Nia Gene Research Articles

A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.

CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for non-selectable target mutations. This becomes challenging when many sequential modifications are required in a single cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, non-selectable genes. We demonstrate that this 'Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)' technology enables an essentially limitless series of genetic modifications to be introduced into a single cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.

Development and validation of a ddPCR assay to detect and quantify tobacco DNA in smoke and smokeless tobacco and tobacco-free products

The last decade, smoke and smokeless products claiming to be tobacco-free, including herbal cigarettes and herbal shisha, became available on the European market and gained popularity. This study proposes a new digital droplet PCR (ddPCR) method, designed based on a previously developed real-time PCR (qPCR) method being currently used by the U.S. Food and Drug Administration (FDA) to specifically detect the presence of tobacco DNA in targeting a sequence from the Nicotiana tabacum nia-1 gene. To ensure a harmonized and reliable control by enforcement laboratories, both of these qPCR and ddPCR methods were then evaluated and validated for their compliance to an international standard. First, the performance of these PCR-based methods was successfully assessed as specific and sensitive, and in line with minimum performance requirements from international standard. Secondly, the transferability to external laboratory was confirmed for these PCR-based methods. Finally, the applicability of these PCR-based methods was demonstrated using 7 ground tobacco reference materials from the Tobacco Research Center (TRC) Toronto University as well as 6 commercial smokeless and tobacco-free smoke and smokeless products. Based on this study, the previously developed qPCR method was confirmed as complying with international standard, ensuring a efficient and harmonize use by enforcement laboratories for tobacco control on the European market. Moreover, this study proposed to enforcement laboratories the possibility to use a ddPCR method, enabling the simultaneous detection and absolute quantification of tobacco DNA as well as a limited impact of PCR inhibitors.

Enhanced nitrate reductase activity offers Arabidopsis ecotype Landsberg erecta better salt stress resistance than Col-0.

The nitrogen utilization efficiency of plants varies depending on the plant species. In modern agriculture, nitrogen fertilizer is used to increase crop production, with the amount of fertilizer addition increasing steadily worldwide. This study included the two most used ecotypes of Arabidopsis thaliana, Landsberg erecta (Ler) and Col-0, which were used to identify differences at the molecular level. We found that the efficiency of nitrogen utilization and salt stress resistance differed between these two ecotypes of the same species. We demonstrated distinct salt stress resistance between Ler and Col-0 depending on the differences in nitrate level, which was explained by different regulation of the NIA2 gene expression in these two ecotypes. Our results demonstrate that the genes and promoters regulate expression of these genes and contribute to trait differences. Further studies are required on genes and promoter elements for an improved understanding of the salinity stress resistance mechanism in plants.

Low R/FR Ratio Affects Pakchoi’s Growth and Nitrate Content under Excess Nitrate Stress

Nitrate accumulation is one of the main factors of secondary soil salinization in protected horticulture in China. Previous studies have shown that a low red(R)/far-red (FR) ratio can improve the salt tolerance of plants under NaCl stress. However, the effect of a low R/FR ratio on plant growth under nitrate stress is not clear. In order to explore the effect of a low R/FR ratio on the adaptability of pakchoi under high nitrogen stress, the growth index, soluble protein content, soluble sugar content, nitrate content, nitrate reductase activity and Nia2 gene expression of pakchoi’s leaves were measured. The results showed that a high level of nitrogen (the addition of 80 mmol · L−1 NO3− (N80) and 160 mmol · L−1 NO3− (N160)) inhibited the growth of pakchoi and promoted the accumulation of osmoregulation substances and nitrate content, respectively. The reduction of the R/FR ratio under high nitrogen stress (L80) increased the fresh weight of the plants under it by 19.0%, reduced the nitrate content in the leaves by 22.7%, increased the NR activity by 29.9%, and made the Nia2 gene expression more significant, compared with N80. There was a similar mitigation effect of a low R/FR ratio under 160 mmol · L−1 excessive NO3− stress. Therefore, the reduction of the R/FR ratio can effectively control the nitrate content and improve the adaptability of pakchoi under high nitrogen stress. Thus, there is a practical application prospect for a low R/FR ratio for the production of pakchoi under a high level of nitrogen.

Analysis of NIA and GSNOR family genes and nitric oxide homeostasis in response to wheat-leaf rust interaction

Nitric oxide (NO) modulates plant response to biotic and abiotic stresses by S-nitrosylation-mediated protein post-translational modification. Nitrate reductase (NR) and S-nitrosoglutathione reductase (GSNOR) enzymes are essential for NO synthesis and the maintenance of Nitric oxide/S-nitroso glutathione (NO/GSNO) homeostasis, respectively. S-nitrosoglutathione, formed by the S-nitrosylation reaction of NO with glutathione, plays a significant physiological role as the mobile reservoir of NO. The genome-wide analysis identified nine NR(NIA) and three GSNOR genes in the wheat genome. Phylogenic analysis revealed that the nine NIA genes +were clustered into four groups and the 3 GSNORs into two groups. qRT-PCR expression profiling of NIAs and GSNORs was done in Chinese spring (CS), a leaf rust susceptible wheat line showing compatible interaction, and Transfer (TR), leaf rust-resistant wheat line showing incompatible interaction, post-inoculation with leaf rust pathotype 77–5 (121-R-63). All the NIA genes showed upregulation during incompatible interaction in comparison with the compatible reaction. The GSNOR genes showed a variable pattern of expression: the TaGSNOR1 showed little change, whereas TaGSNOR2 showed higher expression during the incompatible response. TaGSNOR3 showed a rise of expression both in compatible and incompatible reactions. Before inoculation and after 72 h of pathogen inoculation, NO localization was studied in both compatible and incompatible reactions. The S-nitrosothiol accumulation, NR, and glutathione reductase activity showed a consistent increase in the incompatible interactions. The results demonstrate that both NR and GSNOR plays significant role in defence against the leaf rust pathogen in wheat by modulating NO homeostasis or signalling.

Arabidopsis Growth-Promotion and Root Architecture Responses to the Beneficial Rhizobacterium Phyllobacterium brassicacearum Strain STM196 Are Independent of the Nitrate Assimilatory Pathway.

Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacterium isolated from roots of oilseed rape, stimulates Arabidopsis growth. We have previously shown that the NRT2.5 and NRT2.6 genes are required for this growth promotion response. Since these genes are members of the NRT2 family of nitrate transporters, the nitrogen assimilatory pathway could be involved in growth promotion by STM196. We address this hypothesis using two nitrate reductase mutants, G5 deleted in the major nitrate reductase gene NIA2 and G′4-3 altered in both NIA1 and NIA2 genes. Both mutants had a reduced growth rate and STM196 failed to increase their biomass production on a medium containing NO3− as the sole nitrogen source. However, they both displayed similar growth promotion by STM196 when grown on an NH4+ medium. STM196 was able to stimulate lateral roots development of the mutants under both nutrition conditions. Altogether, our results indicate that the nitrate assimilatory metabolism is not a primary target of STM196 interaction and is not involved in the root developmental response. The NIA1 transcript level was reduced in the shoots of nrt2.5 and nrt2.6 mutants suggesting a role for this nitrate reductase isoform independently from its role in nitrate assimilation.

Physiological Role of Aerobic Fermentation Constitutively Expressed in an Aluminum-Tolerant Cell Line of Tobacco (Nicotiana tabacum)

Aluminum (Al)-tolerant tobacco cell line ALT301 derived from SL (wild-type) hardly exhibits Al-triggered reactive oxygen species (ROS) compared with SL. Molecular mechanism leading to this phenotype was investigated comparatively with SL. Under normal growth condition, metabolome data suggested the activation of glycolysis and lactate fermentation but the repression of the tricarboxylic acid (TCA) cycle in ALT301, namely aerobic fermentation, which seemed to be transcriptionally controlled partly by higher expression of genes encoding lactate dehydrogenase and pyruvate dehydrogenase kinase. Microarray and gene ontology analyses revealed the upregulation of the gene encoding related to APETALA2.3 (RAP2.3)-like protein, one of the group VII ethylene response factors (ERFVIIs), in ALT301. ERFVII transcription factors are known to be key regulators for hypoxia response that promotes substrate-level ATP production by glycolysis and fermentation. ERFVIIs are degraded under normoxia by the N-end rule pathway of proteolysis depending on both oxygen and nitric oxide (NO), and NO is produced mainly by nitrate reductase (NR) in plants. In ALT301, levels of the NR gene expression (NIA2), NR activity and NO production were all lower compared with SL. Consistently, the known effects of NO on respiratory pathways were also repressed in ALT301. Under Al-treatment condition, NO level increased in both lines but was lower in ALT301. These results suggest that the upregulation of the RAP2.3-like gene and the downregulation of the NIA2 gene and resultant NO depletion in ALT301 coordinately enhance aerobic fermentation, which seems to be related to a higher capacity to prevent ROS production in mitochondria under Al stress.

Effects of nitric oxide on nitrogen metabolism and the salt resistance of rice (Oryza sativa L.) seedlings with different salt tolerances

Salt stress inhibits rice productivity seriously. Nitric oxide (NO) is an endogenous signaling molecule in plants that can improve the resistance of rice to abiotic stresses. Previous studies also showed that nitrogen metabolism is essential for rice stress-tolerance. However, the physiological and molecular mechanisms by how NO affects the nitrogen metabolisms of rice seedlings remain unclear. A hydroponic experiment with two rice varieties, Jinyuan85 (salt tolerant) and Liaojing763 (salt sensitive), was carried out to explore whether NO could alleviate the negative effects of salt stress on nitrogen metabolism and increase salt resistance of rice seedlings. The results showed that (1) the application of NO alleviated the inhibitory effects of salt stress on plant height and biomass accumulation, and increased the nitrogen content of rice leaf. (2) the accumulation of the sucrose and proline was markedly increased in salt stress after application of NO, and peroxidase activities was increased by 107% and 67.7% for Jinyuan85 and Liaojing763, respectively. (3) NO significantly increased the activities of glutamate dehydrogenase, sucrose synthase and sucrose phosphate synthase in both rice varieties under salt stress. (4) Additionally, NO regulated the expression levels of AMT, NIA and SUT genes, but these regulation effects are different with rice varieties and treatments. The results suggested that NO mainly increased the glutamate dehydrogenase and peroxidase activities and sucrose accumulation to enhance the nitrogen metabolism and antioxidative capacity, and alleviated the negative effects of salt stress on rice performance.

Prediction of Host-Derived miRNAs with the Potential to Target PVY in Potato Plants.

Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe. PVY reduces the yield and quality of potato cultivars. During the last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in the PVY genome. The PVY genome is approximately 9 thousand nucleotides, which transcribes the following 6 genes:CI, NIa, NIb-Pro, HC-Pro, CP, and VPg. A total of 343 mature miRNAs were retrieved from the miRBase database and were examined for their target sequences in PVY genes using the minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. The identified potato miRNAs against viral mRNA targets have antiviral activities, leading to translational inhibition by mRNA cleavage and/or mRNA blockage. We found 86 miRNAs targeting the PVY genome at 151 different sites. Moreover, only 36 miRNAs potentially targeted the PVY genome at 101 loci. The CI gene of the PVY genome was targeted by 32 miRNAs followed by the complementarity of 26, 19, 18, 16, and 13 miRNAs. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h, and miR5303d) that could target the CI, NIa, NIb-Pro, HC-Pro, CP, and VPg genes of PVY. The predicted miRNAs can be used for the development of PVY-resistant potato crops in the future.

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2.

Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammonium had no effect on CMT3 methylation, indicating that ammonium negatively regulates CMT3-mediated NIA2 methylation without affecting CMT3 methylation. Ammonium upregulated NIA2 mRNA expression, which was consistent with the repression of NIA2 methylation by ammonium. Ammonium treatment also reduced the overall genome methylation level of wild-type Arabidopsis. Moreover, CMT3 bound to specific promoter and intragenic regions of NIA2. These combined results indicate that ammonium inhibits CMT3-mediated methylation of NIA2 and that of other target genes, and CMT3 selectively binds to target DNA sequences for methylation.

Nutritional stress effects under different nitrogen sources on the genes in microalga Isochrysis zhangjiangensis and the assistance of Alteromonas macleodii in releasing the stress of amino acid deficiency

The expressions of nine nitrogen assimilation-associated genes, NRT2, NAR1, NIA2, NIR, GLN2, GLSF, GSN1, GDH, and AAT2, in the microalga Isochrysis zhangjiangensis were investigated to unveil the effects of limitations of various nitrogen sources (NaNO3 , NH4 Cl, NaNO2 , and an amino acid mixture) on the microalgae. The results demonstrated that the NRT2, NAR1, GLN2, GSN1, and AAT2 genes were highly expressed in lipid-rich microalgae under inorganic nitrogen-deficient conditions and they decreased after nitrogen resupply. Significant increases in the expressions of NAR1, GLN2, and GLSF were found in nitrate-depleted microalgae, whereas significant increases in the expressions of NRT2, NAR1, GLN2, and GSN1 were found in nitrite-depleted microalgae. Significant increases in the expressions of only NRT2 and GSN1 were found in ammonium-depleted microalgae (P<0.05). Except for the NRT2, other genes were expressed at lower levels under amino acid-deficient conditions compared with amino acid-sufficient controls. The expression of the NIA2 gene decreased in nitrogen-depleted microalgae regardless of the initial nitrogen source. However, the results of fatty acid analyses showed that the features of fatty acid profiles followed a similar mode, in which the percentage compositions of C16:0 and C18:1Δ(9) increased in nitrogen-depleted cells and that of C16:1Δ(9) , C18:3Δ(9,12,15) , C18:4Δ(6,9,12,15) , and C18:5Δ(3,6,9,12,15) decreased, regardless of the type of nitrogen source applied. It was also found that the epiphytic bacterium Alteromonas macleodii played a particularly important role in releasing microalgae from the stress of amino acid deficiency. These findings also provide a foundation for regulating microalgal lipid production through manipulation of the nitrogen assimilation-associated genes.

Arabidopsis CMT3 activity is positively regulated by AtSIZ1-mediated sumoylation

The activities of mammalian DNA and histone methyltransferases are regulated by post-translational modifications such as phosphorylation and sumoylation; however, it is unclear how the activities of these enzymes are regulated at the post-translational level in plants. Here, we demonstrate that the DNA methylation activity of Arabidopsis CHROMOMETHYLASE 3 (CMT3) is positively regulated by the E3 SUMO ligase AtSIZ1. The methylation level of the Arabidopsis genome, including transposons, was significantly lower in the siz1-2 mutant than in wild-type plants. CMT3 was sumoylated by the E3 ligase activity of AtSIZ1 through a direct interaction, and the DNA methyltransferase activity of CMT3 was enhanced by this modification. In addition, the methylation levels of a large number of genes, including the nitrate reductase gene NIA2, were lower in siz1-2 and cmt3 plants than in wild-type plants. Furthermore, the CHG methylation activity of CMT3 was specific for NIA2and not NIA1 (the other nitrate reductase gene in Arabidopsis), indicating that CMT3 selectively regulates the CHG methylation levels of target genes. Taken together, our results indicate that the sumoylation of CMT3 is critical for its role in the control of gene expression and that AtSIZ1 positively controls the epigenetic repression of CMT3-mediated gene expression.

HY5 regulates nitrite reductase 1 (NIR1) and ammonium transporter1;2 (AMT1;2) in Arabidopsis seedlings

HY5 (Long Hypocotyles 5) is a key transcription factor in Arabidopsis thaliana that has a pivotal role in seedling development. Soil nitrogen is an essential macronutrient, and its uptake, assimilation and metabolism are influenced by nutrient availability and by lights. To understand the role of HY5 in nitrogen assimilation pathways, we examined the phenotype as well as the expression of selected nitrogen assimilation-related genes in hy5 mutant grown under various nitrogen limiting and nitrogen sufficient conditions, or different light conditions. We report that HY5 positively regulates nitrite reductase gene NIR1 and negatively regulates the ammonium transporter gene AMT1;2 under all nitrogen and light conditions tested, while it affects several other genes in a nitrogen supply-dependent manner. HY5 is not required for light induction of NIR1, AMT1;2 and NIA genes, but it is necessary for high level expression of NIR1 and NIA under optimal nutrient and light conditions. In addition, nitrogen deficiency exacerbates the abnormal root system of hy5. Together, our results suggest that HY5 exhibits the growth-promoting activity only when sufficient nutrients, including lights, are provided, and that HY5 has a complex involvement in nitrogen acquisition and metabolism in Arabidopsis seedlings.

Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

Transfusion of Stored Red Blood Cells Activates an Inflammatory Program in Mouse Spleen That Is Enhanced By Endotoxemia

BACKGROUND: Transfusion of stored red blood cells (RBCs) induces a variable amount of erythrophagocytosis depending on the extent of RBC damage suffered from the storage lesion. Both murine and canine studies show that transfusion of RBCs after prolonged refrigerated storage leads to inflammation, as evidenced by increased post-transfusion levels of circulating pro-inflammatory cytokines. An analogous cytokine response was not observed in healthy humans volunteers. However, underlying inflammation may synergize with the cytokine response to stored RBCs; thus, results with healthy human volunteers may not reflect those with ill patients. Finally, plasma cytokine profiles may not accurately reflect local tissue responses at sites of erythrophagocytosis, such as the liver and spleen.AIMS: A mouse model was used to identify splenic transcriptional programs underlying the inflammatory response to stored RBC transfusions. Furthermore, mice were pretreated with a sub-lethal dose of lipopolysaccharide (LPS) to test the effect of underlying inflammation on the response to transfusion.METHODS: RBCs from 8-12 week old wild-type C57BL/6 male mice donors were collected, leukoreduced, and stored in citrate-phosphate-dextrose-adenine (CPDA)-1. Cohorts of syngeneic mice (n=3 per group) were infused with saline or transfused with one human equivalent unit of fresh RBCs (<24 hours old) or stored RBCs (11 days old). In parallel, cohorts of mice were pretreated with 1 μg of LPS (E. coli 0111:B4) and similarly transfused. Two hours after transfusion, blood and organs were collected and plasma cytokine levels were measured by a multiplex assay. Total RNA from each spleen was processed for hybridization to Affymetrix WT Gene microarrays. Whole genome expression profiles were analyzed using publicly available NIA Array and Gene Set Enrichment Analysis (GSEA) software.RESULTS: The 2-hour post-transfusion RBC recovery averaged 75% for mice transfused older RBCs. Non-LPS treated mice transfused with stored RBCs showed no statistically significant change in any plasma cytokine post-transfusion, however several cytokines were increased by stored RBC transfusion in LPS pretreated mice. Principle Component Analysis revealed a distinct splenic gene expression program associated with stored RBC transfusion in both LPS treated and non-LPS treated mice. GSEA showed significant enrichment of macrophage activation gene sets in spleens from non-LPS treated mice transfused with stored RBCs (see Figure). LPS pre-treatment increased baseline expression of macrophage activation genes, and these genes were further increased by stored RBC transfusion. Specifically, transfusion of stored RBCs acted in synergy with LPS to induce the cytokine genes (Il6 and Il1a), chemokine genes (Ccl2, Ccl3, Cxcl1, Cxcl2), the immediate early gene Fos, and the gene encoding COX-2 (Ptgs2; see Figure). Finally, a set of genes activated by stored blood transfusion independent of inflammation was identified. These genes included oxidative stress inducible genes (Hmox1 and Osgin) as well as GDF-15, which has previously been associated with states of hemolysis or ineffective erythropoieisis such as sickle cell disease and thalassemia.CONCLUSIONS: Taken together, these results suggest that although transfusion of one unit of stored RBCs in mice does not lead to a significant difference in circulating cytokine levels at two-hours post-transfusion, it does lead to activation of a distinct inflammatory transcriptional program in spleen. Moreover, sublethal endotoxemia synergizes with stored RBC transfusions to enhance expression of a specific subset of these inflammatory genes. Thus, stored RBC transfusions may be well tolerated by healthy human volunteers, but have a more detrimental impact on hospitalized patients, particularly those with sepsis and low-grade endotoxinemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Response of nitrate reductase activity and NIA genes expression in roots of Arabidopsis hxk1 mutant treated with selected carbon and nitrogen metabolites

In plants sugar sensing and signal transduction involves pathways dependent or independent on HXK1 as a glucose sensor. Research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. The effect of selected carbon and nitrogen metabolites on nitrate reductase (NR) activity in Arabidopsis thaliana wild type (WT) and hxk1 mutant roots was studied. Exogenously supplied sugar, sucrose (Suc) and organic acid, 2-oxoglutarate (2-OG) led to an increase in the total and actual activity of NR. It was due to both the increase in expression of NIA genes and NR activation state. The stimulatory effect of Suc and 2-OG on nitrate reduction was less pronounced in hxk1 mutant roots with T-DNA insertion in the AtHXK1 gene encoding hexokinase1 (HXK1) and characterized by reduced hexokinase activity and root level of G6P and F6P. On the other hand, it was shown that exogenous glucose did not mimic Suc-mediated NR activation in Arabidopsis roots. Taken together, this data suggest that the Suc signaling pathway might be independent from hexose's sensor dependent mechanism.

Effects of the sequence characteristics of miRNAs on multi-viral resistance mediated by single amiRNAs in transgenic tobacco

Artificial microRNA (amiRNA) has become the preferred viral defence that can be induced in plants. In this study, nine amiRNA target sites were selected that were based on the sequence characteristics of natural miRNAs in the cylindrical inclusion protein (CI), nuclear inclusion a protein (NIa), nuclear inclusion b protein (NIb), and coat protein (CP) genes of Potato virus Y (PVYN). These amiRNAs that exhibited high similarities to the sequences of PVYN and TEV-SD1 were considered. To study the effectiveness of gene silencing in amiRNA-mediated viral resistance, we constructed nine amiRNA plant expression vectors by replacing the functional sequences of miRNA319a precursors with our selected amiRNA sequences. These constructs were subsequently introduced to tobacco plants. A Northern blot assay verified that the nine amiRNA plant expression vectors could successfully express amiRNAs in plants. The analysis of viral resistance demonstrated that these transgenic tobacco plants could effectively inhibit PVYN and TEV-SD1 viral infections. The amiRNA that targeted the NIb and CP genes displayed a higher silencing efficiency than did the amiRNAs targeted CI and NIa genes. Northern blot analysis demonstrated that silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA pathway. That is, the amiRNA and the secondary siRNA mediated the degradation of viral RNA together. Genetic analysis demonstrated that the trait for viral resistance in transgenic plants can be consistently inherited via a single copy of the transgenic sequence. Considering the correlation between the sequence characteristics and the activity of amiRNA, we concluded that a few mismatched bases between the amiRNA and the target sequence could be allowed, particularly the mismatched bases in the 3′ end of the amiRNA.

Regulation of nitrate reduction in Arabidopsis WT and hxk1 mutant under C and N metabolites

As in plants sugar sensing and signal transduction involve pathways dependent or independent on hexokinase 1 (HXK1) as a glucose sensor, research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. An Arabidopsis mutant with T-DNA insertion in the AtHXK1 gene and defects in glucose signaling (hxk1) was used to determine nitrate reductase (NR) activity, NIA genes expression in leaves after 8-h treatment with sugars (glucose and sucrose), organic acids [2-oxoglutarate (2OG)] and amino acids (glutamine and glutamate). Sugars, especially sucrose, caused induction of NR actual activity accompanied by an increase of the NR activation state, indicating the posttranslational nature of the modifications. Those modifications were observed in wild-type (WT) and hxk1 leaves, suggesting that regulation of NR activity by sugars does not involve HXK1 as a glucose sensor. Moreover, sugars enhanced expression of NIA genes. However, a higher level of NIA transcripts did not lead to an increase of total NR activity in sugar-treated plants. This may suggest that posttranslational modification of NR is fundamental regulatory mechanisms controlling NR activity in response to C metabolites. Treatment of plants with 2-OG also modified NR through the posttranslational modifications. Elevation of actual NR activity and the enzyme activation state in WT and hxk1 leaves was observed. Amino acids caused a decrease of NIA gene expression and NR activities in WT and hxk1 leaves indicating that mutation in the hexokinase-dependent glucose signaling pathway did not interrupt the amino acid feedback regulation of NR.

Whole‐plant and organ‐level nitrogen isotope discrimination indicates modification of partitioning of assimilation, fluxes and allocation of nitrogen in knockout lines ofArabidopsis thaliana

The nitrogen isotope composition (δ¹⁵N) of plants has potential to provide time-integrated information on nitrogen uptake, assimilation and allocation. Here, we take advantage of existing T-DNA and γ-ray mutant lines of Arabidopsis thaliana to modify whole-plant and organ-level nitrogen isotope composition. Nitrate reductase 2 (nia2), nitrate reductase 1 (nia1) and nitrate transporter (nrt2) mutant lines and the Col-0 wild type were grown hydroponically under steady-state NO₃⁻ conditions at either 100 or 1000 μM NO₃⁻ for 35 days. There were no significant effects on whole-plant discrimination and growth in the assimilatory mutants (nia2 and nia1). Pronounced root vs leaf differences in δ¹⁵N, however, indicated that nia2 had an increased proportion of nitrogen assimilation of NO₃⁻ in leaves while nia1 had an increased proportion of assimilation in roots. These observations are consistent with reported ratios of nia1 and nia2 gene expression levels in leaves and roots. Greater whole-plant discrimination in nrt2 indicated an increase in efflux of unassimilated NO₃⁻ back to the rooting medium. This phenotype was associated with an overall reduction in NO₃⁻ uptake, assimilation and decreased partitioning of NO₃⁻ assimilation to the leaves, presumably because of decreased symplastic intercellular movement of NO₃⁻ in the root. Although the results were more varied than expected, they are interpretable within the context of expected mechanisms of whole-plant and organ-level nitrogen isotope discrimination that indicate variation in nitrogen fluxes, assimilation and allocation between lines.