of conserved miRNAs-miR398a/b and miR408 in pea plants. This time frame could reflect the changes as closely as possible those during water stress cause visible effects under field condition. It was observed that dehydration strongly down regulates the expression of both miR398a/b and miR408 in pea roots and shoots. The down-regulation of miR398a/b and the up-regulation of potential target genes copper superoxide dismutase, CSD1, highlight the involvement of this miRNA in pea stress response. On the contrary, the mRNA level of cytochrome c oxidase subunit 5 (COX5b) was not changed in roots and shoots of water-stressed plants, compared to control-well hydrated plants. This suggests that COX5b is not target of miR398, or that its expression is regulated by some other mechanism. P1B-ATPase expression increased during water deficit only in shoots of pea; in the roots there was no changes in expression. Our results might help to understand the possible role of investigated miRNAs and their contribution to pea capacity to cope with water deficit

Application of different Lactobacillus strains in production pea seed protein hydrolysates with antioxidative activity

In recent years antioxidant properties of plant derived protein hydrolysates were established. Antioxidative activity of these hydrolysates largely depends on specificity of the proteinase used for hydrolysis, duration of proteinase treatment, as well as composition and molecular weight of hydrolysis derived peptides. In order to explore potential use of nine proteinase containing Lactobacillus strains in production of antioxidative peptides we tested their ability to hydrolyze lyophilized hydrosoluble protein fraction from dehulled and defatted dry pea seeds (Pisum sativum), cultivar NS Mraz. We also developed plant protein based liquid medium for cultivation of lactobacilli which was later used as starter for hydrolysis reaction in larger volume. We found that all nine strains could hydrolyze plant hydrosoluble fraction of pea seed proteins but with different efficiency. We also examined the effect of duration of hydrolysis on antioxidative activity in <10kDa fraction of obtained peptide extracts, using most proteolyticaly active strain, human vaginal isolate Lactobacillus rhamnosus T10. General conclusion was that antioxidative activity was positively correlated with increase of hydrolysis duration and amount of small peptides, which reached up to 34% of total proteins after 24h of hydrolysis treatment

A Rapid and Cost-effective Procedure for Delineation and Utilization of Genomic Microsatellites for Paralleled Genotyping in Vicia faba

Although more than 400 microsatellite loci are currently available for Vicia faba L. (faba bean), an important food and feed grain crop legume, they have not yet been used for comprehensive molecular characterization of this crop. We report a three-step procedure for rapid and cost-effective delineation and utilization of informative genomic nuclear SSRs for paralleled genotyping in faba bean suitable also for other species: (i) pre-selection of loci generating PCR products of expected lengths which are potentially polymorphic (achieved by PCR amplification in bulked samples); (ii) exclusion of loci burdened with persistent null alleles and multilocus amplification products (based on PCR amplification of pre-selected loci in individual genotypes), and (iii) multiplexing. We demonstrate also that genomic SSRs are promising molecular tools for molecular characterization of faba bean required also for crop improvement

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate.

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nataša seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.This is a post-peer-review, pre-copyedit version of an article published in Protoplasma. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00709-018-1250-0