Over-expression of the <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> harpin-encoding gene <Emphasis Type="Italic">hrpZm</Emphasis> confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T₂–T₄ generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.     

Identification and expression pattern analysis of the glucosinolate biosynthetic gene <Emphasis Type="Italic">BoCYP83B1</Emphasis> from broccoli

Glucosinolates are a branch of amino acid-derived metabolites, which are specifically found in Brassicales. In Arabidopsis, tryptophan derived indolic glucosinolates are required for plant defense against a wide range of pathogens and herbivores due to their strong antimicrobial activity and potential signaling function. An important enzyme in indolic glucosinolate biosynthesis pathway is CYP83B1, which oxidizes indole-3-acetaldoxime, a precursor of indole-3-acetic acid (IAA). In this study, we reported isolation and expression characterization of a CYP83B1 gene from Brassica oleracea L. var. italica Plenck, which we termed BoCYP83B1. Overexpression of BoCYP83B1 in Arabidopsis resulted in an altered glucosinolate profile and early flowering phenotype. By expressing the reporter gene β-glucuronidase under the control of the BoCYP83B1 promoter in Arabidopsis, we analyzed the spatial expression pattern of BoCYP83B1 under normal growth conditions as well as in response to several hormones and stresses. The BoCYP83B1 was primarily expressed in vascular tissue through the almost whole plant. It was strongly induced by methyl jasmonate, 1-amino-1-cyclopropanecarboxylic acid, salicylic acid (SA), gibberellin, and IAA, suggesting its involvement in complex signaling pathways. Mannitol, NaCl, UV, and Flagelin 22 significantly up-regulated BoCYP83B1 expression, indicating its possible role in stress response. Interestingly, the response of BoCYP83B1 to SA and NaCl showed tissue specificity. Thus, BoCYP83B1 might have different functions in different tissues.     

Association analysis of alcohol metabolizing enzymes <Emphasis Type="Italic">ADH1B,ADH7, CYP2E1</Emphasis> gene polymorphism with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5′-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B ^* G (+MslI) allele, CYP2E1 ^* C2 (+PstI) allele and CYP2E1 ^* C (-DraI) allele were 8.48 ± 1.86, 1.20 ± 0.69, and 10.00 ± 1.90%, respectively. The ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7 ^* C (-StyI) allele was 44.58 ± 3.21%. A significantly higher frequency of CYP2E1 PstI C2 allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03–20.01). The tendency to significant effect of A1A2 genotype in ADH1B MslI polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0.042) were also revealed. Association of A1A2 genotype in ADH1B MslI polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female <Emphasis Type="Italic">Heterodera glycines</Emphasis> cyst formation during infection of <Emphasis Type="Italic">Glycine max</Emphasis>

Host-mediated (hm) expression of parasite genes as tandem inverted repeats was investigated as a means to abrogate the formation of mature Heterodera glycines (soybean cyst nematode) female cysts during its infection of Glycine max (soybean). A Gateway^®-compatible hm plant transformation system was developed specifically for these experiments in G. max. Three steps then were taken to identify H. glycines candidate genes. First, a pool of 150 highly conserved H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode Caenorhabditis elegans were identified. Second, annotation of those 150 genes on the Affymetrix^® soybean GeneChip^® allowed for the identification of a subset of 131 genes whose expression could be monitored during the parasitic phase of the H. glycines life cycle. Third, a microarray analyses identified a core set of 32 genes with induced expression (>2.0-fold, log base 2) during the parasitic stages of infection. H. glycines homologs of small ribosomal protein 3a and 4 (Hg-rps-3a [accession number CB379877] and Hg-rps-4 [accession number CB278739]), synaptobrevin (Hg-snb-1 [accession number BF014436]) and a spliceosomal SR protein (Hg-spk-1 [accession number BI451523.1]) were tested for functionality in hm expression studies. Effects on H. glycines development were observed 8 days after infection. Experiments demonstrated that 81–93% fewer females developed on transgenic roots containing the genes engineered as tandem inverted repeats. The effect resembles RNA interference. The methodology has been used here as an alternative approach to engineer resistance to H. glycines.     

CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation

This study aimed to explore the regulatory mechanism of metabolism of xenobiotics by cytochrome P450 during the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) and consummate the induction differentiation system of chicken embryonic stem cells (cESCs) into SSCs in vitro. We performed RNA-Seq in highly purified male ESCs, male primordial germ cells (PGCs), and SSCs that are associated with the male germ cell differentiation. Thereinto, the metabolism of xenobiotics by cytochrome P450 was selected and analyzed with Venny among male ESC vs male PGC, male PGC vs SSC, and male ESC vs SSC groups and several candidates differentially expressed genes (DEGs) were excavated. Finally, quantitative real-time PCR (qRT-PCR) detected related DEGs under the condition of retinoic acid (RA) induction in vitro, and the expressions were compared with RNA-Seq. By knocking down CYP1A1, we detected the effect of CYP1A1-mediated metabolism of xenobiotics by cytochrome P450 on male germ cell differentiation by qRT-PCR and immunocytochemistry. Results showed that 17,742 DEGs were found during differentiation of ESCs into SSCs and enriched in 72 differently significant pathways. Thereinto, the metabolism of xenobiotics by cytochrome P450 was involved in the whole differentiation process of ESCs into SSCs and several candidate DEGs: CYP1A1, CYP3A4, CYP2D6, ALDH3B1, and ALDH1A3 were expressed with the same trend with RNA-Seq. Knockdown of CYP1A1 caused male germ cell differentiation under restrictions. Our findings showed that the metabolism of xenobiotics by cytochrome P450 was significantly different during the process of male germ cell differentiation and was persistently activated when we induced cESCs to differentiate into SSCs with RA in vitro, which illustrated that the metabolism of xenobiotics by cytochrome P450 played a crucial role in the differentiation process of ESCs into SSCs.     

CYP701A26 is characterized as an <Emphasis Type="Italic">ent</Emphasis>-kaurene oxidase with putative involvement in maize gibberellin biosynthesis

Objectives

To characterize the ent-kaurene oxidase (KO) involved in maize (Zea mays) gibberellin (GA) biosynthesis.

Results

Two putative KO genes were identified in maize based on the homologous alignment. Biochemical characterization indicated that one of them encoded a cytochrome P450 monooxygenase (P450) CYP701A26, which reacted with ent-kaurene to form ent-kaurenoic acid, the key intermediate of GA biosynthesis. CYP701A26 showed constitutive expression in active growing tissues and no inducible expression, which led to putative designation of CYP701A26 as the ZmKO. CYP701A26 exhibited substrate promiscuity to catalyze oxidation of other labdane related diterpenes. Another maize KO homologue, CYP701A43 did not show any catalytic activities on ent-kaurene or other tested diterpenes. It exhibited inducible gene expression and might accept unknown substrates to play roles in specialized metabolism for stress response.

Conclusions

CYP701A26 was characterized to exhibit ent-kaurene oxidase activity with substrate promiscuity and might be involved in maize GA biosynthesis, and its homologue CYP701A43 did not show such function and might play roles in stress response.

     

Contrasting hydraulic strategies in <Emphasis Type="Italic">Salix psammophila</Emphasis> and <Emphasis Type="Italic">Caragana korshinskii</Emphasis> in the southern Mu Us Desert,China

Salix psammophila and Caragana korshinskii are two common shrubs in the southern Mu Us Desert, China. Their hydraulic strategies for adapting to this harsh, dry desert environment are not yet clear. This study examined the hydraulic transport efficiency, vulnerability to cavitation, and daily embolism refilling in the leaves and stems of these two shrubs during the dry season. In order to gain insight into water use strategies of whole plants, other related traits were also considered, including daily changes in stomatal conductance, leaf mass per area, leaf pressure–volume parameters, wood density and the Huber value. The leaves and stems of S. psammophila had greater hydraulic efficiency, but were more vulnerable to drought-induced hydraulic dysfunction than C. korshinskii. The difference between leaf and stem water potential at 50 % loss of conductivity was 0.12 MPa for S. psammophila and 0.81 MPa for C. korshinskii. Midday stomatal conductance decreased by 74 % compared to that at 8:30 in S. psammophila, whereas no change occurred in C. korshinskii. Daily embolism and refilling occurred in the stems of S. psammophila and leaves of C. korshinskii. These results suggest that a stricter stomatal regulation, daily embolism repair in stems, and a higher stem water capacitance could be partially compensating for the greater susceptibility to xylem embolism in S. psammophila, whereas higher leaf elastic modulus, greater embolism resistance in stems, larger difference between leaf and stem hydraulic safety, and drought-induced leaf shedding in C. korshinskii were largely responsible for its more extensive distribution in arid and desert steppes.     

<Emphasis Type="Italic">GmDim1</Emphasis> Gene Encodes Nucleolar Localized U5-Small Nuclear Ribonucleoprotein in <Emphasis Type="Italic">Glycine max</Emphasis>

The dim1⁺ gene family is essential for G2/M transition during mitosis and encodes a small nuclear ribonucleoprotein that functions in the mRNA splicing machinery of eukaryotes. However, the plant homolog of DIM1 gene has not been defined yet. Here, we identified a gene named GmDim1 positioned on chromosome 9 of soybean (Glycine max (L.) Merr.) with 80% homology to other eukaryotic dim1⁺ family genes. A domain of soybean DIM1 protein was primarily conserved with U5 snRNP protein family and secondarily aligned with mitotic DIM1 protein family. The GmDim1 gene was expressed constitutively in all soybean organs. The transgenic Arabidopsis thaliana (L.) plants overexpressing GmDim1 showed early flowering and stem elongation, produced multiple shoots and continued flowering after the post-flowering stage. DIM1 proteins transiently expressed in onion cells were localized in the nucleus with dense deposition in the nucleolus. Therefore, we propose that the soybean GmDim1 gene is a component of plant U5 snRNP involved in mRNA splicing and normal progress of plant growth.