Validation of internal control for gene expression study in soybean by quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean.</p> <p>Results</p> <p>All ten HKGs displayed a wide range of Ct values in 21 sample pools, confirming that they were variably expressed. GeNorm was used to determine the expression stability of the HGKs in seven series sets. For all the sample pools analyzed, the stability rank was <it>ELF1B</it>, <it>CYP2 </it>> <it>ACT11 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For different tissues under the same developmental stage, the rank was <it>ELF1B</it>, <it>CYP2 </it>> <it>ACT2/7 </it>> <it>UBC2 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>ACT11 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For the developmental stage series, the stability rank was <it>ACT2/7</it>, <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ELF1B </it>> <it>TUB </it>> <it>CYP2 </it>> <it>ACT11 </it>> <it>G6PD </it>> <it>UBQ10</it>. For photoperiodic treatments, the rank was <it>ACT11</it>, <it>ELF1B </it>> <it>CYP2 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For different times of the day, the rank was <it>ELF1A</it>, <it>TUA </it>> <it>ELF1B </it>> <it>G6PD </it>> <it>CYP2 </it>> <it>ACT11 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>UBC2 </it>> <it>UBQ10</it>. For different cultivars and leaves on different nodes of the main stem, the ten HKGs' stability did not differ significantly. ΔCt approach and 'Stability index' were also used to analyze the expression stability in all 21 sample pools. Results from ΔCt approach and geNorm indicated that <it>ELF1B </it>and <it>CYP2 </it>were the most stable HKGs, and <it>UBQ10 </it>and <it>G6PD </it>the most variable ones. Results from 'Stability index' analysis were different, with <it>ACT11 </it>and <it>CYP2 </it>being the most stable HKGs, and <it>ELF1A </it>and <it>TUA </it>the most variable ones.</p> <p>Conclusion</p> <p>Our data suggests that HKGs are expressed variably in soybean. Based on the results from geNorm and ΔCt analysis, <it>ELF1B </it>and <it>CYP2 </it>could be used as internal controls to normalize gene expression in soybean, while <it>UBQ10 </it>and <it>G6PD </it>should be avoided. To achieve accurate results, some conditions may require more than one HKG to be used for normalization.</p

Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics

<p>Abstract</p> <p>Background</p> <p>Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. <it>Superroo</it>t of <it>Lotus corniculatus </it>is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic <it>L. corniculatus </it>plants from <it>Superroot</it>-derived leaves through <it>A. tumefaciens-</it>mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification.</p> <p>Results</p> <p>In the present study, we developed an <it>A. rhizogenes</it>-mediated transformation of <it>Superroot</it>-derived <it>L. corniculatus </it>for gene function investigation, combining the efficient <it>A. rhizogenes</it>-mediated transformation and the rapid regeneration system of <it>Superroot</it>. The transformation system using <it>A. rhizogenes </it>K599 harbouring pGFPGUS<it>Plus </it>was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD₆₀₀= 0.6, and co-cultivation on medium (pH 5.4) at 22°C for 2 days enhanced the transformation frequency significantly. As proof of concept, <it>Superroot</it>-derived <it>L. corniculatus </it>was transformed with a gene from wheat encoding an Na⁺/H⁺antiporter (<it>TaNHX2</it>) using the described system. Transgenic <it>Superroot </it>plants were obtained and had increased salt tolerance, as expected from the expression of <it>TaNHX2</it>.</p> <p>Conclusion</p> <p>A rapid and efficient tool for gene function investigation in <it>L. corniculatus </it>was developed, combining the simplicity and high efficiency of the <it>Superroot </it>regeneration system and the availability of <it>A. rhizogenes</it>-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could reach 92% based on GUS detection. The combination of the highly efficient transformation and the regeneration system of <it>Superroot </it>provides a valuable tool for functional genomics studies in <it>L. corniculatus</it>.</p

Concentration-dependent effects of narciclasine on cell cycle progression in Arabidopsis root tips

<p>Abstract</p> <p>Background</p> <p>Narciclasine (NCS) is an Amaryllidaceae alkaloid isolated from <it>Narcissus tazetta </it>bulbs. NCS has inhibitory effects on a broad range of biological activities and thus has various potential practical applications. Here we examine how NCS represses plant root growth.</p> <p>Results</p> <p>Results showed that the inhibition of NCS on cell division in <it>Arabidopsis </it>root tips and its effects on cell differentiation are concentration-dependent; at low concentrations (0.5 and 1.0 μM) NCS preferentially targets mitotic cell cycle specific/cyclin complexes, whereas at high concentration (5.0 μM) the NCS-stimulated accumulation of Kip-related proteins (KRP1 and RP2) affects the CDK complexes with a role at both G1/S and G2/M phases.</p> <p>Conclusions</p> <p>Our findings suggest that NCS modulates the coordination between cell division and differentiation in <it>Arabidopsis </it>root tips and hence affects the postembryonic development of <it>Arabidopsis </it>seedlings.</p

Cytosolic Glucose-6-Phosphate Dehydrogenase Is Involved in Seed Germination and Root Growth Under Salinity in Arabidopsis

Glucose-6-phosphate dehydrogenase (G6PDH or G6PD) is the key regulatory enzyme in the oxidative pentose phosphate pathway (OPPP). The cytosolic isoforms including G6PD5 and G6PD6 account for the major part of the G6PD total activity in plant cells. Here, we characterized the Arabidopsis single null mutant g6pd5 and g6pd6 and double mutant g6pd5/6. Compared to wild type, the mutant seeds showed a reduced germination rate and root elongation under salt stress. The seeds and seedlings lacking G6PD5 and G6PD6 accumulate more reactive oxygen species (ROS) than the wild type under salt stress. Cytosolic G6PD (cy-G6PD) affected the expression of NADPH oxidases and the G6PD enzymatic activities in the mutant atrbohD/F, in which the NADPH oxidases genes are disrupted by T-DNA insertion and generation of ROS is inhibited, were lower than that in the wild type. The NADPH level in mutants was decreased under salt stress. In addition, we found that G6PD5 and G6PD6 affected the activities and transcript levels of various antioxidant enzymes in response to salt stress, especially the ascorbate peroxidase and glutathione reductase. Exogenous application of ascorbate acid and glutathione rescued the seed and root phenotype of g6pd5/6 under salt stress. Interestingly, the cytosolic G6PD negatively modulated the NaCl-blocked primary root growth under salt stress in the root meristem and elongation zone

Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress

In the present study, we investigated the role of glucose-6-phosphate dehydrogenase (G6PDH) in regulating the levels of reduced form of glutathione (GSH) to the tolerance of calli from two reed ecotypes, Phragmites communis Trin. dune reed (DR) and swamp reed (SR), in a long-term salt stress. G6PDH activity was higher in SR callus than that of DR callus under 50-150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300-600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH contents, glutathione reductase (GR) and glutathione peroxidase (GPX) activities in both salt-stressed calli. After G6PDH activity and expression were reduced in glycerol treatments, GSH contents and GR and GPX activity decreased strongly in both calli. Simultaneously, NaCl-induced hydrogen peroxide (H2O2) accumulation was also abolished. Exogenous application of H2O2 increased G6PDH, GR, and GPX activities and GSH contents in the control conditions and glycerol treatment. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H2O2 accumulation, decreased these enzymes activities and GSH contents. Furthermore, exogenous application of H2O2 abolished the N-acetyl-L-cysteine (NAC)-induced decrease in G6PDH activity, and DPI suppressed the effect of buthionine sulfoximine (BSO) on induction of G6PDH activity. Western-blot analyses showed that G6PDH expression was stimulated by NaCl and H2O2, and blocked by DPI in DR callus. Taken together, G6PDH activity involved in GSH maintenance and H2O2 accumulation under salt stress. And H2O2 regulated G6PDH, GR, and GPX activities to maintain GSH levels. In the process, G6PDH plays a central role

Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley

G6PDH provides intermediate metabolites and reducing power (nicotinamide adenine dinucleotide phosphate, NADPH) for plant metabolism, and plays a pivotal role in the cellular redox homeostasis. In this study, we cloned five G6PDH genes (HvG6PDH1 to HvG6PDH5) from highland barley and characterized their encoded proteins. Functional analysis of HvG6PDHs in E. coli showed that HvG6PDH1 to HvG6PDH5 encode the functional G6PDH proteins. Subcellular localization and phylogenetic analysis indicated that HvG6PDH2 and HvG6PDH5 are localized in the cytoplasm, while HvG6PDH1, HvG6PDH3, and HvG6PDH4 are plastidic isoforms. Analysis of enzymatic activities and gene expression showed that HvG6PDH1 to HvG6PDH4 are involved in responses to salt and drought stresses. The cytosolic HvG6PDH2 is the major isoform against oxidative stress. HvG6PDH5 may be a house-keeping gene. In addition, HvG6PDH1 to HvG6PDH4 and their encoded enzymes responded to jasmonic acid (JA) and abscisic acid (ABA) treatments, implying that JA and ABA are probably critical regulators of HvG6PDHs (except for HvG6PDH5). Reactive oxygen species analysis showed that inhibition of cytosolic and plastidic G6PDH activities leads to increased H2O2 and O2&minus; contents in highland barley under salt and drought stresses. These results suggest that G6PDH can maintain cellular redox homeostasis and that cytosolic HvG6PDH2 is an irreplaceable isoform against oxidative stress in highland barley