ZmNF-YB16 is a basic NF-YB superfamily member and a member of a transcription factor complex composed of NF-YA, NF-YB, and NF-YC in maize. ZmNF-YB16 was transformed into the inbred maize line B104 to produce homozygous overexpression lines. ZmNF-YB16 overexpression improves dehydration and drought stress resistance in maize plants during vegetative and reproductive stages by maintaining higher photosynthesis and increases the maize grain yield under normal and drought stress conditions. Based on the examination of differentially expressed genes between the wild-type (WT) and transgenic lines by quantitative real time PCR (qRT-PCR), ZmNF-YB16 overexpression increased the expression of genes encoding antioxidant enzymes, the antioxidant synthase, and molecular chaperones associated with the endoplasmic reticulum (ER) stress response, and improved protection mechanism for photosynthesis system II. Plants that overexpression ZmNF-YB16 showed a higher rate of photosynthesis and antioxidant enzyme activity, better membrane stability and lower electrolyte leakage under control and drought stress conditions. These results suggested that ZmNF-YB16 played an important role in drought resistance in maize by regulating the expression of a number of genes involved in photosynthesis, the cellular antioxidant capacity and the ER stress response.

High-resolution and automated image analysis of individual roots demonstrated that endogenous nitric oxide (NO) contribute significantly to gravitropism of Arabidopsis roots. Lowering of endogenous NO concentrations strongly reduced and even reversed gravitropism, resulting in upward bending, without affecting root growth rate. Notably, the asymmetric accumulation of NO along the upper and lower sides of roots correlated with a positive gravitropic response. Detection of NO by the specific DAF-FM DA fluorescent probe revealed that NO was higher at the lower side of horizontally-oriented roots returning to initial values 2 h after the onset of gravistimulation. We demonstrate that NO promotes plasma membrane re-localization of PIN2 in epidermal cells, which is required during the early root gravitropic response. The dynamic and asymmetric localization of both auxin and NO is critical to regulate auxin polar transport during gravitropism. Our results collectively suggest that, although auxin and NO crosstalk occurs at different levels of regulation, they converge in the regulation of PIN2 membrane trafficking in gravistimulated roots, supporting the notion that a temporally and spatially coordinated network of signal molecules could participate in the early phases of auxin polar transport during gravitropism.

Soybean (Glycine max L. Merr.) white mold (SWM), caused by Sclerotinia sclerotiorum (Lib) de Barry), is a devastating fungal disease in the Upper Midwest of the United States and southern Canada. Various methods exist to evaluate for SWM resistance and many quantitative trait loci (QTL) with minor effect governing SWM resistance have been identified in prior studies. This study aimed to predict field resistance to SWM using low-cost and efficient greenhouse inoculation methods and to confirm the QTL reported in previous studies. Three related but independent studies were conducted in the field, greenhouse, and laboratory to evaluate for SWM resistance. The first study evaluated 66 soybean plant introductions (PIs) with known field resistance to SWM using the greenhouse drop-mycelium inoculation method. These 66 PIs were significantly (P < 0.043) different for resistance to SWM. However, year was highly significant (P < 0.00001), while PI x year interaction was not significant (P < 0.623). The second study compared plant mortality (PM) of 35 soybean breeding lines or varieties in greenhouse inoculation methods with disease severity index (DSI) in field evaluations. Moderate correlation (r) between PM under drop-mycelium method and DSI in field trials (r = 0.65, p < 0.0001) was obtained. The PM under spray-mycelium was also correlated significantly with DSI from field trials (r = 0.51, p < 0.0018). Likewise, significant correlation (r = 0.62, p < 0.0001) was obtained between PM across greenhouse inoculation methods and DSI across field trials. These findings suggest that greenhouse inoculation methods could predict the field resistance to SWM. The third study attempted to validate 33 QTL reported in prior studies using seven populations that comprised a total of 392 F4 : 6 lines derived from crosses involving a partially resistant cultivar "Skylla," five partially resistant PIs, and a known susceptible cultivar "E00290." The estimates of broad-sense heritability (h2) ranged from 0.39 to 0.66 in the populations. Of the seven populations, four had h2 estimates that were significantly different from zero (p < 0.05). Single marker analysis across populations and inoculation methods identified 11 significant SSRs (p < 0.05) corresponding to 10 QTL identified by prior studies. Thus, these five new PIs could be used as new sources of resistant alleles to develop SWM resistant commercial cultivars.

Mychonastes afer HSO-3-1 is a potential producer of nervonic acid, which could be accumulated to 2-3% of dry cell weight. Improving the productivity of nervonic acid is critical to promote the commercialization of this product. In this study, 1-naphthylacetic acid (NAA) and tea polyphenol (TP) were selected as bioactive additives to stimulate the growth of M. afer. Supplementing NAA in the early growth stage and TP in the middle and late growth stage led to improved lipid accumulation in M. afer. The cultures supplemented with TP at the late growth stage maintained higher photosynthetic efficiency than the control groups without TP. Furthermore, the intracellular reactive oxygen species (ROS) accumulations in M. afer supplemented with 500 mg/L of TP was 63% lower than the control group. A linear relationship (R2= 0.899) between the values of Fv/Fm and ROS accumulation was established. We hypothesize supplement of bioactive additives at different growth stage could promote the cell growth rate and nervonic acid productivity of M. afer by retrieving intracellular ROS level. Further analysis of photosynthetic system II (PSII) protein in M. afer cultured in presence of NAA and TP indicated the levels of D1 and D2 proteins, the core skeleton proteins of PSII, showed 33.3 and 25.6% higher than the control group. CP43 protein, a critical module in PSII repair cycle, decreased significantly. These implied that TP possesses the function of slowing down the damage of PSII by scavenging excess intracellular ROS.

Barley stripe rust is caused by Puccinia striiformis f.sp. hordei, (Psh), occurs worldwide, and is a major disease in South Asia. The aim of this work was to identify and estimate effects of loci underlying quantitative resistance to rust at seedling and adult plant stages. HI-AM panel of 261 barley genotypes consisting of released cultivars from North and South America, Europe, Australia, advanced breeding lines, and local landraces from ICARDA barley program were screened at seedling and adult plant stages for resistance to Psh. Seedling resistance was evaluated with the five prevalent Psh races in India. Screening for the adult plant stage resistance was also performed in two different locations by inoculating with a mixture of the five races used for seedling screeing. The panel was genotyped using DaRT-Seq high-throughput genotyping platform. The genome-wide association mapping (GWAM) showed a total of 45 QTL located across the seven barley chromosomes for seedling resistance to the five races and 18 QTL for adult plant stage resistance. Common QTL for different races at seedling stage were found on all chromosomes except on chromosome 1H. Four common QTL associated with seedling and adult plant stage resistance were found on chromosomes 2, 5, and 6H. Moreover, one of the QTL located on the long arm of chromosome 5H showed stable effects across environments for adult plant stage resistance. Several QTL identified in this study were also reported before in bi-parental and association mapping populations studies validating current GWAM. However 15 new QTL were found at adult plant stage on all chromosomes except the 4H, explaining up to 36.79% of the variance. The promising QTL detected at both stages, once validated, can be used for MAS in Psh resistance breeding program globally.

A well-developed root system in rice and other crops can ensure plants to efficiently absorb nutrients and water. Auxin is a key regulator for various aspect of root development, but the detailed molecular mechanisms by which auxin controls crown root development in rice are not understood. We show that overexpression of a YUC gene, which encodes the rate-limiting enzyme in auxin biosynthesis, causes massive proliferation of crown roots. On the other hand, we find that disruption of TAA1, which functions upstream of YUC genes, greatly reduces crown root development. We find that YUC overexpression-induced crown root proliferation requires the presence of the transcription factor WOX11. Moreover, the crown rootless phenotype of taa1 mutants was partially rescued by overexpression of WOX11. Furthermore, we show that WOX11 expression is induced in OsYUC1 overexpression lines, but is repressed in the taa1 mutants. Our results indicate that auxin synthesized by the TAA/YUC pathway is necessary and sufficient for crown root development in rice. Auxin activates WOX11 transcription, which subsequently drives crown root initiation and development, establishing the YUC-Auxin-WOX11 module for crown root development in rice.

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5'-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells.

Elemental selenium is one of the dominant selenium species in soil, but the mechanism of its uptake by plants is still unclear. In this study, nanoparticles of elemental selenium (SeNPs) with different sizes were prepared, and their uptake and transformation in wheat (Triticum aestivum L.) were analyzed in hydroponic experiments by HPLC-ICP-MS. We found that the SeNPs can be absorbed by wheat seedlings, and the process is energy independent. The addition of aquaporins inhibitor caused 92.5 and 93.4% inhibition of chemosynthesized SeNPs (CheSeNPs) and biosynthesized SeNPs (BioSeNPs) absorption by wheat roots, respectively. The 40 nm SeNPs uptake by wheat roots was 1.8-fold and 2.2-fold higher than that of 140 and 240 nm, respectively. The rate of SeNPs uptake in wheat was much slower than that of selenite [Se (IV)], and CheSeNPs were more efficiently absorbed than BioSeNPs. The SeNPs were rapidly oxidized to Se (IV) and converted to organic forms [selenocystine (SeCys2), se-methyl-selenocysteine (MeSeCys), and selenomethionine (SeMet)] after they were absorbed by wheat roots. Additionally, we demonstrated that the aquaporin function in some way is related to the absorption of SeNPs. The particle size and synthesis method of the SeNPs affected their uptake rates by plants. Taken together, our results provide a deep understanding of the SeNPs uptake mechanism in plants.

Nrat1 is a member of the natural resistance-associated macrophage protein (Nramp) family of metal ion transporters in all organisms. Different from other Nramp members capable of transporting divalent metals, Nrat1 specifically transports trivalent aluminum (Al) ion. However, molecular mechanism underlying the Al transport selectivity of Nrat1 remains unknown. Here, we performed structure-function analyses of Nrat1 and other Nramp members to gain insights into the determinants of ion selectivity. A phylogenetic analysis showed that plant Nramp transporters could be divided into five groups. OsNrat1 was found in one of the individual clades and clustered with SbNrat1 and ZmNrat1 on the evolutionary tree. Structural modeling revealed that Nrat1 transporters adopted a common LeuT fold shared by many Nramp-family transporters that likely employed an identical transport mechanism. Sequence alignment and evolutionary conservation analysis of amino acids identified a metal-permeation pathway of Nrat1 centered at the metal binding site. The metal binding site of Nrat1 was characterized by two conserved sequence motifs, i.e., the Asp-Pro-Ser-Asn motif (motif A) and the Ala-Ile-Ile-Thr motif (motif B). Replacement of the Ala-Met-Val-Met motif B of the OsNramp3 manganese (Mn) transporter to that of Nrat1 resulted in a partial gain of Al transport activity and a total loss of Mn in yeast. Conversely, substitution of the motif B of OsNrat1 with that of OsNramp3 altered the Al transport activity. These observations indicated the metal binding site, particularly the motif B, as a key determinant of Al selectivity of Nrat1.

Reference genome sequences have become key platforms for genetics and breeding of the major crop species. Sugarcane is probably the largest crop produced in the world (in weight of crop harvested) but lacks a reference genome sequence. Sugarcane has one of the most complex genomes in crop plants due to the extreme level of polyploidy. The genome of modern sugarcane hybrids includes sub-genomes from two progenitors Saccharum officinarum and S. spontaneum with some chromosomes resulting from recombination between these sub-genomes. Advancing DNA sequencing technologies and strategies for genome assembly are making the sugarcane genome more tractable. Advances in long read sequencing have allowed the generation of a more complete set of sugarcane gene transcripts. This is supporting transcript profiling in genetic research. The progenitor genomes are being sequenced. A monoploid coverage of the hybrid genome has been obtained by sequencing BAC clones that cover the gene space of the closely related sorghum genome. The complete polyploid genome is now being sequenced and assembled. The emerging genome will allow comparison of related genomes and increase understanding of the functioning of this polyploidy system. Sugarcane breeding for traditional sugar and new energy and biomaterial uses will be enhanced by the availability of these genomic resources.

Chemical derivatives of artemisinin, a sesquiterpene lactone produced by Artemisia annua, are the active ingredient in the most effective treatment for malaria. Comprehensive phytochemical analysis of two contrasting chemotypes of A. annua resulted in the characterization of over 80 natural products by NMR, more than 20 of which are novel and described here for the first time. Analysis of high- and low-artemisinin producing (HAP and LAP) chemotypes of A. annua confirmed the latter to have a low level of DBR2 (artemisinic aldehyde Δ11(13) reductase) gene expression. Here we show that the LAP chemotype accumulates high levels of artemisinic acid, arteannuin B, epi-deoxyarteannuin B and other amorpha-4,11-diene derived sesquiterpenes which are unsaturated at the 11,13-position. By contrast, the HAP chemotype is rich in sesquiterpenes saturated at the 11,13-position (dihydroartemisinic acid, artemisinin and dihydro-epi-deoxyarteannunin B), which is consistent with higher expression levels of DBR2, and also with the presence of a HAP-chemotype version of CYP71AV1 (amorpha-4,11-diene C-12 oxidase). Our results indicate that the conversion steps from artemisinic acid to arteannuin B, epi-deoxyarteannuin B and artemisitene in the LAP chemotype are non-enzymatic and parallel the non-enzymatic conversion of DHAA to artemisinin and dihyro-epi-deoxyarteannuin B in the HAP chemotype. Interestingly, artemisinic acid in the LAP chemotype preferentially converts to arteannuin B rather than the endoperoxide bridge containing artemisitene. In contrast, in the HAP chemotype, DHAA preferentially converts to artemisinin. Broader metabolomic and transcriptomic profiling revealed significantly different terpenoid profiles and related terpenoid gene expression in these two morphologically distinct chemotypes.

Alternating glacial and interglacial periods during the Quaternary have dramatically affected the distribution and population genetic structure of plant and animal species throughout the northern hemisphere. Surprisingly, little is known about the post-glacial recolonization history of wetland herbaceous perennials that are widely distributed in the understory of deciduous or mixed deciduous-evergreen forests in eastern North America. In this study, we investigated infraspecific variation among 32 populations of skunk cabbage, Symplocarpus foetidus, to test the hypothesis that the extant species diversity of skunk cabbage is the result of a post-glacial range expansion from southern refugia during the Quaternary Ice Age. A total of 4041 base pairs (bp) of the chloroplast intergenic spacer region (cpDNA) was sequenced from 485 individuals sampled from glaciated (18 populations, 275 individuals) and unglaciated (14 populations, 210 individuals) regions east and west of the Appalachian Mountains. Haplotype number, haplotype diversity, and nucleotide diversity were calculated, and genetic variation within and among populations was assessed by analysis of molecular variance (AMOVA). The geographic pattern of genetic differentiation was further investigated with a spatial analysis of molecular variance (SAMOVA). A total of eight haplotypes and three genetic groups (SAMOVA) were recovered and a much higher haplotype number (eight haplotypes) and haplotype diversity (0.7425) was observed in unglaciated compared to glaciated populations (five haplotypes, haplotype diversity = 0.6099). All haplotypes found in glaciated regions represented a subset of haplotypes found in unglaciated regions. Haplotypes of S. foetidus likely diverged during the Tertiary (mid-Miocene and late Pliocene), predating the last glacial maximum (LGM). Predictions based on ecological niche modeling (ENM) suggested that there was considerably less suitable habitat for skunk cabbage during the LGM, and the habitat range was further south compared to the current distribution. Reduced variation and a subset of haplotypes in glaciated regions suggest a founder effect associated with range expansion via long-distance seed dispersal. Our results do not support the "Driftless Area" scenario for the northern refugium, rather the data suggest a "Northeastern" refugium near the southernmost extent of the LGM.

Avoidance of disadvantageous genetic correlations among growth duration and yield traits is critical in developing crop varieties that efficiently use light and energy resources and produce high yields. To understand the genetic basis underlying the correlations among heading date and three major yield traits in rice, we investigated the four traits in a diverse and representative core collection of 266 cultivated rice accessions in both long-day and short-day environments, and conducted the genome-wide association study using 4.6 million single nucleotide polymorphisms (SNPs). There were clear positive correlation between heading date and grain number per panicle, and negative correlation between grain number per panicle and panicle number, as well as different degrees of correlations among other traits in different subspecies and environments. We detected 47 pleiotropic genes in 15 pleiotropic quantitative trait loci (pQTLs), 18 pleiotropic genes containing 37 pleiotropic SNPs in 8 pQTLs, 27 pQTLs with r2 of linkage disequilibrium higher than 0.2, and 39 pairs of interactive genes from 8 metabolic pathways that may contribute to the above phenotypic correlations, but these genetic bases were different for correlations among different traits. Distributions of haplotypes revealed that selection for pleiotropic genes or interactive genes controlling different traits focused on genotypes with weak effect or on those balancing two traits that maximized production but sometimes their utilization strategies depend on the traits and environment. Detection of pQTLs and interactive genes and associated molecular markers will provide an ability to overcome disadvantageous correlations and to utilize the advantageous correlations among traits through marker-assisted selection in breeding.

Targeting and translocation of proteins to the appropriate subcellular compartments are crucial for cell organization and function. Newly synthesized proteins are transported to mitochondria with the assistance of complex targeting sequences containing either an N-terminal pre-sequence or a multitude of internal signals. Compared with experimental approaches, computational predictions provide an efficient way to infer subcellular localization of a protein. However, it is still challenging to predict plant mitochondrially localized proteins accurately due to various limitations. Consequently, the performance of current tools can be improved with new data and new machine-learning methods. We present MU-LOC, a novel computational approach for large-scale prediction of plant mitochondrial proteins. We collected a comprehensive dataset of plant subcellular localization, extracted features including amino acid composition, protein position weight matrix, and gene co-expression information, and trained predictors using deep neural network and support vector machine. Benchmarked on two independent datasets, MU-LOC achieved substantial improvements over six state-of-the-art tools for plant mitochondrial targeting prediction. In addition, MU-LOC has the advantage of predicting plant mitochondrial proteins either possessing or lacking N-terminal pre-sequences. We applied MU-LOC to predict candidate mitochondrial proteins for the whole proteome of Arabidopsis and potato. MU-LOC is publicly available at http://mu-loc.org.

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.

The occurrence of algal blooms in drinking water sources and recreational water bodies have been increasing and causing severe environmental problems worldwide, particularly when blooms dominated by Microcystis spp. Bloom prediction and early warning mechanisms are becoming increasingly important for preventing harmful algal blooms in freshwater ecosystems. Chlorophyll fluorescence parameters (CFpars) have been widely used to evaluate growth scope and photosynthetic efficiency of phytoplankton. According to our 2-year monthly monitor datasets in Lake Erhai, a simple but convenient method was established to predict Microcystis blooms and algal cell densities based on a CFpar representing maximal photochemical quantum yield of Photosystems II (PSII) of algae. Generalized linear mixed models, used to identify the key factors related to the phytoplankton biomass in Lake Erhai, showed significant correlations between Chl a concentration and both the light attenuation coefficient and water temperature. We fitted seasonal trends of CFpars (Fv/Fm and ΔF/Fm') and algal cell densities into the trigonometric regression to predict their seasonal variations and the autocorrelation function was applied to calculate the time lag between them. We found that the time lag only existed between Fv/Fm from blue channel and algal cell densities even both Fv/Fm and ΔF/Fm' show the significant non-linear dynamics relationships with algal cell densities. The peak values of total algal cell density, cyanobacteria density and Microcystis density followed the foregoing peak value of Fv/Fm from blue channel with a time lagged around 40 days. Therefore, we could predict the possibilities of Microcystis bloom and estimate the algal cell densities in Lake Erhai ahead of 40 days based on the trends of Fv/Fm values from blue channel. The results from our study implies that the corresponding critical thresholds between Fv/Fm value and bloom occurrence, which might give new insight into prediction of cyanobacteria blooms and provide a convenient and efficient way for establishment of early warning of cyanobacteria bloom in eutrophic aquatic ecosystems.

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant-endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant-endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype.

Papaya has been reported to elicit IgE-mediated hypersensitivity via pollen inhalation and fruit consumption. Certain papaya sensitive patients with food allergy were found to experience recurrent respiratory distresses even after quitting the consumption of fruits. This observation prompted us to investigate the allergens commonly present in fruits and pollen grains of papaya. A discovery approach consisting of immunoproteomic detection followed by molecular characterization led to the identification of a novel papaya allergen designated as Cari p 1. This allergen was detected as a 56 kDa IgE-reactive protein from pollen as well as fruit proteome through serological analysis. The protein was identified as an endopolygalacturonase by tandem mass spectrometry. Full length Cari p 1 cDNA was isolated from papaya pollen, cloned in expression vector, and purified as recombinant allergen. The recombinant protein was monomeric and displayed pectinolytic activity. Recombinant Cari p 1 reacted with IgE-antibodies of all the papaya sensitized patient sera. In addition to IgE-reactivity, rCari p 1 displayed allergenic activity by stimulating histamine release from IgE-sensitized granulocytes. CD-spectroscopy of rCari p 1 revealed the presence of predominantly β-sheet characters. The melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational IgE-epitopes characteristic of inhalant allergens in addition to the linear IgE-epitopes of food allergens. The expression of this allergen in papaya fruits was detected by immunoblot with anti-Cari p 1 rabbit IgG and reconfirmed by PCR. In an in vivo mouse model, rCari p 1 exhibited a comparable level of inflammatory responses in the lung and duodenum tissues explaining the dual role of Cari p 1 allergen in respiratory sensitization via pollen inhalation and sensitization of gut mucosa via fruit consumption. Purified rCari p 1 can be used a marker allergen for component-resolved molecular diagnosis. Further immunological studies on Cari p 1 are warranted to design immunotherapeutic vaccine for the clinical management of papaya allergy.

Soybean (Glycine max) seed yields rely on the efficiency of photosynthesis, which is poorly understood in soybean. Chlorophyll, the major light harvesting pigment, is crucial for chloroplast biogenesis and photosynthesis. Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX in the first committed and key regulatory step of chlorophyll biosynthesis. It consists of three types of subunits, ChlI, ChlD, and ChlH. To gain a better knowledge of chlorophyll biosynthesis in soybean, we analyzed soybean Mg-chelatase subunits and their encoding genes. Soybean genome harbors 4 GmChlI genes, 2 GmChlD genes, and 3 GmChlH genes, likely evolved from two rounds of gene duplication events. The qRT-PCR analysis revealed that GmChlI, GmChlD, and GmChlH genes predominantly expressed in photosynthetic tissues, but the expression levels among paralogs are different. In silicon promoter analyses revealed these genes harbor different cis-regulatory elements in their promoter regions, suggesting they could differentially respond to various environmental and developmental signals. Subcellular localization analyses illustrated that GmChlI, GmChlD, and GmChlH isoforms are all localized in chloroplast, consistent with their functions. Yeast two hybrid and bimolecular fluorescence complementation (BiFC) assays showed each isoform has a potential to be assembled into the Mg-chelatase holocomplex. We expressed each GmChlI, GmChlD, and GmChlH isoform in Arabidopsis corresponding mutants, and results showed that 4 GmChlI and 2 GmChlD isoforms and GmChlH1 could rescue the severe phenotype of Arabidopsis mutants, indicating that they maintain normal biochemical functions in vivo. However, GmChlH2 and GmChlH3 could not completely rescue the chlorotic phenotype of Arabidopsis gun5-2 mutant, suggesting that the functions of these two proteins could be different from GmChlH1. Considering the differences shown on primary sequences, biochemical functions, and gene expression profiles, we conclude that the paralogs of each soybean Mg-chelatase subunit have diverged more or less during evolution. Soybean could have developed a complex regulatory mechanism to control chlorophyll content to adapt to different developmental and environmental situations.

Although cytoskeleton is a driving force for cell division and growth in higher plants, there is little evidence about its components in parasitic angiosperms. Microtubules and actin filaments in cells of shoot apical meristem and root-like structure of stem holoparasites European (C. europaea L.) and Eastern (C. monogyna Vahl.) dodders, as well as in prehaustorium, the specific organ adapted to parasitism, were visualized for the first time by immunolabeling and fluorescence microscopy. The significance of cytoskeletal elements during germination and prehaustorium formation was addressed by treatments with taxol, oryzalin, latrunculin B, cytochalasin B/D, jasplakinolide, and 2,3-butanedione monoxime. In shoot apical meristem many dividing cells were observed, in contrast to root-like structure, devoid of cell divisions. Cortical microtubules were oriented transversely and/or obliquely, while actin filaments were randomly distributed in cells of both organs. Furthermore, longitudinal cortical microtubules were present in digitate cells of prehaustorium, and transverse arrays were found in its file cells. Long and short random actin filaments were also observed in prehaustorium cells. Thus, it was shown that the cytoskeleton in dodder shoot cells is organized in a similar way to non-parasitic dicots, while cytoskeletal organization has some peculiarities in quickly senescing root-like structure and prehaustorium.