RNA Isolation Method in Marginal Crops with High Agronomic Potential.

Ribonucleic Acid (RNA) isolation is a basic technique in the field of molecular biology. The purpose of RNA isolation is to acquire pure and complete RNA that can be used to evaluate gene expression. Many methods can be used to perform RNA isolation, all of them based on the chemical properties of nucleic acids. However, some of them do not achieve high RNA yields and purity levels when used in a number of marginally studied crops of agronomic importance, such as grain and vegetable amaranth plants. In the method described here, the use of guanidinium thiocyanate and two additional precipitation steps with different reagents designed to obtain high yields and RNA purity levels from diverse plant species employed for plant functional genomics studies is described.

An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor.

The high quality, yield and purity total RNA samples are essential for molecular experiments. However, harvesting high quality RNA in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, different RNA extraction methods, namely TRIzol method, the modified TRIzol method, Kit method and cetyltrimethylammonium bromide (CTAB) method were employed to obtain total RNA from different tissues in L. davidii var. unicolor. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to detect the RNA quality and integrity. Compared with TRIzol, Kit and CTAB methods, the modified TRIzol method obtained higher RNA concentrations from different tissues and the A260/A280 ratios of RNA samples were ranged from 1.97 to 2.27. Thus, the modified TRIzol method was shown to be the most effective RNA extraction protocol in acquiring RNA with high concentrations. Furthermore, the RNA samples isolated by the modified TRIzol and Kit methods were intact, whereas different degrees of degradation happened within RNA samples isolated by the TRIzol and CTAB methods. In addition, the modified TRIzol method could also isolate high-quality RNA from other edible lily bulbs. Taken together, the modified TRIzol method is an efficient method for total RNA isolation from L. davidii var. unicolor.

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5'-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues.

Acquiring high-quality RNA in sufficient amounts is crucial in plant molecular biology and genetic studies. Several methods for RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only a few molecular studies are available for the species. One reason is the difficulty in obtaining RNA due to the content of the samples, which are rich in polyphenols, polysaccharides, and secondary metabolites. Furthermore, there are few studies available for the isolation of RNA from guava or Psidium samples, which hampers advances in the study of the genus. Here, quality and yields of RNA isolates were compared using six extraction protocols: two protocols based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, one protocol which uses the TRIzol reagent, one which applies guanidine thiocyanate lysis buffer followed by organic phase extraction, and two commercial kits (PureLink RNA Mini Kit and RNeasy Plant Mini Kit). The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf, and root), genotypes, and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaves was 203.06 µg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-qPCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments. CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they were compatible for downstream RNA-based applications, besides being simple and cost-effective.

Genome-Wide Analysis Identified a Set of Conserved lncRNAs Associated with Domestication-Related Traits in Rice.

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. However, limited data shows plant long noncoding RNAs (lncRNAs) are also involved in this biological process. In this study, we performed strand-specific RNA sequencing of cultivated rice Oryza sativa ssp. japonica and O. sativa ssp. indica, and their wild progenitor O. rufipogon. We identified a total of 8528 lncRNAs, including 4072 lncRNAs in O. rufipogon, 2091 lncRNAs in japonica rice, and 2365 lncRNAs in indica rice. The lncRNAs expressed in wild rice were revealed to be shorter in length and had fewer exon numbers when compared with lncRNAs from cultivated rice. We also identified a number of conserved lncRNAs in the wild and cultivated rice. The functional study demonstrated that several of these conserved lncRNAs are associated with domestication-related traits in rice. Our findings revealed the feature and conservation of lncRNAs during rice domestication and will further promote functional studies of lncRNAs in rice.

Single-cell RNA sequencing of batch Chlamydomonas cultures reveals heterogeneity in their diurnal cycle phase.

The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.

A transcriptomic analysis of sugarcane response to Leifsonia xyli subsp. xyli infection.

Sugarcane ratoon stunting disease (RSD) caused by Leifsonia xyli subsp. xyli (Lxx) is a common destructive disease that occurs around the world. Lxx is an obligate pathogen of sugarcane, and previous studies have reported some physiological responses of RSD-affected sugarcane. However, the molecular understanding of sugarcane response to Lxx infection remains unclear. In the present study, transcriptomes of healthy and Lxx-infected sugarcane stalks and leaves were studied to gain more insights into the gene activity in sugarcane in response to Lxx infection. RNA-Seq analysis of healthy and diseased plants transcriptomes identified 107,750 unigenes. Analysis of these unigenes showed a large number of differentially expressed genes (DEGs) occurring mostly in leaves of infected plants. Sugarcane responds to Lxx infection mainly via alteration of metabolic pathways such as photosynthesis, phytohormone biosynthesis, phytohormone action-mediated regulation, and plant-pathogen interactions. It was also found that cell wall defense pathways and protein phosphorylation/dephosphorylation pathways may play important roles in Lxx pathogeneis. In Lxx-infected plants, significant inhibition in photosynthetic processes through large number of differentially expressed genes involved in energy capture, energy metabolism and chloroplast structure. Also, Lxx infection caused down-regulation of gibberellin response through an increased activity of DELLA and down-regulation of GID1 proteins. This alteration in gibberellic acid response combined with the inhibition of photosynthetic processes may account for the majority of growth retardation occurring in RSD-affected plants. A number of genes associated with plant-pathogen interactions were also differentially expressed in Lxx-infected plants. These include those involved in secondary metabolite biosynthesis, protein phosphorylation/dephosphorylation, cell wall biosynthesis, and phagosomes, implicating an active defense response to Lxx infection. Considering the fact that RSD occurs worldwide and a significant cause of sugarcane productivity, a better understanding of Lxx resistance-related processes may help develop tools and technologies for producing RSD-resistant sugarcane varieties through conventional and/or molecular breeding.

Transcriptome analysis of the role of autophagy in plant response to heat stress.

Autophagy plays a critical role in plant heat tolerance in part by targeting heat-induced nonnative proteins for degradation. Autophagy also regulates metabolism, signaling and other processes and it is less understood how the broad function of autophagy affects plant heat stress responses. To address this issue, we performed transcriptome profiling of Arabidopsis wild-type and autophagy-deficient atg5 mutant in response to heat stress. A large number of differentially expressed genes (DEGs) were identified between wild-type and atg5 mutant even under normal conditions. These DEGs are involved not only in metabolism, hormone signaling, stress responses but also in regulation of nucleotide processing and DNA repair. Intriguingly, we found that heat treatment resulted in more robust changes in gene expression in wild-type than in the atg5 mutant plants. The dampening effect of autophagy deficiency on heat-regulated gene expression was associated with already altered expression of many heat-regulated DEGs prior to heat stress in the atg5 mutant. Altered expression of a large number of genes involved in metabolism and signaling in the autophagy mutant prior to heat stress may affect plant response to heat stress. Furthermore, autophagy played a positive role in the expression of defense- and stress-related genes during the early stage of heat stress responses but had little effect on heat-induced expression of heat shock genes. Taken together, these results indicate that the broad role of autophagy in metabolism, cellular homeostasis and other processes can also potentially affect plant heat stress responses and heat tolerance.

Single Cell Type Specific RNA Isolation and Gene Expression Analysis in Rice Using Laser Capture Microdissection (LCM)-Based Method.

The success of single cell type-specific gene expression or functional study largely depends on the efficient isolation of high-quality RNA from them. Laser capture microdissection (LCM) is an efficient technique that allows accessing and dissecting out a specific individual cell or cell type from a microscopic heterogeneous tissue in a minimally disruptive way. Here, we describe an efficient and inexpensive LCM-based method for the extraction of RNAs with high yield and integrity from laser-microdissected mesophyll and bundle sheath cells of rice leaf. The integrity of isolated RNA is assessed with bioanalyzer analysis, and the presence of mRNA of a specific gene is validated through RT-PCR. This RNA could further be used for uncovering single cell type-specific gene expression signature using next-generation transcriptome sequence or through regular RT-PCR.

Laser Microdissection of Cells and Isolation of High-Quality RNA After Cryosectioning.

Laser capture microdissection (LCM) has become a powerful technique that allows analyzing gene expression in specific target cells from complex tissues. Widely used in animal research, still few studies on plants have been carried out. We have applied this technique to the plant-nematode interaction by isolating feeding cells (giant cells; GCs) immersed inside complex swelled root structures (galls) induced by root-knot nematodes. For this purpose, a protocol that combines good morphology preservation with RNA integrity maintenance was developed, and successfully applied to Arabidopsis and tomato galls. Specifically, early developing GCs at 3 and 7 days post-infection (dpi) were analyzed; RNA from LCM GCs was amplified and used successfully for microarray assays.

RNA-Binding Protein Immunoprecipitation and High-Throughput Sequencing.

The RNA-binding proteome plays a key role in controlling every step in the life of RNA molecules. Through interaction with dedicated sequence motifs, RNA-binding proteins coordinate processing of cohorts of genes. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In Arabidopsis thaliana, RNA immunoprecipitation followed by reverse transcription-PCR has been widely used to test the association of candidate targets with RNA-binding proteins. The detection of unknown target transcripts requires methods operating at the level of the entire transcriptome. Here, we describe a protocol for RNA immunoprecipitation coupled to the generation of libraries from the co-purified RNAs for high-throughput sequencing. This allows determining RNAs associated with RNA-binding proteins in planta at a global scale.

The first report on molecular cloning, functional expression, purification, and statistical optimization of Escherichia coli-derived recombinant Ficin from Iranian fig tree (Ficus carica cv.Sabz).

The enzyme ficin, abundantly found in the leaves of the common Fig (Ficus carica. L), is a cysteine protease of the plant endopeptidase family. In terms of activity, this enzyme mimics the activity of the papain enzyme. However, the enzyme is more acidic than papain and binds with higher efficiency to its substrate. Ficin is widely used in the food and pharmaceutical industry along with the medical diagnosis. To date, there are no available data on cloning and recombinant production of various isoforms of ficin. In the present study, after the cloning process and optimized expression of ficin in E. coli BL21, by means of the central composite design (CCD) and approach-based response surface methodology (RSM), the recombinant protein was purified using the Ni-sepharose column and gel filtration. The activity of ficin was determined by its ability to hydrolyze the bovine casein enzyme as a substrate. These results showed the presence of different isoforms of ficin in this cultivar that they are distinct in terms of DNA coding sequences. The optimum conditions for maximum production of the recombinant ficin enzyme in E. coli were as follows; a cell density of 1.25, post-induction time 7 h, 10% (w/v) lactose concentration, and shaking at 115 rpm at 24 °C. The concentration of purified product was reported to be 0.27 mg/ml. The optimization procedures increased the amounts of ficin production by approximately 3 folds (0.67 mg/ml) compared with the expiration level (in the absence of optimization). Also, our findings showed that the recombinant ficin was able to hydrolyze casein, denoting the functionality of the enzyme when used in-vitro. The pitfall of cutting-off the young branches of the common fig tree to purify the enzyme from the young shoots was successfully solved in this study.

Transcriptome sequencing and screening of genes related to sex determination of Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim. (TK) is a dioecious plant in the Cucurbitaceae for which different sexes have separate medicinal uses. In order to study the genes related to sex determination, transcriptome sequencing was performed on flower buds of male and female plants using the high-throughput sequencing technology. A total of 145,975 unigenes and 7110 DEGs were obtained. There were 6776 DEGs annotated to 1234 GO terms and enriched to 18 functional groups, including five biological processes related to sugar metabolism. KEGG pathway analysis indicated genes involved in hormone transduction, hormone synthesis and carbohydrate metabolism. Many DEGs of TK are involved in reproductive organ formation, hormone signal transduction and regulatory networks. Combining the results of GO, KEGG and qRT-PCR, 11 sex determining candidate genes of TK were selected, including MYB80, MYB108, CER1, CBL9, ABCB19, SERK1, HSP81-3, ACS9, SEP3, AUX1 and YUC6. The results provide a foundation for the study of sex differentiation in TK.

A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber.

Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7-9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.

A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk.

Authenticity of dried aromatic herbs and herbal powders for the ASU (ayurvedic, siddha, unani) drug formulations is a key of their clinical success. The DNA based authentication is an answer; however, extraction of PCR quality DNA from such material is often problematic due to the presence of various co-extracted PCR inhibitors. Here, we report a novel DNA isolation and purification method utilizing cow skim milk that successfully yields PCR quality DNA from the aromatic herbs and dried herbal powders. The improved method presented in this study could be used as an alternative to successfully extract PCR quality DNA from such plant materials. Further, we present a set of robust matK primers which could be used as plant barcoding resource in future studies.

cDNA Cloning and Functional Analyses of Ashitaba (Angelica keiskei) Sesquiterpene Synthase Genes.

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.

GSM2, a transaldolase, contributes to reactive oxygen species homeostasis in Arabidopsis.

Plants are exposed to various environmental cues that lead to reactive oxygen species (ROS) accumulation. ROS production and detoxification are tightly regulated to maintain balance. Although studies of glucose (Glc) are always accompanied by ROS in animals, the role of Glc in respect of ROS in plants is unclear. We isolated gsm2 (Glc-hypersensitive mutant 2), a mutant with a notably chlorotic-cotyledon phenotype. The chloroplast-localized GSM2 was characterized as a transaldolase in the pentose phosphate pathway. With 3% Glc treatment, fewer or no thylakoids were observed in gsm2 cotyledon chloroplasts than in wild-type cotyledon chloroplasts, suggesting that GSM2 is required for chloroplast protection under stress. gsm2 also showed evaluated accumulation of ROS with 3% Glc treatment and was more sensitive to exogenous H2O2 than the wild type. Gene expression analysis of the antioxidant enzymes in gsm2 revealed that chloroplast damage to gsm2 cotyledons results from the accumulation of excessive ROS in response to Glc. Moreover, the addition of diphenyleneiodonium chloride or phenylalanine can rescue Glc-induced chlorosis in gsm2 cotyledons. This work suggests that GSM2 functions to maintain ROS balance in response to Glc during early seedling growth and sheds light on the relationship between Glc, the pentose phosphate pathway and ROS.

Effect of small coding genes on the circadian rhythms under elevated CO2 conditions in plants.

Increase in atmospheric carbon dioxide (CO2) has a significant effect on plant growth and development. To explore the elevated-CO2 response, we generated transcriptional profiles over a time course (2 h-14 days) of exposure to elevated CO2 in Arabidopsis thaliana. Genes related to photosynthesis were down-regulated and circadian rhythm-related genes were abnormally regulated in the early to middle phase of elevated CO2 exposure. To understand the novel mechanism of elevated CO2 signaling, we focused on 42 unknown small coding genes that showed differential expression patterns under elevated CO2 conditions. Four transgenic plants overexpressing the small coding gene exhibited a growth-defective phenotype under elevated CO2 but not under current CO2. Transcriptome analysis showed that circadian rhythm-related genes were commonly regulated in four transgenic plants. These circadian rhythm-related genes were transcribed in the dark when CO2 concentrations in the leaf was high. Taken together, our identified four small coding genes are likely to participate in elevated CO2 signaling to the circadian rhythm.

A low-bias and sensitive small RNA library preparation method using randomized splint ligation.

Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.