Both epiphytic and endophytic strains of <Emphasis Type="Italic">Rhodococcus fascians</Emphasis> influence transporter gene expression and cytokinins in infected <Emphasis Type="Italic">Pisum sativum</Emphasis> L. seedlings

Some strains of the soil bacterium Rhodococcus fascians maintain an epiphytic life style while others become endophytic. Virulent, endophytic strains cause multiple shoot growth and inhibit root growth of seed-inoculated Pisum sativum L. We were interested in assessing, at the molecular level, the impact of strains of contrasting niche on the emerging shoots and roots of inoculated seeds. The presence of R. fascians was monitored microscopically, endogenous cytokinin and chlorophyll levels were measured, and the expression of genes monitored by RT-qPCR. The expression of the pea sugar transporter genes (SWEET and SUT), amino acid (AAP) transporters and cell wall invertase gene family members, as well as expression of plant and bacterial cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) genes were monitored. Both the virulent strain and the epiphytic strain affected the expression of the transporter genes, with less obvious differences between the strains on the shoot compared with the effect on the root. Strong expression of the R. fascians genes, RfIPT, RfLOG and RfCKX, in pea seedlings at 15 days post inoculation was mirrored by increased expression of transporter gene family members in the plant. However, the elevated levels of isopentenyl adenine-type and zeatin-type cytokinins were not consistently associated with the virulent strain. In conclusion, while both the virulent strain and the epiphytic strain impacted the expression of transporter genes in the shoots and roots, only the virulent strain affected morphology. The inhibited root growth, the greening of the roots, and the expression of the pea response regulators in the infected roots are indicative of a response to cytokinin, but a role for the ‘classical’ cytokinins as virulence determinants was not established.     

Expression analysis of steroid pathway genes revealed positive correlation with diosgenin biosynthesis in <Emphasis Type="Italic">Trillium govanianum</Emphasis>

Trillium govanianum Wall. ex D. Don, popularly known as Nagchhatri, is an important medicinal plant of northwestern Himalayas. No data exist on molecular characteristics of diosgenin biosynthesis, the main chemical component of T. govanianum. HPLC analysis revealed diosgenin content of 2.4 and 0.7% in hydrolyzed rhizome extracts of Chamba and Lahaul and Spiti, respectively. Quantitative real-time PCR analysis of five genes of steroid pathway in the rhizomes of two locations, differing for diosgenin content showed up to 3.2-fold increase in expression level of genes viz. hydroxymethylglutaryl Co-A reductase (HMGR), farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), 26-O-beta-glucosidase (BETA) and cycloartenol synthase (CAS) in rhizomes of Chamba (2.4%) as compared to Lahaul and Spiti (0.7%), thus inferring their role in diosgenin biosynthesis. The present study delivers the first report where an elite chemotype of T. govanianum for diosgenin content was identified and partial sequences of five genes of steroid pathway were cloned and investigated for their potential role in diosgenin biosynthesis in T. govanianum. The result of this study has prospective applications in the genetic improvement of this medicinally important plant species.     

Benzalkonium chloride tolerance of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> strains isolated from a meat processing facility is related to presence of plasmid-borne <Emphasis Type="Italic">bcr</Emphasis>ABC cassette

Listeria monocytogenes is a serious foodborne pathogen capable of persisting in food processing environments. Tolerance to disinfectants used in industrial settings constitutes an important factor of Listeria survival. In the present study, the mechanism of tolerance to benzalkonium chloride (BAC) was investigated in 77 L. monocytogenes isolates from a meat facility. By PCR approach, the mdrL and lde chromosomal efflux pump genes were detected in all isolates. No isolate was positive for qacH and emrE genes. However, the bcrABC cassette was present in 17 isolates of serogroup IIa possessing the same AscI/ApaI pulsotype, the operon being localized on a plasmid. The significant relation of BAC tolerance with bcrABC presence was confirmed as all bcrABC positive isolates showed the highest minimal inhibitory concentration (MIC) values for BAC and increased sensitivity to BAC was observed after plasmid curing. No effect of the efflux pump inhibitor reserpine on BAC tolerance in bcrABC positive strains was observed in contrast to all bcrABC negative strains. Lower ethidium bromide efflux in bcrABC positive isolates compared to bcrABC negative and plasmid-cured L. monocytogenes isolates was observed. The expression of bcrABC genes was BAC-induced. The confirmed effect of bcrABC to increased BAC tolerance, coupled with its plasmid location, may be an important factor in potential dissemination of the biocide resistance among Listeria species. The understanding of molecular mechanisms of biocide tolerance should help to improve control measures to prevent further spread of L. monocytogenes in food production environments with frequent use of BAC.     

A reexamination on the deficiency of riboflavin accumulation in Malpighian tubules in larval translucent mutants of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A variety of insects accumulate high contents of riboflavin (vitamin B₂) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3^oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.     

Influence of Sulfur on Transcription of Genes Involved in Arsenic Accumulation in Rice Grains

Arsenic (As) contamination of rice grains affects millions of people worldwide. In this study, we found that sulfur application (20As+120S) decreased As concentration in rice grains by 44 % compared to grains without sulfur application (20As+0S). Importantly, sulfur application decreased arsenate [As(V)] and arsenite [As(III)] concentration in rice grains significantly, while there was no significant effect on dimethylarsenate (DMA) concentration. To elucidate the molecular basis of As accumulation in rice grains, we performed Illumina sequencing to acquire the differentially expressed genes induced by arsenate and sulfur treatments. By contrast with the control, the expression of 1,000 genes was found to be changed significantly, with 46 genes up-regulated and 954 genes down-regulated in grains grown in arsenate-contaminated soil (20As+0S). Between samples of control and arsenate together with sulfur treatment (20As+120S), 1,169 genes expressed significantly differently, with 16 genes up-regulated and 1,153 genes down-regulated. Sulfur application significantly changed the expression of genes involved in As metabolism in rice grains, significantly down-regulated phosphate transporter gene OsPT23 and aquaporin gene OsTIP4;2, while ABC transporter genes (OsABCG5, OsABCI7_2 and OsABC6) and phytochelatin synthase genes (OsPCS1, OsPCS3 and OsPCS13) were up-regulated. These results provide an insight into the molecular basis of how sulfur assimilation regulates As accumulation in rice grains.     

Expression of Components of the Serotonergic System in Folliculogenesis and Preimplantation Development in Mice

The functions of serotonin include the growth and development regulation of female germ cells as well as early embryo development. RT-PCR analysis of mRNA expression of the genes of the enzymes for synthesis and degradation and transporters and receptors of serotonin during folliculogenesis and preimplantation development of mice was performed to discover the particular mechanisms of these functions. The mRNA of tryptophan hydroxylase tph1 and monoaminoxidase maoa; membrane transporter sert and vesicular transporter vmat2; and serotonin receptors htr1b, htr1d, htr2a, htr5b, and htr7 were revealed in granulosa cells. The expression of mRNA of the aromatic amino acid decarboxylase ddc and the htr2b receptor additionally appears in the yellow body. The expression of mRNA of the genes of the tph2, ddc, and maoa enzymes; the sert, vmat1, and vmat2 transporters; and quite a number of receptors is observed during the preimplantation development, and it is transitory in most of them. The expression of all components and its dynamics suggest that the serotonergic signaling system is functionally active in mouse folliculogenesis and preimplantation development.     

Resistance Mechanism in a Terbinafine-Resistant Strain of <Emphasis Type="Italic">Microsporum canis</Emphasis>

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2–4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.     

A long term evaluation of differential potassium fertilization of a creeping bentgrass putting green

Aims

Buckwheat (Fagopyrum esculentum) is highly tolerant to Al stress, but the molecular mechanisms remain largely unknown. This study aims to investigate a half-type ABC transporter gene (FeSTAR1) with respect to Al tolerance.

Methods

The expression of FeSTAR1 was examined and complementation test in atstar1 mutant was conducted. Furthermore, Al distribution and cell wall polysaccharides content were analyzed.

Results

FeSTAR1 is an ABC transporter protein with nucleotide binding domain, but lack of transmembrane domain. Consistently, FeSTAR1 is a soluble protein, localizing to both cytoplasm and nucleus. Al rapidly and specifically induced FeSTAR1 expression. Heterologous expression of FeSTAR1 in atstar1 rescued its Al tolerance, and exogenous applied UDP-glucose could alleviate Al sensitivity of atstar1 mutant, suggesting the connection between FeSTAR1 and UDP-glucose in terms of Al tolerance. Furthermore, FeSTAR1 complemented lines accumulated less Al in root cell wall than atstar1 mutant. Further cell wall fraction analysis showed that Al was largely confined to cell wall hemicellulose1, at which Al content was significantly lower in complemented lines. Consistent with Al distribution in different cell wall polysaccharides, complemented lines had lower hemicellulose1 content.

Conclusion

Our results indicate that FeSTAR1 is involved in Al resistance via possibly cell wall matrix polysaccharides metabolism in buckwheat.

     

Expression of the sucrose transporter 3 (<Emphasis Type="Italic">HbSUT3</Emphasis>) in rubber tree and its relation to latex yield

Sucrose is not only a precursor of rubber biosynthesis, but it also participates in other latex regeneration between repeated tapping. Sucrose transporter (HbSUT) genes play key roles in sinks and sources of the rubber biosynthesis pathway. HbSUT3 was dominant in expression in latex among the SUT member genes. Therefore, the expression level of HbSUT3 in latex is a potential indicator distinguishing between high- and low-yielding clones. The aim of this research was to assess the potential of this gene in selective breeding to improve latex yield, from the correlation of HbSUT3 expression with latex yield. Four high-yielding clones were sampled in this study and compared with the common RRIM 600 clone as the baseline, paired by the field. Among the putative full-length cDNAs of Hevea sucrose transporters available in the NCBI database, only HbSUT3 was detected. The HbSUT3 gene was overexpressed in the four rubber clones relative to the control, and the NK1 clone had the highest expression level. The expression level of HbSUT3 correlated positively with latex yield but negatively with the sucrose content of latex. Gene expression analysis of rubber seedlings indicated that the bark had higher expression of this gene than the leaves, and the levels correlated with latex yields of these clones. These data provided new candidate selection criteria for use in the early selection of high-yielding rubber clones, necessary for rapid cycle breeding programs.     

A soybean plastidic ATP/ADP transporter gene, <Emphasis Type="Italic">GmAATP</Emphasis>, is involved in carbohydrate metabolism in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression,and activation of their potential target genes in clear cell renal cell carcinoma

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31–48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher’s exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher’s exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman’s correlation coefficient r _s ranging 0.31–0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (r _s ranging 0.35–0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.