Belowground DNA-based techniques: untangling the network of plant root interactions

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Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory

Plant DNA Barcodes Can Accurately Estimate Species Richness in Poorly Known Floras

Extent: 9p.BACKGROUND: Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (~70%) and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology. METHODOLOGY/PRINCIPAL FINDINGS: Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways.Craig Costion, Andrew Ford, Hugh Cross, Darren Crayn, Mark Harrington and Andrew Low

Suppression of MMP-2 Attenuates TNF-α Induced NF-κB Activation and Leads to JNK Mediated Cell Death in Glioma

BACKGROUND: Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. METHODOLOGY/PRINCIPAL FINDINGS: MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKβ and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKβ and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. CONCLUSION/SIGNIFICANCE: In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo

Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF–atpH, and psbK–psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85–100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK–psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69–71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems

MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients