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In plants, the vascular tissue contains the enucleated sieve tubes facilitating long-distance transport of nutrients, hormones, and proteins. In addition, several mRNAs and small interfering RNAs/microRNAs were shown to be delivered via sieve tubes whose content is embodied by the phloem sap (PS). A number of these phloem transcripts are transported from source to sink tissues and function at targeted tissues. To gain additional insights into phloem-delivered RNAs and their potential role in signaling, we isolated and characterized PS RNA molecules distinct from microRNAs/small interfering RNAs with a size ranging from 30 to 90 bases. We detected a high number of full-length and phloem-specific fragments of noncoding RNAs such as tRNAs, ribosomal RNAs, and spliceosomal RNAs in the PS of pumpkin (Cucurbita maxima). In vitro assays show that small quantities of PS RNA molecules efficiently inhibit translation in an unspecific manner. Proof of concept that PS-specific tRNA fragments may interfere with ribosomal activity was obtained with artificially produced tRNA fragments. The results are discussed in terms of a functional role for long distance delivered noncoding PS RNAs.

MicroRNAs (miRNAs) are a distinct class of small RNAs in plants that not only regulate biological processes but also regulate response to environmental stresses. The toxic heavy metal cadmium (Cd) induces expression of several miRNAs in rapeseed (Brassica napus), but it is not known on a genome-wide scale how the expression of miRNAs and their target genes, is regulated by Cd. In this study, four small RNA libraries and four degradome libraries were constructed from Cd-treated and non-Cd-treated roots and shoots of B. napus seedlings. Using high-throughput sequencing, the study identified 84 conserved and non-conserved miRNAs (belonging to 37 miRNA families) from Cd-treated and non-treated B. napus, including 19 miRNA members that were not identified before. Some of the miRNAs were validated by RNA gel blotting. Most of the identified miRNAs were found to be differentially expressed in roots/shoots or regulated by Cd exposure. The study simultaneously identified 802 targets for the 37 (24 conserved and 13 non-conserved) miRNA families, from which there are 200, 537, and 65 targets, belonging to categories I, II, and III, respectively. In category I alone, many novel targets for miRNAs were identified and shown to be involved in plant response to Cd.

Plant height is a major trait affecting yield potential in rice. Using a large-scale hybrid transcription factor approach, we identified the novel MYB-like transcription factor OsMPH1 (MYB-like gene of Plant Height 1), which is involved in the regulation of plant height in rice. Overexpression of OsMPH1 leads to increases of plant height and grain yield in rice, while knockdown of OsMPH1 leads to the opposite phenotypes. Microscopy of longitudinal stem sections indicated that a change in internode cell length resulted in the change in plant height. RNA sequencing (RNA-seq) analysis of transgenic rice lines showed that multiple genes related to cell elongation and cell wall synthesis, which are associated with plant height and yield phenotypes, exhibited an altered expression profile. These results imply that OsMPH1 might be involved in specific recognition and signal transduction processes related to plant height and yield formation, providing further insights into the mechanisms underlying the regulation of plant height and providing a candidate gene for the efficient improvement of rice yield.

MicroRNAs (miRNAs) play important roles in response of plants to biotic and abiotic stresses. Aluminum (Al) toxicity is a major factor limiting plant growth in acidic soils. However, there has been limited report on the involvement of miRNAs in response of plants to toxic Al³⁺. To identify Al³⁺-responsive miRNAs at wholegenome level, high-throughput sequencing technology was used to sequence libraries constructed from root apices of the model legume plant Medicago truncatula treated with and without Al³⁺. High-throughput sequencing of the control and two Al³⁺-treated libraries led to generation of 17.1, 14.1 and 17.4 M primary reads, respectively. We identified 326 known miRNAs and 21 new miRNAs. Among the miRNAs, expression of 23 miRNAs was responsive to Al³⁺, and the majority of Al³⁺-responsive mRNAs was down-regulated. We further classified the Al³⁺-responsive miRNAs into three groups based on their expression patterns: rapid-responsive, late-responsive and sustained-responsive miRNAs. The majority of Al³⁺-responsive miRNAs belonged to the 'rapid-responsive' category, i.e. they were responsive to short-term, but not long-term Al³⁺ treatment. The Al³⁺-responsive miRNAs were also verified by quantitative real-time PCR. The potential targets of the 21 new miRNAs were predicted to be involved in diverse cellular processes in plants, and their potential roles in Al³⁺-induced inhibition of root growth were discussed. These findings provide valuable information for functional characterization of miRNAs in Al³⁺ toxicity and tolerance.

Populus euphratica , a typical hydro-halophyte, is ideal for studying salt stress responses in woody plants. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that fulfilled an important post-transcriptional regulatory function. MiRNA may regulate tolerance to salt stress but this has not been widely studied in P . euphratica . In this investigation, the small RNAome, degradome and transcriptome were studied in salt stress treated P . euphratica by deep sequencing. Two hundred and eleven conserved miRNAs between Populus trichocarpa and P . euphratica have been found. In addition, 162 new miRNAs, belonging to 93 families, were identified in P . euphratica . Degradome sequencing experimentally verified 112 targets that belonged to 51 identified miRNAs, few of which were known previously in P . euphratica . Transcriptome profiling showed that expression of 15 miRNA-target pairs displayed reverse changing pattern under salt stress. Together, these results indicate that, in P . euphratica under salt stress, a large number of new miRNAs could be discovered, and both known and new miRNA were functionally cleaving to their target mRNA. Expression of miRNA and target were correspondingly induced by salt stress but that it was a complex process in P . euphratica .

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥ 1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Seaweeds represent a promising resource for food as well as pharmaceutical and cosmetic applications. However, genomic and transcriptomic research on these organisms remains underdeveloped, partly due to technical challenges in DNA/RNA extraction and ribosomal RNA (rRNA) depletion. These challenges are particularly acute in species lacking standardized protocols. This study systematically evaluates RNA isolation and rRNA depletion protocols for RNA sequencing (RNA-seq) across 11 commercially relevant edible seaweed species, encompassing six brown (Heterokontophyta), four red (Rhodophyta), and one green (Chlorophyta) alga. Seven RNA extraction protocols (three CTAB-based and four spin-column-based) were compared for RNA yield, integrity, and purity. Brown seaweeds generally yielded superior RNA with CTAB-based methods, whereas red and green seaweeds performed better with chaotropic-salt-based spin-column methods. Additionally, three commercial rRNA depletion kits (Ribo-Zero Plant, riboPOOL, and RiboFree) were assessed for each seaweed species separately in a large matrix-style RNA-seq experiment (43 samples) to determine their effectiveness in removing rRNA compared to undepleted controls. Both RiboFree and riboPOOL significantly outperformed Ribo-Zero Plant, yielding post-depletion rRNA mapping rates of only 6, 9, and 19%, respectively. This study provides practical guidelines for selecting RNA isolation and ribodepletion methods tailored to specific seaweed taxa and supports the development of transcriptomic tools for seaweed research.

Background Sclerotinia sclerotiorum causes stem rot in Brassica napus , which leads to lodging and severe yield losses. Although recent studies have explored significant progress in the characterization of individual S. sclerotiorum pathogenicity factors, a gap exists in profiling gene expression throughout the course of S. sclerotiorum infection on a host plant. In this study, RNA-Seq analysis was performed with focus on the events occurring through the early (1 h) to the middle (48 h) stages of infection. Results Transcript analysis revealed the temporal pattern and amplitude of the deployment of genes associated with aspects of pathogenicity or virulence during the course of S. sclerotiorum infection on Brassica napus . These genes were categorized into eight functional groups: hydrolytic enzymes, secondary metabolites, detoxification, signaling, development, secreted effectors, oxalic acid and reactive oxygen species production. The induction patterns of nearly all of these genes agreed with their predicted functions. Principal component analysis delineated gene expression patterns that signified transitions between pathogenic phases, namely host penetration, ramification and necrotic stages, and provided evidence for the occurrence of a brief biotrophic phase soon after host penetration. Conclusions The current observations support the notion that S. sclerotiorum deploys an array of factors and complex strategies to facilitate host colonization and mitigate host defenses. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B. napus by S. sclerotiorum and provides information for further characterization of genes involved in the S. sclerotiorum -host plant interactions.

Plant microRNAs (miRNAs) are noncoding and endogenous key regulators that play significant functions in regulating plant responses to stress, and plant growth and development. Heat stress is a critical abiotic stress that reduces the yield and quality of flowering Chinese cabbage ( Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). However, limited information is available on whether miRNAs are involved in the regulation of heat stress in B. campestris . A high-throughput sequencing approach was used to identify novel and conserved heat-responsive miRNAs in four small RNA libraries of flowering Chinese cabbage using leaves collected at 0 h, 1 h, 6 h and 12 h after a 38 °C heat-stress treatment. The analysis identified 41 conserved miRNAs (belonging to 19 MIR families), of which MIR156, MIR159, MIR168, MIR171 and MIR1885 had the most abundant molecules. Prediction and evaluation of novel miRNAs using the unannotated reads resulted in 18 candidate miRNAs. Differential expression analysis showed that most of the identified miRNAs were downregulated in heat-treated groups. To better understand functional importance, bioinformatic analysis predicted 432 unique putative target miRNAs involved in cells, cell parts, catalytic activity, cellular processes and abiotic stress responses. Furthermore, the Kyoto Encyclopedia of Genes and Genomes maps of flowering Chinese cabbage identified the significant role of miRNAs in stress adaptation and stress tolerance, and in several mitogen-activated protein kinases signaling pathways including cell death. This work presents a comprehensive study of the miRNAs for understanding the regulatory mechanisms and their participation in the heat stress of flowering Chinese cabbage.

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.