Only the smaller GFP variants moved beyond this zone . To add further complexity to protein trafficking and regulation, phosphorylation, and glycosylation are required for pumpkin CmPP16 to interact and form a stable complex with the mobility-endowing protein, Nt-NCAPP1, prior its phloem trafficking . Discrepancies in observed mobility from one study to another could be attributed to phosphorylation and glycosylation since earlier studies did not take these post-translational, covalent modifications into consideration. Two groups have demonstrated that non-endogenous proteins are retained in the root stock. The Gastrodia antifungal protein expressed by transgenic plum root stocks under the control of the constitutive CaMV35S promoter was identified in roots by immunoblot, but not in the soft shoot or leaf tissues of grafted, WT scions. This suggested that GAFP-1 was not moving into the WT-scion tissues of transgrafted plum trees . In the other example, transgenic watermelon root stocks over-expressing a cucumber mottle mosaic virus coat protein gene were transgrafted with WT watermelon. Protein expression and mRNA levels were detected in the transgenic root stock but not in the non-transgenic scion . Detection limits of the techniques utilized were not reported in either of these studies. A poke weed antiviral protein was expressed in transgenic Nicotiana tabacum root stocks and provided resistance to potato virus X in NN and nn grafted non-transgenic scions. However, the antiviral protein was detected only in the root stocks and not in the grafted scion tissues . The basis for resistance expression in this situation is not clear. Protein translocation from a transgenic root stock to a WTscion will likely depend on the species and/or type of protein in the transgene construct. Should proteins encoded by transgenes manage to migrate to the scion,plants in pots ideas their longevity is a consideration. For example, NPTII and GUS proteins have estimated half-lives of 6–7 min and 36 h, respectively, in planta .
If NPTII were translocated to scions it would be lost rapidly, but the GUS protein would not be reduced to 1% of the initial level accumulated in scions for 10 days. Research on the production of proteins encoded by transgenes in root stocks for delivery to scions arguably is more advanced than analogous work with the use of nucleic acids. For example, researchers at the University of Florida have engineered grape root stocks that deliver hybrid lytic peptides to control bacterial and fungal diseases .Work in our lab has shown that delivery of a protein that inhibits microbial maceration of plant cell walls is possible . While advances to date have focused on delivery of single gene products with specific functions to scions, future advances may target transport of transcription factors that influence expression of multiple genes,which could coordinate concerted scion responses to complex challenges such as pathogens, pests, or abiotic stresses.Proteins that are delivered to and function in the apoplast can provide protection against pathogens, particularly those pathogens that target the cell wall. In many plant–microbe or plant– pathogen interactions, the plant cell walls are a major obstacle to colonization or expansion within plant tissues. To overcome this barrier, most fungal pathogens produce a variety of enzymes, which degrade the host cell wall. Polygalacturonases are often the first enzymes secreted during the infections . PGs cleave α- linkages between d-galacturosyl residues in pectic homogalacturonan, causing cell separation and tissue maceration. Botrytis cinerea expresses six PGs during infection and growth on plant hosts and the PG-inhibiting protein produced in pear fruit , inhibits some but not all of these PGs . Given the importance of PGs in pest and pathogen interactions with plants, it is not surprising that PGIPs are components of the defenses against invasion by pathogens and pests . Tomato foliar and ripe fruit resistance to the fungal pathogen, B. cinerea, is improved about 40% by the constitutive over-expression of pPGIP in tomatoes . The Miridae insect, Lygus hesperus, produces PGs that cause damage to alfalfa and cotton flflorets and PGIPs can inhibit these PGs and may, therefore, reduce the damage to plant tissues . The nematode, Meloidogyne incognitacausing root knot disease expresses PGs , but it is not known if they can be inhibited by PGIPs. PGIPs expressed in root stocks, therefore, are potential anti-pathogen proteins thatcould be delivered from the root stock to the scion in transgrafted plants. Our work has shown that pPGIP expression reduces the effects of Pierce’s Disease in grapevines, caused by the bacterium, Xylella fastidiosabecause it inhibits the X. fastidiosa virulence factor, PG .
As with other vascular pathogens, the X. fastidiosa PG contributes to disease development by digesting the polysaccharides in the pit membranes of the xylem network. When intact, these so-called “membranes” help to prevent the pathogen’s vessel-to-vessel spread from the initial sites of infection of grapevines . Because pPGIP inhibits the X. fastidiosa PG and because pPGIP can enter the xylem, PGIPs in the xylem of both the root stock and the scion could provide protection against other PG-utilizing pathogens in the water transport system. We have observed that when pPGIP-expressing transgenic plants are used as root stocks onto which non-expressing scions are grafted, the pPGIP protein, but not the pPGIP-encoding nucleic acids, are exported to the scion, crossing the graft union via the xylem system . In grafted tomato plants expressing pPGIP in the root stock, pPGIP protein has been detected in scion leaves . Similarly, in grafted grapevines, we have observed the pPGIP protein in the wild-type scion tissue grafted onto pPGIP-expressing root stocks . Furthermore, we have observed that expression of pPGIP in root stocks reduces pathogen damage in scion tissues . Thus, defense factors in roots can be made available to scions via grafting, improving the vigor, quality, and pathogen/pest resistance of the food-producing scion and its crop.DNA barcoding is an effective tool to identify many plant species rapidly and accurately. However, there is no single universal barcode that can be successfully used to identify all plants to the species level. Consequently, two alternative strategies have been proposed to distinguish among plant species: the first one is the use of complete chloroplast genomes, named ‘super-barcoding’, and the second one is an approach that involves searching for mutational hotspots, or using comparative plastid analyses to find loci with suitable species-level divergence. Analyses of entire chloroplast genome sequences provide an effective way to develop both of these strategies. In most angiosperms, the chloroplast genomes are inherited maternally and have a consistent structure, including two inverted repeats , one large and one small single copy region. Te chloroplast genome always contains 110–130 genes that exhibit a range of levels of polymorphism. Thus, chloroplast genome sequence data are extremely valuable for studies of plant population genetics, phylogeny reconstruction, species identification, and genome evolution.
The Ranunculaceae is a large family, which includes approximately 59 genera and 2500 species. Many plants of Ranunculaceae are pharmaceutically important. The genus Pulsatilla Adans. consists of about 40 species which are distributed in temperate subarctic and mountainous areas of the Northern Hemisphere. There are always long, soft hairs covering plants of Pulsatilla species. Most of the fowers of Pulsatilla are large and showy, and therefore the genus has horticultural importance. The fowers are solitary and bisexual. In one flower, there are always six tepals, numerous stamens and carpels, with the outermost stamens resembling degenerated petals, excluding P. kostyczewii. In China, there are eleven species of Pulsatilla. Some species of Pulsatilla have been used in traditional Chinese medicine for many years, such as for “detoxifcation” or “blood-cooling”, because Pulsatilla species contain numerous secondary metabolites, including phytosterols, triterpenoid saponins and anthocyanins. At the same time, all members of Pulsatilla produced the lactone protoanemonin. In Europe, some species of Pulsatilla are rare, endangered and endemic. Those taxa are protected due to their small populations and disappearing localities,container size for blueberries and those species have been placed on the Red Lists of Endangered Species. Taxonomically, Pulsatilla is an especially complex and challenging group. In all treatments published before, three subgenera have been recognized: subgenus Kostyczewianae , subgenus Preonanthus, and the largest subgenus Pulsatilla. However, the intragenic morphological variability of Pulsatilla was especially complicated. Te recognition and identifcation of wild Pulsatilla species is particularly difcult based on traditional approaches. Molecular markers are significant to explore the phylogenetic relationships of the genus Pulsatilla. Phylogenetic relationships between Pulsatilla and closely related genera have been dedicated during the past years. Previous studies have attempted to identify these species among Pulsatilla with universal molecular markers, but the species resolution was relatively low. In this study, we present seven complete cp genomes from two subgenera of Pulsatilla obtained through next-generation sequencing and genomic comparative analyses with four previously published cp genome sequences of Pulsatilla from NCBI, with Anemoclema glaucifolium as the out group. We identify microsatellites , larger repeat sequences, and highly variable regions, with the aim of developing DNA barcodes and testing the feasibility of phylogenetic analyses of Pulsatilla using the chloroplast genome.In most angiosperms, the IR regions of cp genomes of angiosperms are highly conserved, but the expansion and contraction of IR region boundaries are ever present. At the same time, several lineages of land plant chloroplast genomes show great structural rearrangement, even loss of IR regions or some gene families. The expansion and contraction in IRs are significant evolutionary events, because they can change gene content and chloroplast genome size. Expansion of the IRs has been reported in Araceae. Sometimes, the size of LSC increases and that of SSC decreases, becoming only 7000 bp in Pothos. At the same time, a linear chloroplast genome was also reported in some groups, e.g. maize. Expansion and contraction of the IR regions can also lead to duplication of certain genes or conversion of duplicate genes to single copy, respectively. Changes in the size of the IRs can also cause rearrangement of the genes in the SSC as recently observed in Zantedeschia. The Pulsatilla chloroplast genomes were compared to previously published data and showed typical Anemoneae genome structure. As reported for Anemoclema, Anemone, Clematis and Hepatica, the IR regions of genus Pulsatilla are roughly 4.4 kb longer than those of other genera of the family Ranunculaceae, such as Aconitum, Coptis, Talictrum, Megaleranthis, Ranunculus, and Trollius. The gene orders located within the IR-SSC and IR-LSC boundaries are similar among tribe Anemoneae but diferent from those of other genera of Ranunculaceae . We compared the IR/SC boundary regions of Pulsatilla, and the junction positions are very similar and conserved within genus Pulsatilla. In the four boundary regions of seven Pulsatilla cp genomes, the LSC/IRa and IRb/LSC border was in the intergenic region, and the adjacent genes is rps36, rps8 and rps4, respectively. The genes ycf1 andψycf1 have crossed the SSC/IRb and IRa/SSC boundary, respectively, which was also found in Monsteroideae. The pseudogene ycf1 has been found in other groups. The IR regions were highly conserved, with nucleotide diversity values in those regions less than 2%.Chloroplast genome markers, especially several universal chloroplast regions, have been widely used in plant systematics and identifcation at multiple taxonomic levels. Highly suitable polymorphic chloroplast loci have been identifed and designed as unique markers in diferent groups. However, relationships within the genus Pulsatilla have not been well resolved because of the lowpolymorphism of these universal markers. In order to facilitate identification of closely related species of Pulsatilla, we sought to identify highly variable regions of the chloroplast genome, as previously described. As a result, we identified nine divergent hot spot regions, including six intergenic spacer regions and four protein-coding regions. Most commonly employed loci, e.g. trnL-trnF, trnH-psbA were not selected in our finding. The nine highly variable regions included 684 variable sites, including 181 indels. However, these indels are not suitable for the phylogenetic inference because Maximum likelihood model used only substitutions not indels. Their nucleotide diversity values ranged from 0.00802 to 0.02212. The region of ccsA-ndhF showed the highest variability, the next most variable regions were rps4-rps16, ndhC-trnV, and psbE-petL. Te diversity level of two protein-coding regions was the lowest. Among the nine divergent hotspot regions, the ndhI is difcult to align. There are large numbers of indels in ndhI and the intergenic spacer between ndhI and ndhG, these regions were not considered suitable for the phylogenetic inference of the Pulsatilla. Thus, we selected eight regions, four in the LSC and four in the SSC, with relatively high variability as potential molecular markers for the study of species identification and phylogeny in Pulsatilla.