Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests.

Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae).

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure.

The evolutionary conservation of rps3 introns and rps19-rps3-rpl16 gene cluster in Adiantum capillus-veneris mitochondria.

Ferns are a large and evolutionarily critical group of vascular land plants for which quite limited mitochondrial gene content and genome organization data are, currently, available. This study reports that the gene for the ribosomal protein S3 (rps3) is preserved and physically clustered to an upstream rps19 and a downstream overlapping rpl16 locus in the mitochondrial DNA of the true fern Adiantum capillus-veneris L. Sequence analysis also revealed that the rps3 gene is interrupted by two cis-splicing group II introns, like the counterpart in lycopod and gymnosperm representatives. A preliminary polymerase chain reaction (PCR) survey confirmed a scattered distribution pattern of both the rps3 introns also in other fern lineages. Northern blot and reverse transcription (RT)-PCR analyses demonstrated that the three ribosomal protein genes are co-transcribed as a polycistronic mRNA and modified by RNA editing. Particularly, the U-to-C type editing amends numerous genomic stop codons in the A. capillus-veneris rps19, rps3 and rpl16 sequences, thus, assuring the synthesis of complete and functional polypeptides. Collectively, the findings from this study further expand our knowledge of the mitochondrial rps3 architecture and evolution, also, bridging the significant molecular data gaps across the so far underrepresented ferns and all land plants.

A phytochrome/phototropin chimeric photoreceptor of fern functions as a blue/far-red light-dependent photoreceptor for phototropism in Arabidopsis.

In the fern Adiantum capillus-veneris, the phototropic response of the protonemal cells is induced by blue light and partially inhibited by subsequent irradiation with far-red light. This observation strongly suggests the existence of a phytochrome that mediates this blue/far-red reversible response; however, the phytochrome responsible for this response has not been identified. PHY3/NEO1, one of the three phytochrome genes identified in Adiantum, encodes a chimeric photoreceptor composed of both a phytochrome and a phototropin domain. It was demonstrated that phy3 mediates the red light-dependent phototropic response of Adiantum, and that phy3 potentially functions as a phototropin. These findings suggest that phy3 is the phytochrome that mediates the blue/far-red response in Adiantum protonemata. In the present study, we expressed Adiantum phy3 in a phot1 phot2 phototropin-deficient Arabidopsis line, and investigated the ability of phy3 to induce phototropic responses under various light conditions. Blue light irradiation clearly induced a phototropic response in the phy3-expressing transgenic seedlings, and this effect was fully inhibited by simultaneous irradiation with far-red light. In addition, experiments using amino acid-substituted phy3 indicated that FMN-cysteinyl adduct formation in the light, oxygen, voltage (LOV) domain was not necessary for the induction of blue light-dependent phototropism by phy3. We thus demonstrate that phy3 is the phytochrome that mediates the blue/far-red reversible phototropic response in Adiantum. Furthermore, our results imply that phy3 can function as a phototropin, but that it acts principally as a phytochrome that mediates both the red/far-red and blue/far-red light responses.

Phylogenetic relationships of Chinese Adiantum based on five plastid markers.

Adiantum consists of about 150-200 species mostly with a pantropical distribution, yet the classifications of Adiantum have been based primarily on regional studies. Confounding the clarity of reconstructing the evolutionary history of Adiantum is that previous molecular phylogenetic studies suggest that a separate and distinctive clade, the vittarioids, may be derived from within Adiantum. Five plastid markers (atpA, atpB, rbcL, trnL-F and rps4-trnS) are employed to assess the monophyly of Adiantum, and construct the molecular phylogeny of Chinese Adiantum. Our analyses support the monophyly of Adiantum. All temperate Adiantum species form a clade nested within the pantropical grade, suggesting a tropical origin of Adiantum. Six main clades are supported within Chinese Adiantum, which are only partially consistent with Lin's classification of the genus. Series Caudata is polyphyletic with series Gravesiana nested within one subgroup of series Caudata. The prolonged whip-like stolon at the apex of the fronds is the defining character for series Caudata, but it may have evolved multiple times. Adiantum reniforme with the simple fronds is sister to series Venusta, which has a decompound lamina with many flabellate to cuneate segments. Series Veneri-capilliformia is not monophyletic, with A. capillus-veneris sister to series Flabellulata except for A. diaphanum, and A. edentulum sister to series Pedata. Series Flabellulata is biphyletic with A. diaphanum nested within the pantropical grade. The phylogeny suggests that convergent evolution in frond architecture has occurred in Adiantum.

Polyphyly of the fern family Tectariaceae sensu Ching: insights from cpDNA sequence data.

Tectariaceae are a pantropical fern family of about 20 genera, among which 8 are distributed in China. The morphological distinctiveness of the family is widely recognized, yet relatively little systematic research has been conducted on members of Tectariaceae. Phylogenetic analyses of chloroplast DNA sequence data (rbcL and atpB) from 15 species representing all 8 genera in China were carried out under parsimony criteria and Bayesian inference. The phylogenetic reconstructions indicated that the fern family Tectariaceae as traditionally circumscribed are polyphyletic. Ctenitis, Dryopsis, Lastreopsis clustered with and should be included within the newly-defined Dryopteridaceae, and Pleocnemia is also tentatively assigned to it. A narrowly monophyletic Tectariaceae is identified, which includes Ctenitopsis, Hemigramma, Pteridrys, Quercifilix, and Tectaria. In the single rbcL analysis, Arthropteris clustered with the above-mentioned monophyletic Tectariaceae. Although further investigations are still needed to identify infrafamilial relationships within the monophyletic Tectariaceae and to redefine several problematic genera, we propose a working concept here that better reflects the inferred evolutionary history of this group.

The evolution of chloroplast RNA editing.

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.

The fern Adiantum capillus-veneris lacks stomatal responses to blue light.

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.