RNA-seq analysis reveals narrow differential gene expression in MEP and MVA pathways responsible for phytochemical divergence in extreme genotypes of Thymus daenensis Celak.

BACKGROUND: Here, we investigated the underlying transcriptional-level evidence behind phytochemical differences between two metabolically extreme genotypes of Thymus daenensis. The genotypes 'Zagheh-11' (thymol/carvacrol type, poor in essential oil [EO] [2.9%] but rich in triterpenic acids) and 'Malayer-21' (thymol type and rich in EO [3.8%]) were selected from an ongoing breeding program and then clonally propagated for further experimental use. MATERIALS AND METHODS: GC-MS, GC-FID, and HPLC-PDA were utilized to monitor the fluctuation of secondary metabolites at four phenological stages (vegetative, bud burst, early, and full-flowering stages). The highest phytochemical divergence was observed at early flowering stage. Both genotypes were subjected to mRNA sequencing (approximately 100 million paired reads) at the aforementioned stage. The expression patterns of four key genes involved in the biosynthesis of terpenoids were also validated using qRT-PCR. RESULTS: Carvacrol content in 'Zagheh-11' (26.13%) was approximately 23 times higher than 'Malayer-21' (1.12%). Reciprocally, about 10% higher thymol was found in 'Malayer-21' (62.15%). Moreover, the concentrations of three major triterpenic acids in 'Zagheh-11' were approximately as twice as those found in 'Malayer-21'. Transcriptome analysis revealed a total of 1840 unigenes that were differentially expressed, including terpene synthases, cytochrome P450, and terpenoid backbone genes. Several differentially expressed transcription factors (such as MYB, bZIP, HB-HD-ZIP, and WRKY families) were also identified. These results suggest that an active cytosolic mevalonate (MVA) pathway may be linked to higher levels of sesquiterpenes, triterpenic acids, and carvacrol in 'Zagheh-11'. The chloroplastic pathway of methyl erythritol phosphate (MEP) may have also contributed to a higher accumulation of thymol in Malayer-21. Indeed, 'Zagheh-11' showed higher expression of certain genes (HMGR, CYP71D180, ß-amyrin 28-monooxygenase, and sesquiterpene synthases) in the MVA pathway, while some genes in the MEP pathway (including DXR, ispG, and γ-terpinene synthase) were distinctly expressed in Malayer-21. Future efforts in metabolic engineering of MVA/MEP pathways may benefit from these findings to produce increased levels of desired secondary metabolites at commercial scale.

Optimizing an efficient ensemble approach for high-quality de novo transcriptome assembly of Thymus daenensis.

Non-erroneous and well-optimized transcriptome assembly is a crucial prerequisite for authentic downstream analyses. Each de novo assembler has its own algorithm-dependent pros and cons to handle the assembly issues and should be specifically tested for each dataset. Here, we examined efficiency of seven state-of-art assemblers on ~ 30 Gb data obtained from mRNA-sequencing of Thymus daenensis. In an ensemble workflow, combining the outputs of different assemblers associated with an additional redundancy-reducing step could generate an optimized outcome in terms of completeness, annotatability, and ORF richness. Based on the normalized scores of 16 benchmarking metrics, EvidentialGene, BinPacker, Trinity, rnaSPAdes, CAP3, IDBA-trans, and Velvet-Oases performed better, respectively. EvidentialGene, as the best assembler, totally produced 316,786 transcripts, of which 235,730 (74%) were predicted to have a unique protein hit (on uniref100), and also half of its transcripts contained an ORF. The total number of unique BLAST hits for EvidentialGene was approximately three times greater than that of the worst assembler (Velvet-Oases). EvidentialGene could even capture 17% and 7% more average BLAST hits than BinPacker and Trinity. Although BinPacker and CAP3 produced longer transcripts, the EvidentialGene showed a higher collinearity between transcript size and ORF length. Compared with the other programs, EvidentialGene yielded a higher number of optimal transcript sets, further full-length transcripts, and lower possible misassemblies. Our finding corroborates that in non-model species, relying on a single assembler may not give an entirely satisfactory result. Therefore, this study proposes an ensemble approach of accompanying EvidentialGene pipelines to acquire a superior assembly for T. daenensis.

Normalized Workflow to Optimize Hybrid De Novo Transcriptome Assembly for Non-Model Species: A Case Study in Lilium ledebourii (Baker) Boiss.

A high-quality transcriptome is required to advance numerous bioinformatics workflows. Nevertheless, the effectuality of tools for de novo assembly and real precision assembled transcriptomes looks somewhat unexplored, particularly for non-model organisms with complicated (very long, heterozygous, polyploid) genomes. To disclose the performance of various transcriptome assembly programs, this study built 11 single assemblies and analyzed their performance on some significant reference-free and reference-based criteria. As well as to reconfirm the outputs of benchmarks, 55 BLAST were performed and compared using 11 constructed transcriptomes. Concisely, normalized benchmarking demonstrated that Velvet-Oases suffer from the worst results, while the EvidentialGene strategy can provide the most comprehensive and accurate transcriptome of Lilium ledebourii (Baker) Boiss. The BLAST results also confirmed the superiority of EvidentialGene, so it could capture even up to 59% more (than Velvet-Oases) unique gene hits. To promote assembly optimization, with the help of normalized benchmarking, PCA and AHC, it is emphasized that each metric can only provide part of the transcriptome status, and one should never settle for just a few evaluation criteria. This study supplies a framework for benchmarking and optimizing the efficiency of assembly approaches to analyze RNA-Seq data and reveals that selecting an inefficient assembly strategy might result in less identification of unique gene hits.

Complete chloroplast genome of Lilium ledebourii (Baker) Boiss and its comparative analysis: lights into selective pressure and adaptive evolution.

Lilium ledebourii (Baker) Boiss is a rare species, which exhibits valuable traits. However, before its genetic diversity and evolutionary were uncovered, its wild resources were jeopardized. Moreover, some ambiguities in phylogenetic relationships of this genus remain unresolved. Therefore, obtaining the whole chloroplast sequences of L. ledebourii and its comparative analysis along with other Lilium species is crucial and pivotal to understanding the evolution of this genus as well as the genetic populations. A multi-scale genome-level analysis, especially selection pressure, was conducted. Detailed third­generation sequencing and analysis revealed a whole chloroplast genome of 151,884 bp, with an ordinary quadripartite and protected structure comprising 37.0% GC. Overall, 113 different genes were recognized in the chloroplast genome, consisting of 30 distinct tRNA genes, four distinct ribosomal RNAs genes, and 79 unique protein-encoding genes. Here, 3234 SSRs and 2053 complex repeats were identified, and a comprehensive analysis was performed for IR expansion and contraction, and codon usage bias. Moreover, genome-wide sliding window analysis revealed the variability of rpl32-trnL-ccsA, petD-rpoA, ycf1, psbI-trnS-trnG, rps15-ycf1, trnR, trnT-trnL, and trnP-psaJ-rpl33 were higher among the 48 Lilium cp genomes, displaying higher variability of nucleotide in SC regions. Following 1128 pairwise comparisons, ndhB, psbJ, psbZ, and ycf2 exhibit zero synonymous substitution, revealing divergence or genetic restriction. Furthermore, out of 78 protein-coding genes, we found that accD and rpl36 under positive selection: however, at the entire-chloroplast protein scale, the Lilium species have gone through a purifying selection. Also, a new phylogenetic tree for Lilium was rebuilt, and we believe that the Lilium classification is clearer than before. The genetic resources provided here will aid future studies in species identification, population genetics, and Lilium conservation.

Phytosulfokine α (PSKα) delays senescence and reinforces SUMO1/SUMO E3 ligase SIZ1 signaling pathway in cut rose flowers (Rosa hybrida cv. Angelina).

Roses are widely used as cut flowers worldwide. Petal senescence confines the decorative quality of cut rose flowers, an impressively considerable economic loss. Herein, we investigated the SUMO1/SUMO E3 ligase SIZ1 signaling pathway during bud opening, and petal senescence of cut rose flowers. Our results exhibited that the higher expression of SUMO1 and SUMO E3 ligase SIZ1 during bud opening was accompanied by lower endogenous H2O2 accumulation arising from higher expression and activities of SOD, CAT, APX, and GR, promoting proline accumulation by increasing P5CS expression and activity and enhancing GABA accumulation by increasing GAD expression and activity. In harvested flowers, lower expressions of SUMO1 and SUMO E3 ligase SIZ1 during petal senescence were associated with higher endogenous H2O2 accumulation due to lower expression and activities of SOD, CAT, APX, and GR. Therefore, promoting the activity of the GABA shunt pathway as realized by higher expression and activities of GABA-T and SSADH accompanied by increasing OAT expression and activity for sufficiently supply proline in rose flowers during petal senescence might serve as an endogenous antisenescence mechanism for slowing down petals senescence by avoiding endogenous H2O2 accumulation. Following phytosulfokine α (PSKα) application, postponing petal senescence in cut rose flowers could be ascribed to higher expression of SUMO1 and SUMO E3 ligase SIZ1 accompanied by higher expression and activities of SOD, CAT, APX, and GR, higher activity of GABA shunt pathway as realized by higher expression and activities of GAD, GABA-T, and SSADH, higher expression and activities of P5CS and OAT for supplying proline and higher expression of HSP70 and HSP90. Therefore, our results highlight the potential of the PSKα as a promising antisenescence signaling peptide in the floriculture industry for postponing senescence and extending the vase life of cut rose flowers.

Employing exogenous melatonin applying confers chilling tolerance in tomato fruits by upregulating ZAT2/6/12 giving rise to promoting endogenous polyamines, proline, and nitric oxide accumulation by triggering arginine pathway activity.

In the present study, the mechanisms employed by exogenous melatonin applying for conferring chilling tolerance in tomato fruits during storage at 4 °C for 28 days were investigated. Conferring chilling tolerance in tomato fruits in response to exogenous melatonin applying at 100 µM may arise from upregulating SlZAT2/6/12 giving rise to triggering CBF1 gene expression. Employing higher arginine pathway activity in tomato fruits by exogenous melatonin applying demonstrating by higher endogenous polyamines accumulation arising from higher ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) genes expression and enzymes activity, higher endogenous proline accumulation arising from higher pyroline 5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) genes expression and enzymes activity accompanying by lower proline dehydrogenase (PDH) gene expression and enzyme activity and higher endogenous nitric oxide (NO) accumulation arising from higher nitric oxide synthase (NOS) gene expression and enzyme activity may be responsible for keeping safe membrane integrity.

Mycorrhizal limonium sinuatum (L.) mill. Enhances accumulation of lead and cadmium.

Heavy metals accumulation in soils poses a potential threat to ecosystems, which, in turn, threat human health through food chains. Therefore, remediating polluted sites is important to environment and humanity. In this investigation, statice (L. sinuatum) was exposed to Cd (0, 15, 30, 60 mg kg(-1) soil) or Pb (0, 100, 150, 300 mg kg(-1) soil) in a pot experiment to assess its tolerance to each metal and study its phytoaccumulation capability. The benefits of mycorrhization (mixture of Glomus mosseae and G. intraradices) were also studied simultaneously. Single exposure to Cd or Pb reduced the plant growth, but statice was still relatively tolerant to both metals. The plants accumulated both metals in their roots; little was translocated to the shoots. Total Pb and total Cd accumulated by the roots was approximately 2 and 3 times higher in mycorrhizal than non-mycorrhizal plants (49 versus 147 and 595 versus 956 µg plant(-1)) respectively; however, mycorrhization alleviated metal phytotoxicity. The results suggest that statice is a potential candidate to be used as an ornamental plant in lead and cadmium polluted sites, mainly inoculated with arbuscular mycorrhizae. Besides that, it would be useful as a Pb or Cd controlling agent by means of phytostabilization.