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Abstract Cadmium (Cd) contamination poses significant risks to agricultural productivity and human health, particularly through its accumulation in staple crops such as bread wheat (Triticum aestivum L.). This study evaluated Cd accumulation and tolerance among six bread wheat cultivars exposed to six Cd concentrations (0, 2.5, 5, 10, 15, 20, and 25 mg kg−1 soil). Phenotypic assessments and quantitative real-time PCR (qRT-PCR) were conducted to analyze the expression patterns of TaNRAMP and TaZIP genes in various tissues and developmental stages of wheat, which play crucial roles in Cd uptake and transport. Results demonstrated significant variability in Cd accumulation. The Barat cultivar exhibited the lowest accumulation in grain (ranging from 0.21 to 8.8 mg kg−1) and the highest tolerance. In contrast, Kavir and Pishtaz displayed elevated Cd levels in both grain and straw, while Parsi accumulated more Cd in straw at lower concentrations (56.9 mg kg−1 in Cd concentration of 10 mg kg−1 soil). The gene expression analysis revealed that most cultivars showed increased expression of TaNRAMP genes, particularly TaNRAMP2 in Cd concentration of 10 mg kg−1 soil, which facilitates Cd uptake from the soil, and TaZIP genes, such as TaZIP4 and TaZIP7, involved in transporting Cd within the plant. Notably, the expression of TaZIP1 was significantly lower in cultivars with high Cd accumulation, suggesting a potential regulatory mechanism for Cd tolerance. Furthermore, cultivars exhibiting higher Cd levels correlated with increased expression of stress-responsive genes, indicating a broader response to Cd stress. These findings highlight Barat’s potential for bread-making applications due to its low Cd accumulation, while Morvarid and Pishtaz which show reduced Cd content in the straw even under high Cd exposure are better suited for animal feed. This research underscores the genetic variability of wheat cultivars in response to Cd stress and provides essential insights into the molecular mechanisms underlying Cd accumulation, offering valuable information for breeding programs aimed at developing Cd-tolerant varieties to ensure food security in contaminated regions.

Abstract Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.

Abstract Background The auxin/indole-3-acetic acid (Aux/IAA) gene family is a crucial element of the auxin signaling pathway, significantly influencing plant growth and development. Hence, we conducted a comprehensive investigation of Aux/IAAs gene family using the Sp75 and Monoe-Viroflay genomes in spinach. Results A total of 24 definitive Aux/IAA genes were identified, exhibiting diverse attributes in terms of amino acid length, molecular weight, and isoelectric points. This diversity underscores potential specific roles within the family, such as growth regulation and stress response. Structural analysis revealed significant variations in gene length and molecular weight. These variations indicate distinct roles within the Aux/IAA gene family. Chromosomal distribution analysis exhibited a dispersed pattern, with chromosomes 4 and 1 hosting the highest and lowest numbers of Aux/IAA genes, respectively. Phylogenetic analysis grouped the identified genes into distinct clades, revealing potential evolutionary relationships. Notably, the phylogenetic tree highlighted specific gene clusters suggesting shared genetic ancestry and potential functional synergies within spinach. Expression analysis under NAA treatment unveiled gene-specific and time-dependent responses, with certain genes exhibiting distinct temporal expression patterns. Specifically, SpoIAA5 displayed a substantial increase at 2 h post-NAA treatment, while SpoIAA7 and SpoIAA9 demonstrated continuous rises, peaking at the 4-hour time point. Conclusions These observations indicate a complex interplay of gene-specific and temporal regulation in response to auxin. Moreover, the comparison with other plant species emphasized both shared characteristics and unique features in Aux/IAA gene numbers, providing insights into the evolutionary dynamics of this gene family. This comprehensive characterization of Aux/IAA genes in spinach not only establishes the foundation for understanding their specific functions in spinach development but also provides a valuable resource for experimental validation and further exploration of their roles in the intricate network of auxin signaling pathways.

Abstract The complex trait of yield is controlled by several quantitative trait loci (QTLs). Given the global water deficit issue, the development of rice varieties suitable for non-flooded cultivation holds significant importance in breeding programs. The powerful approach of Meta-QTL (MQTL) analysis can be used for the genetic dissection of complicated quantitative traits. In the current study, a comprehensive MQTL analysis was conducted to identify consistent QTL regions associated with drought tolerance and yield-related traits under water deficit conditions in rice. In total, 1087 QTLs from 134 rice populations, published between 2000 to 2021, were utilized in the analysis. Distinct MQTL analysis of the relevant traits resulted in the identification of 213 stable MQTLs. The confidence interval (CI) for the detected MQTLs was between 0.12 and 19.7 cM. The average CI of the identified MQTLs (4.68 cM) was 2.74 times narrower compared to the average CI of the initial QTLs. Interestingly, 63 MQTLs coincided with SNP peak positions detected by genome-wide association studies for yield and drought tolerance-associated traits under water deficit conditions in rice. Considering the genes located both in the QTL-overview peaks and the SNP peak positions, 19 novel candidate genes were introduced, which are associated with drought response index, plant height, panicle number, biomass, and grain yield. Moreover, an inclusive MQTL analysis was performed on all the traits to obtain “Breeding MQTLs”. This analysis resulted in the identification of 96 MQTLs with a CI ranging from 0.01 to 9.0 cM. The mean CI of the obtained MQTLs (2.33 cM) was 4.66 times less than the mean CI of the original QTLs. Thirteen MQTLs fulfilling the criteria of having more than 10 initial QTLs, CI

Foliar application of growth regulators seems to alleviate the adverse effects of abiotic stresses in plants through different mechanisms such as antioxidative and gene expression regulation. As an important abiotic stress, salinity has an enormous negative impact on crop plants such as bread wheat. In the present study, exogenous strigolactone (GR24) was considered for its potential stress-mitigating effect in two contrasting wheats cultivars (‘Arta’ and ‘Karchia’) grown under 2,053,100 mM NaCl stress (10 dS m−1). Yield-related traits, oxidative (APX, POX, CAT, H2O2, MDA, GB and Proline), ionic indices (Na+ and K+ content in leaf and root), and the expression of a selected set of oxidative stress related genes (TaAPX, TaGPX, TaP5CS, TaNHX2, TaSOS1, TaHAK, TaAKT2 and TaHKT2;1) were surveyed in the flowering stage. Strigolactone application (10 μM) had a significant effect on the induction and improvement of biochemical and ionic features such as reducing the electrolyte leakage, H2O2 and MDA or increasing the grain yield, total protein, proline, GB and antioxidant enzymes (APX, POX) activity, of salinity-stressed plants. The gene expression analysis with real-time PCR showed transcriptional variation of selected genes in response to salt stress and GR24. Improved homeostasis in Na+ and K+ and their ratio was found to be correlated with the expression of the antiporter genes and the consequent reduction of ion toxicity. The findings of this study can improve our knowledge about complex mechanism of strigolactone plant hormone during salt stress in crop plans to mitigation of salinity stress.

Abstract Background Lettuce (Lactuca sativa L.) is considered the most important vegetable in the leafy vegetable group. However, bolting affects quality, gives it a bitter taste, and as a result makes it inedible. Bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components. Although bolting/flowering responsive genes have been identified in most sensitive and non-sensitive species, non-coding RNA molecules like long non-coding RNAs (lncRNAs) have not been investigated in lettuce. Hence, in this study, potential long non-coding RNAs that regulate flowering /bolting were investigated in two lettuce strains S24 (resistant strain) and S39 (susceptible strain) in different flowering times to better understand the regulation of lettuce bolting mechanism. For this purpose, we used two RNA-seq datasets to discover the lncRNA transcriptome profile during the transition from vegetative to reproductive phase. Results For identifying unannotated transcripts in these datasets, a 7-step pipeline was employed to filter out these transcripts and terminate with 293 novel lncRNAs predicted by PLncPRO and CREMA. These transcripts were then utilized to predict cis and trans flowering-associated targets and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Computational predictions of target gene function showed the involvement of putative flowering-related genes and enrichment of the floral regulators FLC, CO, FT, and SOC1 in both datasets. Finally, 17 and 18 lncRNAs were proposed as competing endogenous target mimics (eTMs) for novel and known lncRNA miRNAs, respectively. Conclusion Overall, this study provides new insights into lncRNAs that control the flowering time of plants known for bolting, such as lettuce, and opens new windows for further study.

Abstract Background Spinach is a beneficial annual vegetable species and sensitive to the bolting or early flowering, which causes a large reduction in quality and productivity. Indeed, bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components. Although some key flowering responsive genes have been identified in spinach, non-coding RNA molecules like long non-coding RNAs (lncRNAs) were not investigated yet. Herein, we used bioinformatic approaches to analyze the transcriptome datasets from two different accessions Viroflay and Kashan at two vegetative and reproductive stages to reveal novel lncRNAs and the construction of the lncRNA-mRNA co-expression network. Additionally, correlations among gene expression modules and phenotypic traits were investigated; day to flowering was chosen as our interesting trait. Results In the present study, we identified a total of 1141 lncRNAs, of which 111 were differentially expressed between vegetative and reproductive stages. The GO and KEGG analyses carried out on the cis target gene of lncRNAs showed that the lncRNAs play an important role in the regulation of flowering spinach. Network analysis pinpointed several well-known flowering-related genes such as ELF, COL1, FLT, and FPF1 and also some putative TFs like MYB, WRKY, GATA, and MADS-box that are important regulators of flowering in spinach and could be potential targets for lncRNAs. Conclusions This study is the first report on identifying bolting and flowering-related lncRNAs based on transcriptome sequencing in spinach, which provides a useful resource for future functional genomics studies, genes expression researches, evaluating genes regulatory networks and molecular breeding programs in the regulation of the genetic mechanisms related to bolting in spinach.

Abstract Background Bolting refers to the early flowering stem production on agricultural and horticultural crops before harvesting. Indeed, bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components, which cause a large reduction in the quality and productivity of vegetable crops like spinach. However, little is known about the signaling pathways and molecular functions involved in bolting mechanisms in spinach. The genetic information regarding the transition from vegetative growth to the reproductive stage in spinach would represent an advantage to regulate bolting time and improvement of resistant cultivars to minimize performance loss. Results To investigate the key genes and their genetic networks controlling spinach bolting, we performed RNA-seq analysis on early bolting accession Kashan and late-bolting accession Viroflay at both vegetative and reproductive stages and found a significant number of differentially expressed genes (DEGs) ranging from 195 to 1230 in different comparisons. These genes were mainly associated with the signaling pathways of vernalization, photoperiod/circadian clock, gibberellin, autonomous, and aging pathways. Gene ontology analysis uncovered terms associated with carbohydrate metabolism, and detailed analysis of expression patterns for genes of Fructose-1, 6-bisphosphate aldolase, TREHALOSE-6-PHOSPHATE SYNTHASE 1, FLOWERING PROMOTING FACTOR 1, EARLY FLOWERING, GIGANTEA, and MADS-box proteins revealed their potential roles in the initiating or delaying of bolting. Conclusion This study is the first report on identifying bolting and flowering-related genes based on transcriptome sequencing in spinach, which provides insight into bolting control and can be useful for molecular breeding programs and further study in the regulation of the genetic mechanisms related to bolting in other vegetable crops.

Phalaenopsis is one of the most well-known genera of the orchid family and has relatively good growth due to its high adaptability. The Phalaenopsis breeding program and full investigation of progenies takes three to five years, which can be reduced by using molecular markers. In this research, in order to decrease the process of selecting superior genotypes, progenies obtained from crosses between 5 different cultivars were examined using IRAP and RAPD markers. Among 299 bands produced in RAPD, 86% of the bands were polymorphic. The average number of polymorphic bands was 13.5 bands per primer and the minimum genetic similarity (43%) was obtained between 'Sevilla'x'Sevilla' and 'Manila'x'Bombay' hybrids, while the maximum similarity (72%) was found between 'Sevilla'x 'Okayama' and 'Okayama'x'Sevilla' hybrids based on Nei similarity coefficient. From 6 selected IRAP primer combinations, 83 bands were produced, among them 72 bands were considered polymorphic bands. The highest ratio of polymorphism was obtained by 3'LTRLTR6150, 3'LTR-3'LTR primers combination and the lowest by Sukkula -3'LTR. The maximum genetic similarity, 82%, using IRAP marker was observed between 'Sevilla'x'Okayama' and 'Sevilla'x'Manila' hybrids and the lowest amount, 32%, was obtained between 'Sevilla'x'Sevilla' and 'Manila'x'Bombay' hybrids, indicating the genetic proximity and distance, respectively, of the studied genotypes. Recombined genotypes obtained in this research, which had different band patterns with their parents, can be used for breeding programs and introducing new cultivars. [ABSTRACT FROM AUTHOR]

Abstract Allium is one of the well-known genera of the Amaryllidaceae family, which contains over 780 species. Onions, garlic, leeks, and shallots are the most important species of this genus. Allium hirtifolium (shallot) is a rich source of proteins, carbohydrates, lipids, amino acids, and bioactive compounds such as organic sulfur compounds with an expansive range of biological activities and medicinal attributes. To identify the putative compounds and genes involved in the organic sulfur pathway, we applied GC–MS and RNA-seq techniques for the bulb, stem, and flower tissues of A. hirtifolium. The essential oil analysis revealed the maximum amount of sulfur compounds in stem against flower and bulb tissues. Transcriptome profiling showed 6155, 6494, and 4259 DEGs for bulb vs. flower, bulb vs. stem, and flower vs. stem, respectively. Overall, more genes were identified as being up-regulated rather than down-regulated in flower tissue compared to the stem and bulb tissues. Our findings in accordance with other results from different papers, suggest that carbohydrates are vital to bulb formation and development because a high number of identified DEGs (586 genes) were mapped to carbohydrate metabolism. This study has detected the genes in the organic sulfur pathway and indicated that the alliinase gene shows a high variability among different tissues. In general, this study formed a useful genomic resource data to explore tissue-specific sulfur pathway in A. hirtifolium, which is helpful for functional breeding.

Soybean (Glycine max) is a major plant protein source and oilseed crop. However, plant-parasitic nematodes (PPNs) affect its annual yield. In the current study, in order to better understand the regulation of defense mechanism against PPNs in soybean, we investigated the role of long non-coding RNAs (lncRNAs) in response to two nematode species, Heterodera glycines (SCN: soybean cyst nematode) and Rotylenchulus reniformis (reniform). To this end, two publicly available RNA-seq data sets (SCN data set and RAD: reniform-associated data set) were employed to discover the lncRNAome profile of soybean under SCN and reniform infection, respectively. Upon identification of unannotated transcripts in these data sets, a seven-step pipeline was utilized to sieve these transcripts, which ended up in 384 and 283 potential lncRNAs in SCN data set and RAD, respectively. These transcripts were then used to predict cis and trans nematode-related targets in soybean genome. Computational prediction of target genes function, some of which were also among differentially expressed genes, revealed the involvement of putative nematode-responsive genes as well as enrichment of multiple stress responses in both data sets. Finally, 15 and six lncRNAs were proposed to be involved in microRNA-mediated regulation of gene expression in soybean in response to SNC and reniform infection, respectively. Collectively, this study provides a novel insight into the signaling and regulatory network of soybean-pathogen interactions and opens a new window for further research.

Cold-acclimated and non-acclimated contrasting Camelina (Camelina sativa L.) biotypes were investigated for changes in stress-associated biomarkers, including antioxidant enzyme activity, lipid peroxidation, protein, and proline content. In addition, a well-known freezing tolerance pathway participant known as C-repeat/DRE-binding factors (CBFs), an inducer of CBF expression (ICE1), and a cold-regulated (COR6.6) genes of the ICE-CBF-COR pathway were studied at the transcriptional level on the doubled-haploid (DH) lines. Freezing stress had significant effects on all studied parameters. The cold-acclimated DH34 (a freezing-tolerant line) showed an overall better performance under freezing stress than non-acclimated plants. The non-cold-acclimated DH08 (a frost-sensitive line) showed the highest electrolyte leakage after freezing stress. The highest activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) was also detected in non-acclimated plants, whereas the cold-acclimated plants showed lower enzyme activities upon stress treatment. Cold acclimation had a significantly positive effect on the total protein and proline content of stressed plants. The qRT-PCR analysis revealed significant differences in the expression and cold-inducibility of CsCBF1-3, CsICE1, and CsCOR6.6 genes among the samples of different treatments. The highest expression of all CBF genes was recorded in the non-acclimated frost-tolerant biotype after freezing stress. Interestingly a significantly higher expression of COR6.6 was detected in cold-acclimated samples of both frost-sensitive and -tolerant biotypes after freezing stress. The presented results provide more insights into freezing tolerance mechanisms in the Camelina plant from both a biochemical point of view and the expression of the associated genes.

Common bean (Phaseolus vulgaris L.) is a major source of proteins and one of the most important edible foods for more than three hundred million people in the world. The common bean plants are frequently attacked by spider mite (Tetranychus urticae Koch), leading to a significant decrease in plant growth and economic performance. The use of resistant cultivars and the identification of the genes involved in plant-mite resistance are practical solutions to this problem. Hence, a comprehensive study of the molecular interactions between resistant and susceptible common bean cultivars and spider mite can shed light into the understanding of mechanisms and biological pathways of resistance. In this study, one resistant (Naz) and one susceptible (Akhtar) cultivars were selected for a transcriptome comparison at different time points (0, 1 and 5 days) after spider mite feeding. The comparison of cultivars in different time points revealed several key genes, which showed a change increase in transcript abundance via spider mite infestation. These included genes involved in flavonoid biosynthesis process; a conserved MYB-bHLH-WD40 (MBW) regulatory complex; transcription factors (TFs) TT2, TT8, TCP, Cys2/His2-type and C2H2-type zinc finger proteins; the ethylene response factors (ERFs) ERF1 and ERF9; genes related to metabolism of auxin and jasmonic acid (JA); pathogenesis-related (PR) proteins and heat shock proteins.

Camelina (Camelina sativa (L.) Crantz) is an emerging industrial crop from the Brassicaceae family, with its seed oil and cake being used for food, feed, and fuel applications. In this study, the relationships between economically important agronomic traits including seed yield (SY), days to maturity (DM), 1000-seed weight (TSW), seed protein content (PC), seed oil content (OC), and fatty acid composition in 136 doubled haploid (DH) camelina lines were investigated under rain-fed conditions in two consecutive years. There was prominent diversity among the studied DH lines for the agronomic traits such as seed yield, erucic acid, omega3, protein content, etc. Based on the Pearson correlation analysis of the data, SY was positively correlated with DM and OC, and negatively correlated with TSW, PC, and linolenic acid (C18:3) content. The positive relationships of the main characteristics, relevant to industrial applications, suggest the feasibility of developing new higher-yielding camelina cultivars with high seed oil content. The high seed yield of some camelina lines (DH044 and DH075) during the two growing seasons showed the potential of the lines. On the other hand, the contrasting genotypes for key traits in this study promised a favorable source to develop the superior breeding lines with higher seed yield and food/nonfood traits. Therefore, it can be concluded that the diversity of camelina DH lines traits is crucial for developing new cultivars. Furthermore, the present study reports some significant correlations among the DH lines, which may be useful for the current and future camelina breeding program.

Chelidonium majus is a traditional medicinal plant, which commonly known as a rich resource for the major benzylisoquinoline alkaloids (BIAs), including morphine, sanguinarine, and berberine. To understand the biosynthesis of C. majus BIAs, we performed de novo transcriptome sequencing of its leaf and root tissues using Illumina technology. Following comprehensive evaluation of de novo transcriptome assemblies produced with five programs including Trinity, Bridger, BinPacker, IDBA-tran, and Velvet/Oases using a series of k-mer sizes (from 25 to 91), BinPacker was found to produce the best assembly using a k-mer of 25. This study reports the results of differential gene expression (DGE), functional annotation, gene ontology (GO) analysis, classification of transcription factor (TF)s, and SSR and miRNA discovery. Our DGE analysis identified 6,028 transcripts that were up-regulated in the leaf, and 4,722 transcripts that were up-regulated in the root. Further investigations showed that most of the genes involved in the BIA biosynthetic pathway are significantly expressed in the root compared to the leaf. GO analysis showed that the predominant GO domain is "cellular component", while TF analysis found bHLH to be the most highly represented TF family. Our study further identified 10 SSRs, out of a total of 39,841, that showed linkage to five unigenes encoding enzymes in the BIA pathway, and 10 conserved miRNAs that were previously not detected in this plant. The comprehensive transcriptome information presented herein provides a foundation for further explorations on study of the molecular mechanisms of BIA synthesis in C. majus.

In this study, the various concentrations of casein hydrolysate (25, 50, 75, 100 mg/L) and L-phenylalanine (50, 100, 150, 200 µM/l) were incorporated in MS containing 15 µM BA plus 5 µM 2,4-D for enhancement of secondary metabolites in cell culture of Spilanthes acmella. The presence of casein hydrolysate in the nutrient medium improved the growth of cell biomass and the production of scopoletin. The addition of casein hydrolysate up to 75 mg/L stimulated the accumulation of scopoletin, but increasing excess 75 mg/L the level of casein hydrolysate reduced the production of scopoletin. The addition of L-phenylalanine in the nutrient medium was found to be more effective for production of secondary metabolite in S. acmella. The addition of 50 µM/L of L-phenylalanine in the medium increased scopoletin content to 27.12 ± 0.58 µg/g dry weight, compared to the scopoletin content of control at 7.89 ± 0.61 µg/g dry weight. The highest accumulation of scopoletin was observed in the 100 µM/L L-phenylalanine in cell suspension, which was 4.51 times more than the control. As a result, using moderate concentration of L-phenylalanine was ideal for the production of scopoletin. In general, casein hydrolysate was more effective than L-phenylalanine for production of scopoletin and growth of cell biomass in the cell culture of S. acmella.