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Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Background The B-BOX (BBX) proteins have important functions in regulating plant growth and development. In plants, the BBX gene family has been identified in several plants, such as rice, Arabidopsis and tomato. However, there still lack a genome-wide survey of BBX genes in pear. Results In the present study, a total of 25 BBX genes were identified in pear ( Pyrus bretschneideri Rehd.). Subsequently, phylogenetic relationship, gene structure, gene duplication, transcriptome data and qRT-PCR were conducted on these BBX gene members. The transcript analysis revealed that twelve PbBBX genes (48%) were specifically expressed in pear pollen tubes. Furthermore, qRT-PCR analysis indicated that both PbBBX4 and PbBBX13 have potential role in pear fruit development, while PbBBX5 should be involved in the senescence of pear pollen tube. Conclusions This study provided a genome-wide survey of BBX gene family in pear, and highlighted its roles in both pear fruits and pollen tubes. The results will be useful in improving our understanding of the complexity of BBX gene family and functional characteristics of its members in future study.

Priming plants with beneficial microbes can establish rapid and robust resistance against numerous pathogens. Here, compelling evidence is provided that the treatment of rapeseed plants with Trichoderma harzianum OMG16 and Bacillus velezensis FZB42 induces defence activation against Verticillium longisporum infection. The relative expressions of the JA biosynthesis genes LOX2 and OPR3, the ET biosynthesis genes ACS2 and ACO4 and the SA biosynthesis and signalling genes ICS1 and PR1 were analysed separately in leaf, stem and root tissues using qRT-PCR. To successfully colonize rapeseed roots, the V. longisporum strain 43 pathogen suppressed the biosynthesis of JA, ET and SA hormones in non-primed plants. Priming led to fast and strong systemic responses of JA, ET and SA biosynthesis and signalling gene expression in each leaf, stem and root tissue. Moreover, the quantification of plant hormones via UHPLC-MS analysis revealed a 1.7- and 2.6-fold increase in endogenous JA and SA in shoots of primed plants, respectively. In roots, endogenous JA and SA levels increased up to 3.9- and 2.3-fold in Vl43-infected primed plants compared to non-primed plants, respectively. Taken together, these data indicate that microbial priming stimulates rapeseed defence responses against Verticillium infection and presumably transduces defence signals from the root to the upper parts of the plant via phytohormone signalling.

Low temperature is a significant abiotic stress factor that not only impacts plant growth, development, yield, and quality but also constrains the geographical distribution of numerous wild plants. Kohlrabi (Brassica oleracea L. var. caulorapa L.) belongs to the Brassicaceae family and has a short growing period. In this study, a total of 196,642 unigenes were obtained from kohlrabi seedlings at low temperatures; of these, 52,836 unigenes were identified as differentially expressed genes. Transcription factor family members ARR-B, C3H, B3-ARF, etc. that had a high correlation with biochemical indicators related to low temperature were identified. A total of nineteen BocARR-B genes (named BocARR-B1–BocARR-B19) were obtained, and these genes were distributed unevenly across seven chromosomes. Nineteen BocARR-B genes searched four conserved motifs and were divided into three groups. The relative expression level analysis of 19 BocARR-B genes of kohlrabi showed obvious specificity in different tissues. This study lays a foundation and provides new insight to explain the low-temperature resistance mechanism and response pathways of kohlrabi. It also provides a theoretical basis for the functional analysis of 19 BocARR-B transcription factor gene family members.

Aims Phosphorus (P) mobility depends on the availability of water in soil, both of which are limited resources for crop production. We studied the mechanisms governing tolerance to P-deficiency and drought stress in contrasting mungbean accessions. Methods Tolerant (IC333090 and IC507340) and sensitive (IC488526 and EC397142) mungbean accessions were grown in soil under four treatments: control (sufficient P, irrigated), P-deficiency (no P, irrigated), drought (sufficient P, irrigation withheld), combined stress (no P, irrigation withheld), followed by a 48 h recovery. Results Drought stress reduced the relative water content and membrane stability index, affecting overall plant growth. The tolerant accessions maintained significantly higher root growth, leaf area, and biomass under combined stress (P-deficiency and drought) than the sensitive accessions, mainly due to enhanced nutrient uptake and symbiotic N 2 -fixation. The combined stress also increased osmolyte concentration, antioxidative compounds, and the scavenging activity of antioxidant enzymes in tolerant accessions. Recovery from drought stress significantly reduced osmolyte concentration. Transcript abundance of candidate genes related to P-deficiency and drought under individual and combined stress, was significantly higher in leaves of IC333090 than IC488526. IC333090 recovered from drought stress better than IC488526 due to the enhanced expression of stress-responsive genes. Conclusions Physiological traits, such as the accumulation of total soluble sugars and reduced glutathione, ascorbate, and scavenging activity of antioxidant enzymes, facilitated by the differential expression of stress-responsive genes impart cross-tolerance to P-deficiency and drought stress in tolerant mungbean accessions. Hence, accession IC333090 is potential genetic stock for tolerance to drought, P-deficiency, and combined stresses.

Correct selection of the reference gene(s) is the most important step in gene expression analysis. The aims of this study were to identify and evaluate the panel of possible reference genes in neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP) obtained from human-induced pluripotent stem cells (hiPSC). The stability of expression of genes commonly used as the reference in cells during neural differentiation is variable and does not meet the criteria for reference genes. In the present work, we evaluated the stability of expression of 16 candidate reference genes using the four most popular algorithms: the ΔCt method, BestKeeper, geNorm and NormFinder. All data were analysed using the online tool RefFinder to obtain a comprehensive ranking. Our results indicate that NormFinder is the best tool for reference gene selection in early stages of hiPSC neural differentiation. None of the 16 tested genes is suitable as reference gene for all three stages of development. We recommend using different genes (panel of genes) to normalise RT–qPCR data for each of the neural differentiation stages.

Background Past research has shown that virus-induced phytoene desaturase ( PDS ) gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a pale-yellow fruit phenotype. Although the PDS gene is often used as a marker for gene silencing in tomatoes, little is known about the role of PDS in fruit ripening. In this study, we investigated whether the pepper PDS gene silenced endogenous PDS genes in the fruit of two tomato cultivars, Dotaerang Plus and Legend Summer. Results We found that the pepper PDS gene successfully silenced endogenous PDS in tomato fruit at a silencing frequency of 100% for both cultivars. A pale-yellow silenced area was observed over virtually the entire surface of individual fruit due to the transcriptional reduction in phytoene desaturase ( PDS ), zeta-carotene ( ZDS ), prolycopene isomerase ( CrtlSO ), and beta-carotene hydroxylase ( CrtR - b2 ), which are the carotenoid biosynthesis genes responsible for the red coloration in tomatoes. PDS silencing also affected the expression levels of the fruit-ripening genes Tomato AGAMOUS-LIKE1 ( TAGL1 ), RIPENING INHIBITOR ( RIN ), pectin esterase gene ( PE ), lipoxygenase ( LOX ), FRUITFULL1/FRUITFUL2 ( FUL1/FUL2 ), and the ethylene biosynthesis and response genes 1-aminocyclopropane-1-carboxylate oxidase 1 and 3 ( ACO1 and ACO3 ) and ethylene-responsive genes ( E4 and E8 ). Conclusion These results suggest that PDS is a positive regulator of ripening in tomato fruit, which must be considered when using it as a marker for virus-induced gene silencing (VIGS) experiments in order to avoid fruit-ripening side effects.

Papaver armeniacum hairy roots were induced by four Rhizobium rhizogenes strains on three expiants (shoot, root, and hypocotyl). Also, the effects of two concentrations (100 and 200 μM) of methyl jasmonate (MJ) and salicylic acid (SA) were assessed on productions of papaverine, noscapine, thebaine, morphine, and codeine and expression of some related genes (TYDC, DBOX, BBE, SalAT, T60DM, and COR) in P. armeniacum L. hairy root culture at 24 and 48 h after elicitation. R. rhizogenes strain C58C1 induced the highest hairy root rate on hypocotyl expiant. Application of 100 μM MJ resulted in the highest contents of thebaine, codeine, and morphine by enhancing the expression of SalAT, COR, and T6ODM genes, respectively, while application of 100 μM SA resulted in the highest contents of papaverine and noscapine by upregulating DBOX and BBE genes, respectively. 100 μM MJ can be used as an effective elicitor in P. armeniacum hairy root culture to increase studied morphinan alkaloids. Also, SA can be suggested for enhancing papaverine and noscapine contents in P. armeniacum hairy root culture. It may be due to that there is a SA- and MJ-signaling crosstalk, which results in reciprocal antagonism between SA and MJ signaling pathways. The effects of MJ and SA elicitors on benzylisoquinoline alkaloids (BIAs) production were level-dependent.

Current industrial carbon monoxide (CO) production depends mainly on fossil fuels. Composting offers a sustainable alternative, where CO can be produced by compost‐inhabiting bacteria synthesizing the CO dehydrogenase (CODH) enzyme. This study aimed to characterize the microbial community and, for the first time, to investigate the expression of the cooS gene encoding CODH during biowaste composting under laboratory conditions. Composting was performed at mesophilic (45°C) and thermophilic (70°C) temperatures to evaluate CO production under conditions known to stimulate elevated CO emissions. The results confirmed biotechnological potential for microbial CO production within the mesophilic range, potentially reducing costs compared with thermophilic composting. Although the study revealed higher CO concentrations under thermophilic conditions (average 208 ppm; maximum 818 ppm) compared to mesophilic conditions (average 102 ppm; maximum 499 ppm), the compost obtained at 45°C exhibited a more diverse and balanced microbial community than that observed at higher temperatures. In the 45°C compost, bacteria previously reported as CODH or CO producers were identified, including Bacillus licheniformis and representatives of Alphaproteobacteria and Streptomyces. The cooS gene expression analysis showed a significant increase during composting at 45°C, reaching an 11,016‐fold rise within the week. The expression was likely induced by CO and not inhibited by oxygen. This first report demonstrating cooS expression during composting confirmed the biological origin of CO under mesophilic conditions through the activity of anaerobic Ni, Fe‐CODH. This study demonstrates, for the first time, the expression of the cooS gene during composting, confirming microbial CO production under mesophilic conditions. The results highlight composting as a novel waste‐to‐biochemicals process, offering a sustainable, low‐carbon pathway for generating carbon monoxide via bacterial CO dehydrogenase activity.

Tanacetum parthenium, commonly known as feverfew, is a perennial medicinal herb renowned for its therapeutic properties, particularly attributed to the sesquiterpene lactone parthenolide. This study explores the regulatory impact of Trichostatin A (TSA), an epigenetic modulator, on the expression of genes associated with parthenolide biosynthesis and accumulation in T. parthenium. We assessed the expression profiles of key genes, namely Germacrene A synthase (GAS), Parthenolide synthase (PTS), and Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) with qRT-PCR. The experimental approach involved treating feverfew plants with varying concentrations of TSA through seed pretreatment under in vitro conditions and foliar spray on in vivo-cultured plants. Our findings indicate that specific concentrations of TSA, notably 0.5 µM and 1 µM, positively modulate the expression of GAS and PTS genes, thereby influencing the final concentration of parthenolide. This research provides valuable insights into the potential of TSA as a strategic tool for enhancing secondary metabolite production in medicinal plants.Key messageTrichostatin A (TSA), a typical histone deacetylase inhibitor, can increase the production of parthenolide (a major sesquiterpene lactone) in the in vivo plants and in vtiro seedlings of feverfew.