Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach.

The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42-100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5'-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.

Stress responses and nickel and zinc accumulation in different accessions of Stellaria media (L.) Vill. in response to solution pH variation in hydroponic culture.

In most non-hyperaccumulating plants, Ni and Zn uptake is negatively correlated with soil pH, however, few studies so far have investigated how pH influences the activity and uptake of Ni and Zn in plants grown in a hydroponic system, which generally allows culture variables to be singularly manipulated. In this study, the non-accumulator Stellaria media (L.) Vill. (Caryophyllaceae) had opposite trends of Ni and Zn uptake along a pH gradient (between 5 and 8 for Zn and between 5 and 6.5 for Ni), when grown in hydroponics. In all treatments, the solution metal concentration was fixed at 0.1 mM Ni or 0.55 mM Zn. Nickel accumulation increased with increasing pH with an average concentration in shoots of 167 µg/gDW at pH 5 and of 250 µg/gDW at pH 6.5. In contrast, Zn accumulation decreased with increasing pH, with an average concentration in shoots varying from 1640 µg/gDW, at pH 5, to 435 µg/gDW at pH 8. Assessment of total polyphenol and flavonoid contents and of antioxidant activity showed that these parameters were positively correlated with Ni or Zn accumulation in S. media shoots, while photosynthetic pigments content and root and shoot biomass were negatively correlated with Ni and Zn accumulation. The study was carried out on five different S. media populations, which did not show differences in relation to the accumulation of metals and synthesis of antioxidant compounds, nonetheless showing a different biomass production under control conditions.

Effects of mutual intercropping on the cadmium accumulation in accumulator plants Stellaria media, Malachium aquaticum, and Galium aparine.

Intercropping affects heavy-metal uptake in plants. In this study, three cadmium (Cd)-accumulator species (Stellaria media, Malachium aquaticum, and Galium aparine) were intercropped together in Cd-contaminated soil to study the effects of intercropping on co-remediation. Mutual intercropping significantly increased the biomasses of S. media, M. aquaticum, and G. aparine compared with their respective monocultures. The photosynthetic pigment contents of three species were not significantly affected by mutual intercropping. Mutual intercropping did not increase the Cd contents in roots and shoots of G. aparine, but it increased the Cd contents in roots of S. media and M. aquaticum. It also decreased the Cd contents in shoots of S. media and M. aquaticum. Only G. aparine intercropped with M. aquaticum and three-species intercropping increased Cd-accumulation levels in whole plants of each species. Only S. media intercropped with M. aquaticum and three-species intercropping increased the Cd accumulation at the whole plant level in every plant in a single pot, with S. media intercropped with M. aquaticum showing the greatest increase. Therefore, S. media intercropped with M. aquaticum and three-species intercropping may improve the efficiency of phytoremediation of Cd-contaminated soil, with S. media intercropped with M. aquaticum representing the best combination.

Defense peptide repertoire of Stellaria media predicted by high throughput next generation sequencing.

Being perfectly adapted to diverse environments, chickweed (Stellaria media (L.) Vill), a ubiquitous garden weed, grows widely in Europe and North America. As opposed to the model plants, many weeds, and S. media in particular, have been poorly studied, although they are likely to contain promising components of immunity and novel resistance genes. In this study, for the first time RNA-seq analysis of healthy and infected with Fusarium oxysporum chickweed seedlings, as well as de novo transcriptome assembly and annotation, are presented. Note, this research is focused on antimicrobial peptides (AMPs), the major components of plant immune system. Using custom software developed earlier, 145 unique putative AMPs (pAMPs) including defensins, thionins, hevein-like peptides, snakins, alpha-hairpinins, LTPs, and cysteine-rich peptides with novel cysteine motifs were predicted. Furthermore, changes in AMP expression profile in response to fungal infection were traced. In addition, the comparison of chickweed AMP repertoire with those of other Caryophyllaceae plants whose transcriptomes are presently available is made. As a result, alpha-hairpinins and hevein-like peptides which display characteristic modular structure appear to be specific AMPs distinguishing S. media from Dianthus caryophyllus, Silene vulgaris, and Silene latifolia. Finally, revealing several AMPs with proven antimicrobial activity gives opportunity to conclude that the presented method of AMP repertoire analysis reveals highly active AMPs playing vital role in plant immunity.

[Effect of drought stress on growth and physiological-biochemical characteristics of Stellaria dichotoma].

A pot experiment was conducted to study effect of drought stress on leaf physiological characteristics and growth of one year old Stellaria dichotoma seedlings. The result showed that plant height and shoot dry weight significantly decreased with decrease in soil water content; however, root length and root dry weight increased at light drought stress and decreased at severe drought stress. The result also showed that with the decrease of soil water content, proline content in S. dichotoma leaves decreased then increase, while solube protein content decreased. Activities of SOD and POD in S. dichotoma leaves significantly decreased as soil water content decreased, while activity of CAT significantly decreased at severe drought stress. Membrane permeability in S. dichotoma leaves increased, while MDA content decreased then increased as soil water decreased. These results suggest that S. dichotoma had osmotic stress resistance ability and reactive oxygen scavenging capacity at light drought stress, which caused S. dichotoma growth was no inhibited at a certain extent drought stress.

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution.

Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C(1)X3C(2)XnC(3)X3C(4)), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C(1)-C(4) and C(2)-C(3). Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal "tail" regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S-S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.

Purification and characterization of a novel anti-HSV-2 protein with antiproliferative and peroxidase activities from Stellaria media.

A novel antiviral protein, designated as Stellarmedin A, was purified from Stellaria media (L.) Vill. (Caryophyllaceae) by using ammonium sulfate precipitation, cation-exchange chromatography system. Gel electrophoresis analysis showed that Stellarmedin A is a highly basic glycoprotein with a molecular weight of 35.1 kDa and an isoelectric point of ∼8.7. The N-terminal 14-amino acid sequence, MGNTGVLTGERNDR, is similar to those of other plant peroxidases. This protein inhibited herpes simplex virus type 2 (HSV-2) replication in vitro with an IC50 of 13.18 µg/ml and a therapeutic index exceeding 75.9. It was demonstrated that Stellarmedin A affects the initial stage of HSV-2 infection and is able to inhibit the proliferation of promyelocytic leukemia HL-60 and colon carcinoma LoVo cells with an IC50 of 9.09 and 12.32 µM, respectively. Moreover, Stellarmedin A has a peroxidase activity of 36.6 µmol/min/mg protein, when guaiacol was used as substrate. To our knowledge, this is the first report about an anti-HSV-2 protein with antiproliferative and peroxidase activities from S. media.

Isolation and characterization of PHYC gene from Stellaria longipes: differential expression regulated by different red/far-red light ratios and photoperiods.

We have cloned and characterized the phytochrome C ( PHYC) gene from Stellaria longipes. The PHYC gene is composed of a 110-bp 5'-untranslated leader sequence, a 3,342-bp coding region, and a 351-bp 3'-untranslated sequence. The Stellaria PHYC contains three long introns within the coding region at conserved locations as in most angiosperm PHY genes. DNA blot analysis indicates that the Stellaria genome contains a single copy of PHYC. Stellaria PHYC shares 60%, 58%, and 57% deduced amino acid identities with rice, Sorghum, and Arabidopsis PHYC, respectively. Phylogenetic analysis indicates that Stellaria PHYC is located in the dicot branch, but is divergent from Arabidopsis PHYC. The Stellaria PHYC is constitutively expressed in different plant organs, though the level of PHYC gene transcript in roots is slightly higher than in flowers, leaves, and stems. When 2-week old seedlings grown in the dark were exposed to constant white light, PHYC mRNA quickly accumulates within 1-12 h. When plants grown in darkness for 7 days were exposed to different red/far-red light (R/FR) ratios, the levels of PHYC mRNA at R/FR = 0.7 are much lower than under R/FR = 3.5. The levels of PHYC mRNA under short-day (SD) photoperiod are higher than under long-day (LD) photoperiod. Plants under SD conditions do not elongate, and are only about 1.7 cm tall at 19 days. In contrast, plants under LD conditions elongate with an average height of 21.2 cm at 19 days. The plants do not flower under SD conditions, but do so at 18-19 days under LD conditions. These results indicate that under SD conditions the high level of PHYC mRNA may inhibit stem elongation and flower initiation. In contrast, under LD conditions the high level of PHYC mRNA may promote stem elongation and flowering.