Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

Relationship between bovine oocytes developmental competence and mRNA expression of apoptotic and mitochondrial genes following the change of vitrification temperatures and cryoprotectant concentrations

The present study analyzed the relationship between bovine oocytes developmental competence and mRNA expression of apoptotic and mitochondrial genes following the change of vitrification temperatures (VTs) and cryoprotectant agent concentrations (CPAs). Cumulus oocyte complexes were randomly divided into five groups: control, vitrified in liquid nitrogen (LN; −196 °C) with 5.6 M CPAs (LN 5.6 M), LN with 6.6 M CPAs (LN 6.6 M), liquid helium (LHe; −269 °C) with 5.6 M CPAs (LHe 5.6 M), and LHe with 6.6 M CPAs (LHe 6.6 M). After vitrification and warming, oocytes of vitrified and control groups were subjected to in vitro maturation (IVM), in vitro fertilization and in vitro culture. The blastocyst rate in LHe 5.6 M group was the highest among the four vitrified groups (13.7% vs. 9.4%, 1.3%, and 8.4%; P < 0.05). The mRNA expression level of 8 apoptotic- and 12 mitochondria-related genes were detected through qRT-PCR after IVM. Lower VT (LHe, −269 °C) positively affected the mRNA expression levels of apoptotic genes (BAD, BID, BTK, TP53, and TP53I3) and mitochondrial genes (COX6B1, DERA, FIS1, NDUFA1, NDUFA4, PRDX2, SLC25A5, TFB1M, and UQCRB), and reduced oxidative stress from freezing. Decreased CPAs (5.6 M) positively affected mRNA expression levels of apoptotic genes (BAD, BCL2A1, BID, and CASP3) in LHe vitrification but negatively affected apoptotic genes (BAD, BAX, BID, BTK, and BCL2A1) in LN vitrification. In conclusion, decreased VTs and CPAs in LHe vitrification may increase the blastocyst rate by changing the mRNA expression levels of these apoptotic and mitochondrial genes for the vitrified oocytes.     

Expression of genes involved in the anthocyanin biosynthesis pathway in white and red fruits of Fragaria pentaphylla and genetic variation in the dihydroflavonol-4-reductase gene

Structural and regulatory genes control fruit colors in plants. Real-time quantitative PCR results showed significantly higher expression levels of structural genes (FpCHS, FpDFR, FpANS, and FpUFGT) as well as of the regulatory gene MYB10 in red fruits of Fragaria pentaphylla compared to white fruits. These genes were strongly associated with anthocyanin accumulation within fruits. The full-length sequence of the FpDFR gene in red fruits of F. pentaphylla had a length of 2080 bp, was separated by five introns, and shared 95% homology with the F. vesca DFR sequence. Twenty-seven SNPs were detected in the FpDFR gDNA sequences between red and white fruits. Among these, transition substitutions were more frequent than transversions (66.7% vs. 33.3%), and a larger number of nucleotide variants existed in introns compared to exons (70.4% vs. 29.6%). A Chi-square test showed only three SNPs significantly associated with fruit color. Combined with structural analyses of the FpDFR protein and an expression analysis of the anthocyanin pathway genes, these results indicate that trans-regulation might contribute to color control in F. pentaphylla.     

Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.     

Stability of Expression of Reference Genes Among Different Lentil (Lens culinaris) Genotypes Subjected to Cold Stress, White Mold Disease, and Aphanomyces Root Rot

Precise quantification of differences in gene expression between plants requires the use of “reference” genes, which are stably expressed across different lines and treatments and serve as endogenous controls for normalizing gene expression data. The objectives of this study were to determine the expression stability of several reference genes across five different lentil varieties subjected to either cold stress, inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease, or inoculation with Aphanomyces euteiches, the causal agent of Aphanomyces root rot. Expression stability was examined in the stems and leaves of plants subjected to cold stress or inoculation with S. sclerotiorum and in the roots of plants inoculated with A. euteiches. Real-time PCR assays (SYBR Green) were designed for six different genes: translation initiation factor (TIF), 18S rRNA, actin, β-tubulin-2, β-tubulin-3, and glyceraldehyde 3-phosphate dehydrogenase. TIF, actin, and 18S rRNA tended to be the most stably expressed genes, with expression stability (M) values less than 0.5 during cold stress and inoculation with A. euteiches. Two reference genes were required to normalize data from plants exposed to cold stress or inoculated with A. euteiches. The reference genes exhibited the lowest expression stability in plants inoculated with S. sclerotiorum, for which five reference genes were required to normalize data. The reference genes reported in this study appear to have a promise for examining gene expression in lentil foliar and root tissues in response to diverse abiotic and biotic factors.     

Cellulose synthase genes that control the fiber formation of flax (Linum usitatissimum L.)

Four cellulose synthase genes were identified by analysis of their class-specific regions (CSRII) in plants of fiber flax during the “rapid growth” stage. These genes were designated as LusCesA1, LusCesA4, LusCesA7 and LusCesA9. LusCesA4, LusCesA7, and LusCesA9 genes were expressed in the stem; LusCesA1 and LusCesA4 genes were expressed in the apex part of plants; and the LusCesA4 gene was expressed in the leaves of fiber flax. The expression of the LusCesA7 and LusCesA9 genes was specific to the stems of fiber flax. These genes may influence the quality of the flax fiber.     

Altered Expression of Immune-Related Genes in Children with Down Syndrome

Individuals with Down syndrome (DS) have a high incidence of immunological alterations with increased susceptibility to bacterial and viral infections and high frequency of different types of hematologic malignancies and autoimmune disorders. In the current study, we profiled the expression pattern of 92 immune-related genes in peripheral blood mononuclear cells (PBMCs) of two different groups, children with DS and control children, to identify differentially expressed genes that might be of pathogenetic importance for the development and phenotype of the immunological alterations observed in individuals with DS. PBMCs samples were obtained from six DS individuals with karyotypically confirmed full trisomy 21 and six healthy control individuals (ages 2–6 years). Gene expression was profiled in duplicate according to the manufacturer''s instructions provided by commercially available TaqMan Human Immune Array representing 92 immune function genes and four reference genes on a 96-plex gene card. A set of 17 differentially expressed genes, not located on chromosome 21 (HSA21), involved in immune and inflammatory pathways was identified including 13 genes (BCL2, CCL3, CCR7, CD19, CD28, CD40, CD40LG, CD80, EDN1, IKBKB, IL6, NOS2 and SKI) significantly down-regulated and four genes (BCL2L1, CCR2, CCR5 and IL10) significantly up-regulated in children with DS. These findings highlight a list of candidate genes for further investigation into the molecular mechanism underlying DS pathology and reinforce the secondary effects of the presence of a third copy of HSA21.     

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.     

Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L⁻¹ to 3.16 g L⁻¹, with a molecular weight range of 1.40×10⁶–1.83×10⁶ Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10⁶ U mL⁻¹), the production of HA was substantially increased from 5.96 g L⁻¹ to 19.38 g L⁻¹. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10³–1.42×10⁶ Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.     

Cloning and Expression of Multiple Cytochrome P450 Genes: Induction by Fipronil in Workers of the Red Imported Fire Ant (Solenopsis invicta Buren)

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the “first line of defense” when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL⁻¹ (median lethal dose) and 0.56 μg mL⁻¹ (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant.     

Expression of selected genes involved in cadmium detoxification in tobacco plants grown on a sulphur-amended metal-contaminated field

We investigated the effect of a Cd solubilising soil treatment on the expression of genes regulating Cd uptake and detoxification in field-grown tobacco plants. Tobacco plants were grown on a heavy-metal contaminated soil to which elemental sulphur was applied to increase the phytoextraction of Cd. The expression of tobacco gene homologues for A. thaliana MRP3, PDR8, ATM3, Sultr1, LAST, APR2, APR3, GSHI, GSHII, and NAS3 was assayed by qualitative RT-PCR.Increased root and shoot Cd concentrations were associated with up-regulation of the putative Cd transporters and the genes involved in sulphur assimilation in root tissues. This is consistent with previous studies using hydroponics. However, unlike the previous studies, most of the genes tested in the leaves were unaffected by Cd concentration.These differences may be due to the more complex stress situation that plants experienced here under field conditions. Moreover, unlike in hydroponic studies, our plants were sampled at maturity and not in the seedling stage. Our results indicate that hydroponic or agar experiments are useful predictors of effects that may be expected in the field. However, there is a need for studies investigating gene expression in response to multiple stresses representative for field conditions at later developmental stages.     

Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.