Evolution of Transcript Abundance is Influenced by Indels in Protein Low Complexity Regions.

Protein Protein low complexity regions (LCRs) are compositionally biased amino acid sequences, many of which have significant evolutionary impacts on the proteins which contain them. They are mutationally unstable experiencing higher rates of indels and substitutions than higher complexity regions. LCRs also impact the expression of their proteins, likely through multiple effects along the path from gene transcription, through translation, and eventual protein degradation. It has been observed that proteins which contain LCRs are associated with elevated transcript abundance (TAb), despite having lower protein abundance. We have gathered and integrated human data to investigate the co-evolution of TAb and LCRs through ancestral reconstructions and model inference using an approximate Bayesian calculation based method. We observe that on short evolutionary timescales TAb evolution is significantly impacted by changes in LCR length, with insertions driving TAb down. But in contrast, the observed data is best explained by indel rates in LCRs which are unaffected by shifts in TAb. Our work demonstrates a coupling between LCR and TAb evolution, and the utility of incorporating multiple responses into evolutionary analyses.

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

Low Complexity Regions in Mammalian Proteins are Associated with Low Protein Abundance and High Transcript Abundance.

Low Complexity Regions (LCRs) are present in a surprisingly large number of eukaryotic proteins. These highly repetitive and compositionally biased sequences are often structurally disordered, bind promiscuously, and evolve rapidly. Frequently studied in terms of evolutionary dynamics, little is known about how LCRs affect the expression of the proteins which contain them. It would be expected that rapidly evolving LCRs are unlikely to be tolerated in strongly conserved, highly abundant proteins, leading to lower overall abundance in proteins which contain LCRs. To test this hypothesis and examine the associations of protein abundance and transcript abundance with the presence of LCRs, we have integrated high-throughput data from across mammals. We have found that LCRs are indeed associated with reduced protein abundance, but are also associated with elevated transcript abundance. These associations are qualitatively consistent across 12 human tissues and nine mammalian species. The differential impacts of LCRs on abundance at the protein and transcript level are not explained by differences in either protein degradation rates or the inefficiency of translation for LCR containing proteins. We suggest that rapidly evolving LCRs are a source of selective pressure on the regulatory mechanisms which maintain steady-state protein abundance levels.

Molecular and physiochemical evaluation of buck semen cryopreserved with antioxidants.

The current study evaluated the physiochemical quality and gene expression profile of post-thawed buck semen after supplementation with antioxidants [melatonin (M), L-carnitine (LC), cysteine (Cys), LC + M, M + Cys, LC + Cys, LC + Cys + M] in comparison with the non-treated control group. Physical and biochemical characteristics of semen were evaluated following freezing and thawing. Transcript abundance of six selected candidate genes was profile using quantitative real-time PCR. The data demonstrated significant enhancement of post-freezing total motility, progressive motility, percentage of live sperm, CASA parameters, plasma membrane and acrosome integrity in all groups supplemented with Cys, LC, M + Cys and LC + Cys compared with the control group. The biochemical analysis of semen indicated that semen groups supplemented with LC and LC + Cys recorded increased levels of GPX and SOD that were coupled with up-regulation of antioxidant genes (SOD1, GPX1 and NRF2) and mitochondrial transcripts (CPT2 and ATP5F1A). Moreover, H2O2 level and DNA fragmentation percentage were reduced compared with other groups. In conclusion, supplementation of Cys alone or in combination with LC positively improved the post-thaw physiochemical properties of rabbit semen through activation of bioenergetics-related mitochondrial genes and cellular antioxidant defence mechanism.

Genome-wide identification and transcriptional characterization of DNA methyltransferases conferring temperature-sensitive male sterility in wheat.

BACKGROUND: DNA methyltransferase (DMT) genes contribute to plant stress responses and development by de novo establishment and subsequent maintenance of DNA methylation during replication. The photoperiod and/or temperature-sensitive genic male sterile (P/TGMS) lines play an important role in hybrid seed production of wheat. However, only a few studies have reported on the effect of DMT genes on temperature-sensitive male sterility of wheat. Although DMT genes have been investigated in some plant species, the identification and analysis of DMT genes in wheat (Triticum aestivum L.) based on genome-wide levels have not been reported. RESULTS: In this study, a detailed overview of phylogeny of 52 wheat DMT (TaDMT) genes was presented. Homoeolog retention for TaDMT genes was significantly above the average retention rate for whole-wheat genes, indicating the functional importance of many DMT homoeologs. We found that the strikingly high number of TaDMT genes resulted mainly from the significant expansion of the TaDRM subfamily. Intriguingly, all 5 paralogs belonged to the wheat DRM subfamily, and we speculated that tandem duplications might play a crucial role in the TaDRM subfamily expansion. Through the transcriptional analysis of TaDMT genes in a TGMS line BS366 and its hybrids with the other six fertile lines under sterile and fertile conditions, we concluded that TaCMT-D2, TaMET1-B1, and TaDRM-U6 might be involved in male sterility in BS366. Furthermore, a correlation analysis showed that TaMET1-B1 might negatively regulate the expression of TaRAFTIN1A, an important gene for pollen development, so we speculated regarding an epigenetic regulatory mechanism underlying the male sterility of BS366 via the interaction between TaMET1-B1 and TaRAFTIN1A. CONCLUSIONS: Our findings presented a detailed phylogenic overview of the DMT genes and could provide novel insights into the effects of DMT genes on TGMS wheat.

The U-to-C RNA editing affects the mRNA stability of nuclear genes in Arabidopsis thaliana.

Cytidine-to-uridine (C-to-U) RNA editing has been generally observed in land plants; however, reverse (U-to-C) RNA editing is a rare phenomenon. In this study, we investigated the U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability, with a special focus on PPR proteins. A previous study showed the extensive occurrence of U-to-C RNA editing in 12-day and 20-dayold Arabidopsis seedlings. Here, we have demonstrated the effects of this "reverse" RNA editing on the mRNA stability for all seven edited genes. We also identified U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. The U-to-C RNA editing sites were found in the untranslated region (3' UTR) of the mature mRNA and may affect its secondary structure. We also examined the correlation between U-to-C RNA editing-related genes and their mRNA abundance. Furthermore, we investigated the effects of U-to-C RNA editing in Arabidopsis using the transcription inhibitor actinomycin D (Act D). The addition of Act D to the seedlings of transgenic Arabidopsis generated by Agrobacterium-mediated transformation showed that single nucleotide base conversion adversely affected the mRNA secondary structure and stability.

System-level analyses of keystone genes required for mammalian tooth development.

When a null mutation of a gene causes a complete developmental arrest, the gene is typically considered essential for life. Yet, in most cases, null mutations have more subtle effects on the phenotype. Here we used the phenotypic severity of mutations as a tool to examine system-level dynamics of gene expression. We classify genes required for the normal development of the mouse molar into different categories that range from essential to subtle modification of the phenotype. Collectively, we call these the developmental keystone genes. Transcriptome profiling using microarray and RNAseq analyses of patterning stage mouse molars show highly elevated expression levels for genes essential for the progression of tooth development, a result reminiscent of essential genes in single-cell organisms. Elevated expression levels of progression genes were also detected in developing rat molars, suggesting evolutionary conservation of this system-level dynamics. Single-cell RNAseq analyses of developing mouse molars reveal that even though the size of the expression domain, measured in the number of cells, is the main driver of organ-level expression, progression genes show high cell-level transcript abundances. Progression genes are also upregulated within their pathways, which themselves are highly expressed. In contrast, a high proportion of the genes required for normal tooth patterning are secreted ligands that are expressed in fewer cells than their receptors and intracellular components. Overall, even though expression patterns of individual genes can be highly different, conserved system-level principles of gene expression can be detected using phenotypically defined gene categories.

Sequence polarity between the promoter and the adjacent gene modulates promoter activity.

Promoter engineering has been employed as a strategy to enhance and optimize the production of bio-products. Availability of promoters with predictable activities is needed for downstream application. However, whether promoter activity remains the same in different gene contexts remains unknown. Six consecutive promoters that have previously been determined to have different activity levels were used to construct six different versions of plasmid backbone pTH1227, followed by inserted genes encoding two polymer-producing enzymes. In some cases, promoter activity in the presence of inserted genes did not correspond to the reported activity levels in a previous study. After removing the inserted genes, the activity of these promoters returned to their previously reported level. These changes were further confirmed to occur at the transcriptional level. Polymer production using our newly constructed plasmids showed polymer accumulation levels corresponding to the promoter activity reported in our study. Our study demonstrated the importance of re-assessing promoter activity levels with regard to gene context, which could influence promoter activity, leading to different outcomes in downstream applications.

Reproductive health and endocrine disruption in smallmouth bass (Micropterus dolomieu) from the Lake Erie drainage, Pennsylvania, USA.

Smallmouth bass Micropterus dolomieu were sampled from three sites within the Lake Erie drainage (Elk Creek, Twentymile Creek, and Misery Bay, an embayment in Presque Isle Bay). Plasma, tissues for histopathological analyses, and liver and testes preserved in RNALater® were sampled from 30 smallmouth bass (of both sexes) at each site. Liver and testes samples were analyzed for transcript abundance with Nanostring nCounter® technology. Evidence of estrogenic endocrine disruption was assessed by the presence and severity of intersex (testicular oocytes; TO) and concentrations of plasma vitellogenin in male fish. Abundance of 17 liver transcripts associated with reproductive function, endocrine activity, and contaminant detoxification pathways and 40 testes transcripts associated with male and female reproductive function, germ cell development, and steroid biosynthesis were also measured. Males with a high rate of TO (87-100%) and plasma vitellogenin were noted at all sites; however, TO severity was greatest at the site with the highest agricultural land cover. Numerous transcripts were differentially regulated among the sites and patterns of transcript abundance were used to better understand potential risk factors for estrogenic endocrine disruption. The results of this study suggest endocrine disruption is prevalent in this region and further research would benefit to identify the types of contaminants that may be associated with the observed biological effects.

Hyperpigmented melanistic skin lesions of smallmouth bass Micropterus dolomieu from the Chesapeake Bay watershed.

Hyperpigmented melanistic skin lesions (HPMLs) of smallmouth bass Micropterus dolomieu are observed in the Potomac and Susquehanna rivers, Chesapeake Bay watershed, USA. Routine, nonlethal population surveys were conducted at 8 sites on the mainstem Susquehanna River and 9 on the Juniata River, a tributary of the Susquehanna River, between 2012 and 2018, and the prevalence of HPMLs was documented. A total of 4078 smallmouth bass were collected from the mainstem Susquehanna River and 6478 from the Juniata River. Lesions were primarily seen in bass greater than 200 mm, and prevalence in the Susquehanna River (8%) was higher (p < 0.001) than in the Juniata River (2%). As part of ongoing fish health monitoring projects, smallmouth bass were collected at additional sites, primarily tributaries of the Susquehanna (n = 758) and Potomac (n = 545) rivers between 2013 and 2018. Prevalence in the Susquehanna River (13%) was higher (p < 0.001) than the Potomac (3%). Microscopically, HPMLs were characterized by an increased number of melanocytes in the epidermis or within the dermis and epidermis. RNAseq analyses of normal and melanistic skin identified 3 unique sequences in HPMLs. Two were unidentified and the third was a viral helicase (E1). Transcript abundance in 16 normal skin samples and 16 HPMLs showed upregulation of genes associated with melanogenesis and cell proliferation in HPMLs. The E1 transcript was detected in 12 of the 16 melanistic areas but in no samples from normal skin. Further research will be necessary to identify the putative new virus and determine its role in melanocyte proliferation.

How do cryptochromes and UVR8 interact in natural and simulated sunlight?

Cryptochromes (CRYs) and UV RESISTANCE LOCUS 8 (UVR8) photoreceptors perceive UV-A/blue (315-500 nm) and UV-B (280-315 nm) radiation in plants, respectively. While the roles of CRYs and UVR8 have been studied in separate controlled-environment experiments, little is known about the interaction between these photoreceptors. Here, Arabidopsis wild-type Ler, CRYs and UVR8 photoreceptor mutants (uvr8-2, cry1cry2 and cry1cry2uvr8-2), and a flavonoid biosynthesis-defective mutant (tt4) were grown in a sun simulator. Plants were exposed to filtered radiation for 17 d or for 6 h, to study the effects of blue, UV-A, and UV-B radiation. Both CRYs and UVR8 independently enabled growth and survival of plants under solar levels of UV, while their joint absence was lethal under UV-B. CRYs mediated gene expression under blue light. UVR8 mediated gene expression under UV-B radiation, and in the absence of CRYs, also under UV-A. This negative regulation of UVR8-mediated gene expression by CRYs was also observed for UV-B. The accumulation of flavonoids was also consistent with this interaction between CRYs and UVR8. In conclusion, we provide evidence for an antagonistic interaction between CRYs and UVR8 and a role of UVR8 in UV-A perception.

Redox-regulation of germination during imbibitional oxidative and chilling stress in an indica rice cultivar (Oryza sativa L., Cultivar Ratna).

Imbibitional oxidative stress of different magnitude, imposed by treatment with different titer of H2O2 (both elevated, 20 mM and low, 500 µM) to an indica rice cultivar (Oryza sativa L., Cultivar Ratna) caused formation of differential redox cues at the metabolic interface, as evident from significant alteration of ROS/antioxidant ratio, efficacy of ascorbate-glutathione cycle, radical scavenging property, modulation of total thiol content and expression of oxidative membrane protein and lipid damages as biomarkers of oxidative stress. All the redox parameters examined, substantiate the experimental outcome that treatment with elevated concentration of H2O2 caused serious loss of redox homeostasis and germination impairment, whereas low titre H2O2 treatment not only restored redox homeostasis but also improve germination and post-germinative growth. The inductive pulse of H2O2 (500 µM) exhibited significantly better performance of ascorbate-glutathione pathway, which was otherwise down-regulated significantly in 20 mM H2O2 treatment-raised seedlings. A comparison between imbibitional chilling stress-raised experimental rice seedlings with 20 mM H2O2 treated rice seedling revealed similar kind of generation of redox cues and oxidative stress response. Further, imbibitional H2O2 treatments in rice also revealed a dose-dependent regulation of expression of genes of Halliwell-Asada pathway enzymes, which is in consonance with the redox metabolic response of germinating rice seeds. In conclusion, a dose-dependent regulation of H2O2 mediated redox cues and redox regulatory properties during germination in rice are suggested, the knowledge of which may be exploited as a promising seed priming technology.

A Salt Overly Sensitive Pathway Member from Brassica juncea BjSOS3 Can Functionally Complement ΔAtsos3 in Arabidopsis.

BACKGROUND: Salt Overly Sensitive (SOS) pathway is a well-known pathway in arabidopsis, essential for maintenance of ion homeostasis and thus conferring salt stress tolerance. In arabidopsis, the Ca2+ activated SOS3 interacts with SOS2 which further activates SOS1, a Na+/H+ antiporter, responsible for removing toxic sodium ions from the cells. In the present study, we have shown that these three components of SOS pathway, BjSOS1, BjSOS2 and BjSOS3 genes exhibit differential expression pattern in response to salinity and ABA stress in contrasting cultivars of Brassica. It is also noticed that constitutive expression of all the three SOS genes is higher in the tolerant cultivar B. juncea as compared to the sensitive B. nigra. In silico interaction of BjSOS2 and BjSOS3 has been reported recently and here we demonstrate in vivo interaction of these two proteins in onion epidermal peel cells. Further, overexpression of BjSOS3 in corresponding arabidopsis mutant ΔAtsos3 was able to rescue the mutant phenotype and exhibit higher tolerance towards salinity stress at the seedling stage. CONCLUSION: Taken together, these findings demonstrate that the B. juncea SOS3 (BjSOS3) protein is a functional ortholog of its arabidopsis counterpart and thus show a strong functional conservation of SOS pathway responsible for salt stress signalling between arabidopsis and Brassica species.

Water-deficit-induced changes in transcription factor expression in maize seedlings.

Plants tolerate water deficits by regulating gene networks controlling cellular and physiological traits to modify growth and development. Transcription factor (TF)-directed regulation of transcription within these gene networks is key to eliciting appropriate responses. In this study, reverse transcription quantitative PCR (RT-qPCR) was used to examine the abundance of 618 transcripts from 536 TF genes in individual root and shoot tissues of maize seedlings grown in vermiculite under well-watered (water potential of -0.02 MPa) and water-deficit conditions (water potentials of -0.3 and -1.6 MPa). A linear mixed model identified 433 TF transcripts representing 392 genes that differed significantly in abundance in at least one treatment, including TFs that intersect growth and development and environmental stress responses. TFs were extensively differentially regulated across stressed maize seedling tissues. Hierarchical clustering revealed TFs with stress-induced increased abundance in primary root tips that likely regulate root growth responses to water deficits, possibly as part of abscisic acid and/or auxin-dependent signaling pathways. Ten of these TFs were selected for validation in nodal root tips of drought-stressed field-grown plants (late V1 to early V2 stage). Changes in abundance of these TF transcripts under a field drought were similar to those observed in the seedling system.

De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids.

Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.

Transcriptomic analysis of differentially expressed genes in the floral transition of the summer flowering chrysanthemum.

BACKGROUND: Chrysanthemum is a leading cut flower species. Most conventional cultivars flower during the fall, but the Chrysanthemum morifolium 'Yuuka' flowers during the summer, thereby filling a gap in the market. To date, investigations of flowering time determination have largely focused on fall-flowering types. Little is known about molecular basis of flowering time in the summer-flowering chrysanthemum. Here, the genome-wide transcriptome of 'Yuuka' was acquired using RNA-Seq technology, with a view to shedding light on the molecular basis of the shift to reproductive growth as induced by variation in the photoperiod. RESULTS: Two sequencing libraries were prepared from the apical meristem and leaves of plants exposed to short days, three from plants exposed to long days and one from plants sampled before any photoperiod treatment was imposed. From the ~316 million clean reads obtained, 115,300 Unigenes were assembled. In total 70,860 annotated sequences were identified by reference to various databases. A number of transcription factors and genes involved in flowering pathways were found to be differentially transcribed. Under short days, genes acting in the photoperiod and gibberellin pathways might accelerate flowering, while under long days, the trehalose-6-phosphate and sugar signaling pathways might be promoted, while the phytochrome B pathway might block flowering. The differential transcription of eight of the differentially transcribed genes was successfully validated using quantitative real time PCR. CONCLUSIONS: A transcriptome analysis of the summer-flowering cultivar 'Yuuka' has been described, along with a global analysis of floral transition under various daylengths. The large number of differentially transcribed genes identified confirmed the complexity of the regulatory machinery underlying floral transition.

Nitrogen assimilation system in maize is regulated by developmental and tissue-specific mechanisms.

KEY MESSAGE: We found metabolites, enzyme activities and enzyme transcript abundances vary significantly across the maize lifecycle, but weak correlation exists between the three groups. We identified putative genes regulating nitrate assimilation. Progress in improving nitrogen (N) use efficiency (NUE) of crop plants has been hampered by the complexity of the N uptake and utilisation systems. To understand this complexity we measured the activities of seven enzymes and ten metabolites related to N metabolism in the leaf and root tissues of Gaspe Flint maize plants grown in 0.5 or 2.5 mM NO3 (-) throughout the lifecycle. The amino acids had remarkably similar profiles across the lifecycle except for transient responses, which only appeared in the leaves for aspartate or in the roots for asparagine, serine and glycine. The activities of the enzymes for N assimilation were also coordinated to a certain degree, most noticeably with a peak in root activity late in the lifecycle, but with wide variation in the activity levels over the course of development. We analysed the transcriptional data for gene sets encoding the measured enzymes and found that, unlike the enzyme activities, transcript levels of the corresponding genes did not exhibit the same coordination across the lifecycle and were only weakly correlated with the levels of various amino acids or individual enzyme activities. We identified gene sets which were correlated with the enzyme activity profiles, including seven genes located within previously known quantitative trait loci for enzyme activities and hypothesise that these genes are important for the regulation of enzyme activities. This work provides insights into the complexity of the N assimilation system throughout development and identifies candidate regulatory genes, which warrant further investigation in efforts to improve NUE in crop plants.

Interaction of plant growth regulators and reactive oxygen species to regulate petal senescence in wallflowers (Erysimum linifolium).

BACKGROUND: In many species floral senescence is coordinated by ethylene. Endogenous levels rise, and exogenous application accelerates senescence. Furthermore, floral senescence is often associated with increased reactive oxygen species, and is delayed by exogenously applied cytokinin. However, how these processes are linked remains largely unresolved. Erysimum linifolium (wallflower) provides an excellent model for understanding these interactions due to its easily staged flowers and close taxonomic relationship to Arabidopsis. This has facilitated microarray analysis of gene expression during petal senescence and provided gene markers for following the effects of treatments on different regulatory pathways. RESULTS: In detached Erysimum linifolium (wallflower) flowers ethylene production peaks in open flowers. Furthermore senescence is delayed by treatments with the ethylene signalling inhibitor silver thiosulphate, and accelerated with ethylene released by 2-chloroethylphosphonic acid. Both treatments with exogenous cytokinin, or 6-methyl purine (which is an inhibitor of cytokinin oxidase), delay petal senescence. However, treatment with cytokinin also increases ethylene biosynthesis. Despite the similar effects on senescence, transcript abundance of gene markers is affected differentially by the treatments. A significant rise in transcript abundance of WLS73 (a putative aminocyclopropanecarboxylate oxidase) was abolished by cytokinin or 6-methyl purine treatments. In contrast, WFSAG12 transcript (a senescence marker) continued to accumulate significantly, albeit at a reduced rate. Silver thiosulphate suppressed the increase in transcript abundance both of WFSAG12 and WLS73. Activity of reactive oxygen species scavenging enzymes changed during senescence. Treatments that increased cytokinin levels, or inhibited ethylene action, reduced accumulation of hydrogen peroxide. Furthermore, although auxin levels rose with senescence, treatments that delayed early senescence did not affect transcript abundance of WPS46, an auxin-induced gene. CONCLUSIONS: A model for the interaction between cytokinins, ethylene, reactive oxygen species and auxin in the regulation of floral senescence in wallflowers is proposed. The combined increase in ethylene and reduction in cytokinin triggers the initiation of senescence and these two plant growth regulators directly or indirectly result in increased reactive oxygen species levels. A fall in conjugated auxin and/or the total auxin pool eventually triggers abscission.

Investigating Circadian Gating of Temperature Responsive Genes.

Understanding gene expression dynamics in the context of the time of day and temperature response is an important part of understanding plant thermotolerance in a changing climate. Performing "gating" experiments under constant conditions and light-dark cycles allows users to identify and dissect the contribution of the time of day and circadian clock to the dynamic nature of stress-responsive genes. Here, we describe the design of specific laboratory experiments in plants (Arabidopsis thaliana and bread wheat, Triticum aestivum) to investigate temporal responses to heat (1 h at 37 °C) or cold (3 h at 4 °C), and we include known marker genes that have circadian-gated responses to temperature changes.

RNA-Seq Data Analysis for Differential Gene Expression Using HISAT2-StringTie-Ballgown Pipeline.

Comparison of transcriptome for candidate gene discovery has become an important tool for biologists. While such studies lack the degree of resolution one gets from well-designed forward or reverse genetic studies, nevertheless, this has been a method of choice for giving coarse insight into the underlying biological processes or mechanisms. This was further accelerated with the availability of sequencing technologies. While many pipelines are available for RNA-seq data analysis, the protocol discussed here will guide the first-time users for conducting routine RNA-seq analysis using whole genome sequence as reference.