Integrated Cytological, Physiological, and Transcriptome Analyses Provide Insight into the Albino Phenotype of Chinese Plum (Prunus salicina).

Albino seedlings that arise during seed reproduction can have a significant impact on plant growth and breeding. In this research, we present the first report of albino occurrences in the seed reproduction process of Prunus salicina and describe the cytological, physiological, and transcriptomic changes observed in albino seedlings. The albino seedlings which were observed in several plum cultivars exhibited abnormal chloroplast ultrastructure and perturbed stomatal structure. Compared to normal seedlings, the photosynthetic pigment contents in albino seedlings decreased by more than 90%, accompanied by significant reductions in several chlorophyll fluorescence parameters. Furthermore, substantially changed photosynthetic parameters indicated that the photosynthetic capacity and stomatal function were impaired in albino seedlings. Additionally, the activities of the antioxidant enzyme were drastically altered against the background of higher proline and lower ascorbic acid in leaves of albino seedlings. A total of 4048 differentially expressed genes (DEGs) were identified through transcriptomic sequencing, and the downregulated DEGs in albino seedlings were greatly enriched in the pathways for photosynthetic antenna proteins and flavonoid biosynthesis. GLK1 and Ftsz were identified as candidate genes responsible for the impaired chloroplast development and division in albino seedlings. Additionally, the substantial decline in the expression levels of examined photosystem-related chloroplast genes was validated in albino seedlings. Our findings shed light on the intricate physiological and molecular mechanisms driving albino plum seedling manifestation, which will contribute to improving the reproductive and breeding efforts of plums.

Supplementation of chestnut tannins in diets can improve meat quality and antioxidative capability in Hu lambs.

Chestnut tannins (CNT), as a source of hydrolyzable tannins, positively affect the antioxidant status of livestock. In the current study, 90 male Hu lambs were used to investigate the effect of dietary CNT intake on growth performance, nutrient digestibility, meat quality and oxidative stability, rumen microbial, and the transcriptomes of muscle and liver. A completely randomized design with three CNT intake levels (0, 0.3%, and 0.6%) was used. Rumen microbial and nutrient digestibility were not significantly altered by CNT intake. Diets with 0.3% CNT intake significantly reduced the shear force, yellowness at 24 h, and C20:2 polyunsaturated fatty acids of lamb meat and malondialdehyde in serum and longissimus thoracis (LT) muscle. Meanwhile, the 0.3% CNT diet significantly increased average daily gain during the 1- 21 days and 64- 90 days, dry matter intake during the 1- 21 days, the slaughter weight, and liver index of lambs. The 0.3% CNT diet significantly increased C26:0 saturated fatty acids, total antioxidant capacity, glutathione peroxidase, superoxide dismutase, and catalase in LT muscle. The meat shelf life of 0.3% CNT and 0.6% CNT groups was prolonged by 8.7 h and 5.4 h, respectively. Transcriptomic analysis revealed that CNT supplementation can induce the expression of antioxidant enzyme gene (CAT, SOD1), and the differentially expressed genes were mainly involved in antioxidant activity, transferase activity, and adenosine triphosphate binding. These results suggest that 0.3% CNT intake can relieve the oxidative stress of lambs, and improve the stability of meat color and meat tenderness, due to the enhanced antioxidative capacity.

Cellular Morphology and Transcriptome Comparative Analysis of Astragalus membranaceus Bunge Sprouts Cultured In Vitro under Different LED Light.

Astragalus membranaceus, the major components of which are saponins, flavonoids, and polysaccharides, has been established to have excellent pharmacological activity. After ginseng, it is the second most used medicinal plant. To examine the utility of A. membranaceus as a sprout crop for plant factory cultivation, we sought to establish a functional substance control model by comparing the transcriptomes of sprouts grown in sterile, in vitro culture using LED light sources. Having sown the seeds of A. membranaceus, these were exposed to white LED light (continuous spectrum), red LED light (632 nm, 1.58 µmol/m2/s), or blue LED light (465 nm, 1.44 µmol/m2/s) and grown for 6 weeks; after which, the samples were collected for transcriptome analysis. Scanning electron microscopy analysis of cell morphology in plants exposed to the three light sources revealed that leaf cell size was largest in those plants exposed to red light, where the thickest stem was observed in plants exposed to white light. The total number of genes in A. membranaceus spouts determined via de novo assembly was 45,667. Analysis of differentially expressed genes revealed that for the comparisons of blue LED vs. red LED, blue LED vs. white LED, and red LED vs. white LED, the numbers of upregulated genes were 132, 148, and 144, respectively. Binding, DNA integration, transport, phosphorylation, DNA biosynthetic process, membrane, and plant-type secondary cell wall biogenesis were the most enriched in the comparative analysis of blue LED vs. red LED, whereas Binding, RNA-templated DNA biosynthetic process, DNA metabolic process, and DNA integration were the most enriched in the comparative analysis of blue vs. white LED, and DNA integration and resolution of meiotic recombination intermediates were the most enrichment in the comparison between red LED vs. white LED. The GO term associated with flavonoid biosynthesis, implying the functionality of A. membranaceus, was the flavonoid biosynthetic process, which was enriched in the white LED vs. red LED comparison. The findings of this study thus indicate that different LED light sources can differentially influence the transcriptome expression pattern of A. membranaceus sprouts, which can provide a basis for establishing a flavonoid biosynthesis regulation model and thus, the cultivation of high-functional Astragalus sprouts.

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.

Transcriptome reveals insights into biosynthesis of ginseng polysaccharides.

BACKGROUND: Ginseng polysaccharides, have been used to treat various diseases as an important active ingredient. Nevertheless, the biosynthesis of ginseng polysaccharides is poorly understood. To elucidate the biosynthesis mechanism of ginseng polysaccharides, combined the transcriptome analysis and polysaccharides content determination were performed on the roots, stems, and leaves collected from four cultivars of ginseng. RESULTS: The results indicated that the total contents of nine monosaccharides were highest in the roots. Moreover, the total content of nine monosaccharides in the roots of the four cultivars were different but similar in stems and leaves. Glucose (Glc) was the most component of all monosaccharides. In total, 19 potential enzymes synthesizing of ginseng polysaccharides were identified, and 17 enzymes were significantly associated with polysaccharides content. Among these genes, the expression of phosphoglucomutase (PGM), glucose-6-phosphate isomerase (GPI), UTP-glucose-1-phosphate uridylyltransferase (UGP2), fructokinase (scrK), mannose-1-phosphate guanylyltransferase (GMPP), phosphomannomutase (PMM), UDP-glucose 4-epimerase (GALE), beta-fructofuranosidase (sacA), and sucrose synthase (SUS) were correlated with that of MYB, AP2/ERF, bZIP, and NAC transcription factors (TFs). These TFs may regulate the expression of genes involved in ginseng polysaccharides synthesis. CONCLUSION: Our findings could provide insight into a better understanding of the regulatory mechanism of polysaccharides biosynthesis, and would drive progress in genetic improvement and plantation development of ginseng.

Identifying Terpenoid Biosynthesis Genes in Euphorbia maculata via Full-Length cDNA Sequencing.

The annual herb Euphorbia maculata L. produces anti-inflammatory and biologically active substances such as triterpenoids, tannins, and polyphenols, and it is used in traditional Chinese medicine. Of these bioactive compounds, terpenoids, also called isoprenoids, are major secondary metabolites in E. maculata. Full-length cDNA sequencing was carried out to characterize the transcripts of terpenoid biosynthesis reference genes and determine the copy numbers of their isoforms using PacBio SMRT sequencing technology. The Illumina short-read sequencing platform was also employed to identify differentially expressed genes (DEGs) in the secondary metabolite pathways from leaves, roots, and stems. PacBio generated 62 million polymerase reads, resulting in 81,433 high-quality reads. From these high-quality reads, we reconstructed a genome of 20,722 genes, in which 20,246 genes (97.8%) did not have paralogs. About 33% of the identified genes had two or more isoforms. DEG analysis revealed that the expression level differed among gene paralogs in the leaf, stem, and root. Whole sets of paralogs and isoforms were identified in the mevalonic acid (MVA), methylerythritol phosphate (MEP), and terpenoid biosynthesis pathways in the E. maculata L. The nucleotide information will be useful for identifying orthologous genes in other terpenoid-producing medicinal plants.

Full-length transcriptome sequencing reveals the molecular mechanism of potato seedlings responding to low-temperature.

BACKGROUND: Potato (Solanum tuberosum L.) is one of the world's most important crops, the cultivated potato is frost-sensitive, and low-temperature severely influences potato production. However, the mechanism by which potato responds to low-temperature stress is unclear. In this research, we apply a combination of second-generation sequencing and third-generation sequencing technologies to sequence full-length transcriptomes in low-temperature-sensitive cultivars to identify the important genes and main pathways related to low-temperature resistance. RESULTS: In this study, we obtained 41,016 high-quality transcripts, which included 15,189 putative new transcripts. Amongst them, we identified 11,665 open reading frames, 6085 simple sequence repeats out of the potato dataset. We used public available genomic contigs to analyze the gene features, simple sequence repeat, and alternative splicing event of 24,658 non-redundant transcript sequences, predicted the coding sequence and identified the alternative polyadenylation. We performed cluster analysis, GO, and KEGG functional analysis of 4518 genes that were differentially expressed between the different low-temperature treatments. We examined 36 transcription factor families and identified 542 transcription factors in the differentially expressed genes, and 64 transcription factors were found in the AP2 transcription factor family which was the most. We measured the malondialdehyde, soluble sugar, and proline contents and the expression genes changed associated with low temperature resistance in the low-temperature treated leaves. We also tentatively speculate that StLPIN10369.5 and StCDPK16 may play a central coordinating role in the response of potatoes to low temperature stress. CONCLUSIONS: Overall, this study provided the first large-scale full-length transcriptome sequencing of potato and will facilitate structure-function genetic and comparative genomics studies of this important crop.

Cold-induced secondary dormancy and its regulatory mechanisms in Beta vulgaris.

The dynamic behaviour of seeds in soil seed banks depends on their ability to act as sophisticated environmental sensors to adjust their sensitivity thresholds for germination by dormancy mechanisms. Here we show that prolonged incubation of sugar beet fruits at low temperature (chilling at 5°C, generally known to release seed dormancy of many species) can induce secondary nondeep physiological dormancy of an apparently nondormant crop species. The physiological and biophysical mechanisms underpinning this cold-induced secondary dormancy include the chilling-induced accumulation of abscisic acid in the seeds, a reduction in the embryo growth potential and a block in weakening of the endosperm covering the embryonic root. Transcriptome analysis revealed distinct gene expression patterns in the different temperature regimes and upon secondary dormancy induction and maintenance. The chilling caused reduced expression of cell wall remodelling protein genes required for embryo cell elongation growth and endosperm weakening, as well as increased expression of seed maturation genes, such as for late embryogenesis abundant proteins. A model integrating the hormonal signalling and master regulator expression with the temperature-control of seed dormancy and maturation programmes is proposed. The revealed mechanisms of the cold-induced secondary dormancy are important for climate-smart agriculture and food security.

Transcriptome analysis of genes expressed in the earthworm Eisenia fetida in response to cadmium exposure.

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.

Diverse Sorghum bicolor accessions show marked variation in growth and transcriptional responses to arbuscular mycorrhizal fungi.

Sorghum is an important crop grown worldwide for feed and fibre. Like most plants, it has the capacity to benefit from symbioses with arbuscular mycorrhizal (AM) fungi, and its diverse genotypes likely vary in their responses. Currently, the genetic basis of mycorrhiza-responsiveness is largely unknown. Here, we investigated transcriptional and physiological responses of sorghum accessions, founders of a bioenergy nested association mapping panel, for their responses to four species of AM fungi. Transcriptome comparisons across four accessions identified mycorrhiza-inducible genes; stringent filtering criteria revealed 278 genes that show mycorrhiza-inducible expression independent of genotype and 55 genes whose expression varies with genotype. The latter suggests variation in phosphate transport and defence across these accessions. The mycorrhiza growth and nutrient responses of 18 sorghum accessions varied tremendously, ranging from mycorrhiza-dependent to negatively mycorrhiza-responsive. Additionally, accessions varied in the number of AM fungi to which they showed positive responses, from one to several fungal species. Mycorrhiza growth and phosphorus responses were positively correlated, whereas expression of two mycorrhiza-inducible phosphate transporters, SbPT8 and SbPT9, correlated negatively with mycorrhizal growth responses. AM fungi improve growth and mineral nutrition of sorghum, and the substantial variation between lines provides the potential to map loci influencing mycorrhiza responses.

Effects of dietary administration of Lactococcus lactis HNL12 on growth, innate immune response, and disease resistance of humpback grouper (Cromileptes altivelis).

Lactic acid bacteria are a common group of probiotics that have been widely studied and used in aquaculture. In the present study, we isolated Lactococcus lactis HNL12 from the gut of wild humpback grouper (Cromileptes altivelis) and explored its probiotic properties. For this purpose, L. lactis HNL12 was added to the commercial fish feed. The results showed that HNL12 had high auto-aggregation ability and strong tolerance to simulated gastrointestinal stress. When C. altivelis consumed a diet containing 0 (control), 106, 108, or 1010 CFU/g HNL12 for four weeks, all of the groupers fed a diet with HNL12 had significantly increased percent weight gain (PWG), especially those fed with 108 CFU/g, which had a PWG of 231.45%. Compared to the control, fish fed with L. lactis HNL12 exhibited significantly increased survival rates following injection with Vibrio harveyi after one month. Immunological analysis showed that C. altivelis fed with HNL12 had (i) enhanced respiratory burst activity of head kidney macrophages, superoxide dismutase, acid phosphatase, and lysozyme activities of serum; (ii) an improved survival rate from 36% to 70%; and (iii) upregulated expression of a broad spectrum of immunity. Meanwhile, de novo transcriptome assembly yielded 89,314 unigenes, which were annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG and KEGG). A total of 307 genes showed significantly different expression between the groups fed with or without added HNL12. GO and KEGG enrichment analyses of the significantly different expression gene categories and pathways were related to infectious diseases, antigen processing and presentation, and other immune system responses. These results indicate that L. lactis HNL12 is effective for enhancing the growth, immunity, and disease resistance of C. altivelis; this study also provides insight into the use of probiotics for commercial applications.

Modular Design of Picroside-II Biosynthesis Deciphered through NGS Transcriptomes and Metabolic Intermediates Analysis in Naturally Variant Chemotypes of a Medicinal Herb, Picrorhiza kurroa.

Picroside-II (P-II), an iridoid glycoside, is used as an active ingredient of various commercial herbal formulations available for the treatment of liver ailments. Despite this, the knowledge of P-II biosynthesis remains scarce owing to its negligence in Picrorhiza kurroa shoots which sets constant barrier for function validation experiments. In this study, we utilized natural variation for P-II content in stolon tissues of different P. kurroa accessions and deciphered its metabolic route by integrating metabolomics of intermediates with differential NGS transcriptomes. Upon navigating through high vs. low P-II content accessions (1.3-2.6%), we have established that P-II is biosynthesized via degradation of ferulic acid (FA) to produce vanillic acid (VA) which acts as its immediate biosynthetic precursor. Moreover, the FA treatment in vitro at 150 µM concentration provided further confirmation with 2-fold rise in VA content. Interestingly, the cross-talk between different compartments of P. kurroa, i.e., shoots and stolons, resolved spatial complexity of P-II biosynthesis and consequently speculated the burgeoning necessity to bridge gap between VA and P-II production in P. kurroa shoots. This work thus, offers a forward looking strategy to produce both P-I and P-II in shoot cultures, a step toward providing a sustainable production platform for these medicinal compounds via-à-vis relieving pressure from natural habitat of P. kurroa.

Transcriptomic analysis links gene expression to unilateral pollen-pistil reproductive barriers.

BACKGROUND: Unilateral incompatibility (UI) is an asymmetric reproductive barrier that unidirectionally prevents gene flow between species and/or populations. UI is characterized by a compatible interaction between partners in one direction, but in the reciprocal cross fertilization fails, generally due to pollen tube rejection by the pistil. Although UI has long been observed in crosses between different species, the underlying molecular mechanisms are only beginning to be characterized. The wild tomato relative Solanum habrochaites provides a unique study system to investigate the molecular basis of this reproductive barrier, as populations within the species exhibit both interspecific and interpopulation UI. Here we utilized a transcriptomic approach to identify genes in both pollen and pistil tissues that may be key players in UI. RESULTS: We confirmed UI at the pollen-pistil level between a self-incompatible population and a self-compatible population of S. habrochaites. A comparison of gene expression between pollinated styles exhibiting the incompatibility response and unpollinated controls revealed only a small number of differentially expressed transcripts. Many more differences in transcript profiles were identified between UI-competent versus UI-compromised reproductive tissues. A number of intriguing candidate genes were highly differentially expressed, including a putative pollen arabinogalactan protein, a stylar Kunitz family protease inhibitor, and a stylar peptide hormone Rapid ALkalinization Factor. Our data also provide transcriptomic evidence that fundamental processes including reactive oxygen species (ROS) signaling are likely key in UI pollen-pistil interactions between both populations and species. CONCLUSIONS: Gene expression analysis of reproductive tissues allowed us to better understand the molecular basis of interpopulation incompatibility at the level of pollen-pistil interactions. Our transcriptomic analysis highlighted specific genes, including those in ROS signaling pathways that warrant further study in investigations of UI. To our knowledge, this is the first report to identify candidate genes involved in unilateral barriers between populations within a species.

Analysis of multiple transcriptomes of the African oil palm (Elaeis guineensis) to identify reference genes for RT-qPCR.

The African oil palm (Elaeis guineensis), which is grown in tropical and subtropical regions, is a highly productive oil-bearing crop. For gene expression-based analyses such as reverse transcription-quantitative real time PCR (RT-qPCR), reference genes are essential to provide a baseline with which to quantify relative gene expression. Normalization using reliable reference genes is critical in correctly interpreting expression data from RT-qPCR. In order to identify suitable reference genes in African oil palm, 17 transcriptomes of different tissues obtained from NCBI were systematically assessed for gene expression variation. In total, 53 putative candidate reference genes with coefficient of variation values <3.0 were identified: 18 in reproductive tissue and 35 in vegetative tissue. Analysis for enriched functions showed that approximately 90% of identified genes were clustered in cell component gene functions, and 12 out of 53 genes were traditional housekeeping genes. We selected and validated 16 reference genes chosen from leaf tissue transcriptomes by using RT-qPCR in sets of cold, drought and high salinity treated samples, and ranked expression stability using statistical algorithms geNorm, Normfinder and Bestkeeper. Genes encoding actin, adenine phosphoribosyltransferase and eukaryotic initiation factor 4A genes were the most stable genes over the cold, drought and high salinity stresses. Identification of stably expressed genes as reference gene candidates from multiple transcriptome datasets was found to be reliable and efficient, and some traditional housekeeping genes were more stably expressed than others. We provide a useful molecular genetic resource for future gene expression studies in African oil palm, facilitating molecular genetics approaches for crop improvement in this species.