Translation Efficiency Test Using Polysome Profiles Under Heat Stress.

Translational control of different genes under heat stress is a critical step for plant adaptation to the environment. Assessing the translational activities of various genes can help us understand the molecular mechanisms underlying plant resilience, contributing to the development of crops with enhanced stress tolerance in the face of global climate change. This paper presents a detailed methodology for assessing translation efficiency through polysome profiling in plants exposed to heat stress. The procedure is divided into three parts: heat stress treatment for Arabidopsis, translation efficiency test using polysome profiles, and calculation of translation efficiency by isolating non-polysomal and polysomal RNA based on the profile. In the first part, Arabidopsis plants are subjected to controlled heat stress conditions to mimic environmental challenges. The treatment involves exposing the plants to high temperatures for specified durations, ensuring consistent and reproducible stress induction. This step is crucial for studying the plant's physiological and molecular responses to heat stress. The second part involves the translation efficiency test using polysome profiling. Polysomes are extracted through sucrose gradient centrifugation, which separates mRNAs based on ribosomal loading. This allows for the examination of ribosome occupancy on mRNAs, providing insights into the translational control mechanisms under stress conditions. In the third part, RNA is isolated from both polysomal and non-polysomal fractions. Spike-in RNA is used to accurately measure the amount of RNA in each fraction. The calculation of translation efficiency is performed by comparing the distribution of mRNAs across these fractions under normal and heat stress conditions. The translation activities of specific genes are further assessed by performing quantitative real-time PCR (qRT-PCR) with ribosome-associated RNA and total RNA. This methodology focuses exclusively on the effects of heat stress, providing a detailed protocol for analyzing translational regulation in plants.

Genome-Wide Identification of microRNAs Associated with Starch Biosynthesis and Endosperm Development in Foxtail Millet.

Foxtail millet is one of the oldest crops, and its endosperm contains up to 70% of starch. Grain filling is an important starch accumulation process associated with foxtail millet yield and quality. However, the molecular mechanisms of grain filling in foxtail millet are relatively unclear. Here, we investigate the genes and regulated miRNAs associated with starch synthesis and metabolism in foxtail millet using high-throughput small RNA, mRNA and degradome sequencing. The regulation of starch synthesis and quality is carried out mainly at the 15 DAA to 35 DAA stage during grain filling. The DEGs between waxy and non-waxy foxtail millet were significant, especially for GBSS. Additionally, ptc-miR169i_R+2_1ss21GA, fve-miR396e_L-1R+1, mtr-miR162 and PC-5p-221_23413 regulate the expression of genes associated with the starch synthesis pathway in foxtail millet. This study provides new insights into the molecular mechanisms of starch synthesis and quality formation in foxtail millet.

Whole-transcriptome analysis and construction of an anther development-related ceRNA network in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

Anther development is precisely regulated by a complex gene network, which is of great significance to plant breeding. However, the molecular mechanism of anther development in Chinese cabbage is unclear. Here, we identified microRNAs (miRNAs), mRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) related to anther development in Chinese cabbage (Brassica campestris L. ssp. pekinensis) to construct competitive endogenous RNA (ceRNA) regulatory networks and provide valuable knowledge on anther development. Using whole-transcriptome sequencing, 9055, 585, 1344, and 165 differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), lncRNAs (DElncRNAs), and circRNAs (DEcircRNAs) were identified, respectively, in the anthers of Chinese cabbage compared with those in samples of the vegetative mass of four true leaves. An anther-related ceRNA regulatory network was constructed using miRNA targeting relationships, and 450 pairs of ceRNA relationships, including 97 DEmiRNA-DEmRNA, 281 DEmiRNA-DElncRNA, and 23 DEmiRNA-DEcircRNA interactions, were obtained. We identified important genes and their interactions with lncRNAs, circRNAs, and miRNAs involved in microsporogenesis, tapetum and callose layer development, pollen wall formation, and anther dehiscence. We analyzed the promoter activity of six predominant anther expression genes, which were expressed specifically in the anthers of Arabidopsis thaliana, indicating that they may play an important role in anther development of Chinese cabbage. This study lays the foundation for further research on the molecular mechanisms of anther growth and development in Chinese cabbage.

Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA.

DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo­electron microscopy structures of the Pol IV­RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV­generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a 'backtracking-triggered RNA channeling' mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.

An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection.

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

Grape Small Auxin Upregulated RNA (SAUR) 041 Is a Candidate Regulator of Berry Size in Grape.

Grape (Vitis vinifera) is an important horticultural crop that can be used to make juice and wine. However, the small size of the berry limits its yield. Cultivating larger berry varieties can be an effective way to solve this problem. As the largest family of auxin early response genes, SAUR (small auxin upregulated RNA) plays an important role in the growth and development of plants. Berry size is one of the important factors that determine grape quality. However, the SAUR gene family's function in berry size of grape has not been studied systematically. We identified 60 SAUR members in the grape genome and divided them into 12 subfamilies based on phylogenetic analysis. Subsequently, we conducted a comprehensive and systematic analysis on the SAUR gene family by analyzing distribution of key amino acid residues in the domain, structural features, conserved motifs, and protein interaction network, and combined with the heterologous expression in Arabidopsis and tomato. Finally, the member related to grape berry size in SAUR gene family were screened. This genome-wide study provides a systematic analysis of grape SAUR gene family, further understanding the potential functions of candidate genes, and provides a new idea for grape breeding.

Multi-tissue transcriptome analysis of two Begonia species reveals dynamic patterns of evolution in the chalcone synthase gene family.

Begonia is an important horticultural plant group, as well as one of the most speciose Angiosperm genera, with over 2000 described species. Genus wide studies of genome size have shown that Begonia has a highly variable genome size, and analysis of paralog pairs has previously suggested that Begonia underwent a whole genome duplication. We address the contribution of gene duplication to the generation of diversity in Begonia using a multi-tissue RNA-seq approach. We chose to focus on chalcone synthase (CHS), a gene family having been shown to be involved in biotic and abiotic stress responses in other plant species, in particular its importance in maximising the use of variable light levels in tropical plants. We used RNA-seq to sample six tissues across two closely related but ecologically and morphologically divergent species, Begonia conchifolia and B. plebeja, yielding 17,012 and 19,969 annotated unigenes respectively. We identified the chalcone synthase gene family members in our Begonia study species, as well as in Hillebrandia sandwicensis, the monotypic sister genus to Begonia, Cucumis sativus, Arabidopsis thaliana, and Zea mays. Phylogenetic analysis suggested the CHS gene family has high duplicate turnover, all members of CHS identified in Begonia arising recently, after the divergence of Begonia and Cucumis. Expression profiles were similar within orthologous pairs, but we saw high inter-ortholog expression variation. Sequence analysis showed relaxed selective constraints on some ortholog pairs, with substitutions at conserved sites. Evidence of pseudogenisation and species specific duplication indicate that lineage specific differences are already beginning to accumulate since the divergence of our study species. We conclude that there is evidence for a role of gene duplication in generating diversity through sequence and expression divergence in Begonia.

Overexpression of antisense phosphatase 2C affords cold resistance in hybrid Populus davidiana × Populus bolleana.

BACKGROUND: Overexpression of the abiotic and biotic stress-resistance genes of the plant signaling pathway is well known for its significant role in the regulation of plant growth and enhancement of the productivity of agricultural land under changing climatic conditions. OBJECTIVES: This research aimed to clone Populus davidiana × Populus bolleana PP2C (PdPP2C) gene and analyze its structure and function, and downregulate PdPP2C by overexpression of its antisense PdPP2C (AS-PdPP2C) gene for enhancing cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. METHODS: PdPP2C was cloned and transformed to identify its function, and its antisense was overexpressed via downregulation to increase the cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. RESULTS: Antisense inhibition of protein phosphatase 2C accelerates the cold acclimation of Poplar (P. davidiana × P. bolleana) gene in terms of antifreeze. CONCLUSION: PdPP2C was expressed in the roots, stems, and leaves of P. davidiana × P. bolleana, and the expression was higher in the leaves. The expression of PdPP2C was also significantly downregulated at low-temperature (0 °C and 4 °C) stress. The relative conductivity and malondialdehyde content of non-transgenic lines were higher than those of AS-PdPP2C lines after 2 days of cold treatment at - 1 °C. The leaves of the transgenic lines were not wilted and showed no chlorosis compared with those of the non-transgenic lines. The AS-PdPP2C transgenic lines also showed higher freezing resistance than the non-transgenic lines. AS-PdPP2C participated in the regulation of freezing resistance.

The Bradyrhizobium diazoefficiens type III effector NopE modulates the regulation of plant hormones towards nodulation in Vigna radiata.

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

Ectopic expression of a novel cold-resistance protein 1 from Brassica oleracea promotes tolerance to chilling stress in transgenic tomato.

Cold stress is considered as one of the major environmental factors that adversely affects the plant growth and distribution. Therefore, there arises an immediate need to cultivate effective strategies aimed at developing stress-tolerant crops that would boost the production and minimise the risks associated with cold stress. In this study, a novel cold-responsive protein1 (BoCRP1) isolated from Brassica oleracea was ectopically expressed in a cold susceptible tomato genotype Shalimar 1 and its function was investigated in response to chilling stress. BoCRP1 was constitutively expressed in all the tissues of B. oleracea including leaf, root and stem. However, its expression was found to be significantly increased in response to cold stress. Moreover, transgenic tomato plants expressing BoCRP1 exhibited increased tolerance to chilling stress (4 °C) with an overall improved rate of seed germination, increased root length, reduced membrane damage and increased accumulation of osmoprotectants. Furthermore, we observed increased transcript levels of stress responsive genes and enhanced accumulation of reactive oxygen species scavenging enzymes in transgenic plants on exposure to chilling stress. Taken together, these results strongly suggest that BoCRP1 is a promising candidate gene to improve the cold stress tolerance in tomato.

Environmental responsiveness of flowering time in cassava genotypes and associated transcriptome changes.

Cassava is an important food security crop in tropical regions of the world. Cassava improvement by breeding is limited by its delayed and poor production of flowers, such that cassava flowering under field conditions indirectly lengthens the breeding cycle. By studying genotype and environment interaction under two Nigerian field conditions (Ubiaja and Ibadan) and three controlled temperature conditions (22°C/18°C, 28/24°C and 34/30°C (day/night)), we found that while early flowering genotypes flowered at similar times and rates under all growing conditions (unfavorable and favorable field and controlled-temperature environments), late flowering genotypes were environmentally sensitive such that they were substantially delayed in unfavorable environments. On the basis of nodes-to-flower, flowering of late genotypes approached the flowering time of early flowering genotypes under relatively cool Ubiaja field conditions and in growth chambers at 22°C, whereas warmer temperatures elicited a delaying effect. Analysis of transcriptomes from leaves of field and controlled-temperature environments revealed that conditions which promote early flowering in cassava have low expression of the flowering repressor gene TEMPRANILLO 1 (TEM1), before and after flowering. Expression data of field plants showed that the balance between flower stimulatory and inhibitory signaling appeared to correlate with flowering time across the environments and genotypes.

A method to analyze time expression profiles demonstrated in a database of chili pepper fruit development.

RNA-Seq experiments allow genome-wide estimation of relative gene expression. Estimation of gene expression at different time points generates time expression profiles of phenomena of interest, as for example fruit development. However, such profiles can be complex to analyze and interpret. We developed a methodology that transforms original RNA-Seq data from time course experiments into standardized expression profiles, which can be easily interpreted and analyzed. To exemplify this methodology we used RNA-Seq data obtained from 12 accessions of chili pepper (Capsicum annuum L.) during fruit development. All relevant data, as well as functions to perform analyses and interpretations from this experiment, were gathered into a publicly available R package: "Salsa". Here we explain the rational of the methodology and exemplify the use of the package to obtain valuable insights into the multidimensional time expression changes that occur during chili pepper fruit development. We hope that this tool will be of interest for researchers studying fruit development in chili pepper as well as in other angiosperms.

Meta-analysis of RNA-Seq studies reveals genes with dominant functions during flower bud endo- to eco-dormancy transition in Prunus species.

In deciduous fruit trees, entrance into dormancy occurs in later summer/fall, concomitantly with the shortening of day length and decrease in temperature. Dormancy can be divided into endodormancy, ecodormancy and paradormancy. In Prunus species flower buds, entrance into the dormant stage occurs when the apical meristem is partially differentiated; during dormancy, flower verticils continue their growth and differentiation. Each species and/or cultivar requires exposure to low winter temperature followed by warm temperatures, quantified as chilling and heat requirements, to remove the physiological blocks that inhibit budburst. A comprehensive meta-analysis of transcriptomic studies on flower buds of sweet cherry, apricot and peach was conducted, by investigating the gene expression profiles during bud endo- to ecodormancy transition in genotypes differing in chilling requirements. Conserved and distinctive expression patterns were observed, allowing the identification of gene specifically associated with endodormancy or ecodormancy. In addition to the MADS-box transcription factor family, hormone-related genes, chromatin modifiers, macro- and micro-gametogenesis related genes and environmental integrators, were identified as novel biomarker candidates for flower bud development during winter in stone fruits. In parallel, flower bud differentiation processes were associated to dormancy progression and termination and to environmental factors triggering dormancy phase-specific gene expression.

Plant transfer RNA-derived fragments: Biogenesis and functions.

Processing of mature transfer RNAs (tRNAs) produces complex populations of tRNA-derived fragments (tRFs). Emerging evidence shows that tRFs have important functions in bacteria, animals, and plants. Here, we review recent advances in understanding plant tRFs, focusing on their biological and cellular functions, such as regulating stress responses, mediating plant-pathogen interactions, and modulating post-transcriptional gene silencing and translation. We also review sequencing strategies and bioinformatics resources for studying tRFs in plants. Finally, we discuss future directions for plant tRF research, which will expand our knowledge of plant non-coding RNAs.

Evolution of plant telomerase RNAs: farther to the past, deeper to the roots.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

Absence of CG methylation alters the long noncoding transcriptome landscape in multiple species.

The noncoding regions throughout the genome are in large part comprised of transposable elements (TEs), some of which are functionalized with long intergenic noncoding RNAs (lincRNAs). DNA methylation is predominantly associated with TEs, but little is known about its contribution to the transcription of lincRNAs. Here, we examine the lincRNA profiles of DNA methylation-related mutants of five species, Arabidopsis, rice, tomato, maize, and mouse, to elucidate patterns in lincRNA regulation under altered DNA methylation status. Significant activation of lincRNAs was observed in the absence of CG DNA methylation rather than non-CG. Our study establishes a working model of the contribution of DNA methylation to regulation of the dynamic activity of lincRNA transcription.

MicroRNAs modulate ethylene induced retrograde signal for rice endosperm starch biosynthesis by default expression of transcriptome.

Control of stage specific spike in ethylene production at anthesis has been a vauable route to potentially enhance genetic ceiling for grain filling of rice spikelet. A number of genes controlling ethylene homeostasis and starch synthesis have been identified so long, but lack of credible information on master modulation of gene expression by miRNAs and their target genes associated with hormonal dynamics obfuscate mechanisms controlling genotype difference in quantum of grain filling. The confusion accounts for consequent shrinkage of options for yield manipulation. In a two by two factorial design, miRNA regulation of spikelet specific grain development in low against high sterile recombinant inbred lines of rice Oryza sativa L. namely CR 3856-62-11-3-1-1-1-1-1-1 (SR 157) and CR 3856-63-1-1-1-1-1-1-1 (SR 159) respectively, and inferior verses superior spikelets were compared during first 10 days after anthesis. Grain filling was poorer in SR159 than SR157 and inferior spikelets in the former were most vulnerable. Between the cultivars, overall expression of unique miRNAs with targets on ethylene pathway genes was higher in SR159 than SR157 and the situation was opposite for auxin pathway genes. Precision analysis in psTarget server database identified up-regulation of MIR2877 and MIR530-5p having Os11t0141000-02 and Os07t0239400-01 (PP2A regulatory subunit-like protein and ethylene-responsive small GTP-binding proteins) and MIR396h having Os01t0643300-02 (an auxin efflux carrier protein) and Os01t0643300-01 (a PIN1-like auxin transport protein), as targets with highest probability at anthesis and 5 days after anthesis respectively, in the inferior spikelet and the fold change values of DGE matched with pattern of gene expression (relative transcript level) in the qRT-PCR studies conducted for relevant miRNAs and protein factors for ethylene and auxin signalling. In conclusion, epigenetic regulation of both auxin and ethylene homeostasis control grain filling of rice spikelet was established, but evidences were more robust for the latter.

Azadirachta indica MicroRNAs: Genome-Wide Identification, Target Transcript Prediction, and Expression Analyses.

MicroRNAs are short, endogenous, non-coding RNAs, liable for essential regulatory function. Numerous miRNAs have been identified and studied in plants with known genomic or small RNA resources. Despite the availability of genomic and transcriptomic resources, the miRNAs have not been reported in the medicinal tree Azadirachta indica (Neem) till date. Here for the first time, we report extensive identification of miRNAs and their possible targets in A. indica which might help to unravel their therapeutic potential. A comprehensive search of miRNAs in the A. indica genome by C-mii tool was performed. Overall, 123 miRNAs classified into 63 families and their stem-loop hairpin structures were predicted. The size of the A. indica (ain)-miRNAs ranged between 19 and 23 nt in length, and their corresponding ain-miRNA precursor sequence MFEI value averaged as -1.147 kcal/mol. The targets of ain-miRNAs were predicted in A. indica as well as Arabidopsis thaliana plant. The gene ontology (GO) annotation revealed the involvement of ain-miRNA targets in developmental processes, transport, stress, and metabolic processes including secondary metabolism. Stem-loop qRT-PCR was carried out for 25 randomly selected ain-miRNAs and differential expression patterns were observed in different A. indica tissues. Expression of miRNAs and its targets shows negative correlation in a dependent manner.

Characterization and Expression Analysis of Resistance Gene Analogues in Elite Sugarcane Genotypes.

BACKGROUND: Resistance Gene Analogues (RGAs) are an important source of disease resistance in crop plants and have been extensively studied for their identification, tagging and mapping of Quantitative Trait Loci (QTLs). Tracking these RGAs in sugarcane can be of great help for the selection and screening of disease resistant clones. OBJECTIVE: In the present study expression of different Resistance Gene Analogues (RGAs) was assessed in indigenous elite sugarcane genotypes which include resistant, highly resistant, susceptible and highly susceptible to disease infestation. METHODS: Total cellular DNA and RNA were isolated from fourteen indigenous elite sugarcane genotypes. PCR, semi-quantitative RT PCR and real time qPCR analyses were performed. The resultant amplicons were sequence characterized, chromosomal localization and phylogenetic analysis were performed. RESULTS: All of the 15 RGA primers resulted in amplification of single or multiple fragments from genomic DNA whereas only five RGA primers resulted in amplification from cDNA. Sequence characterization of amplified fragments revealed 86-99% similarity with disease resistance proteins indicating their potential role in disease resistance response. Phylogenetic analysis also validated these findings. Further, expression of RGA-012, RGA-087, RGA-118, RGA-533 and RGA-542 appeared to be upregulated and down regulated in disease resistant and susceptible genotypes, respectively, after inoculation with Colletotrichum falcatum. CONCLUSION: RGAs are present in most of our indigenous genotypes. Anyhow, differential expression of five RGAs indicated that they have some critical role in disease resistance. So, the retrieved results can not only be employed to devise molecular markers for the screening of disease resistant genotypes but can also be used to develop disease resistant plants through transgenic technology.

Genome-Wide Identification of Copper Stress-Regulated and Novel MicroRNAs in Mulberry Leaf.

Copper (Cu) is an essential trace element for plant growth and development. It is widely involved in respiration, photosynthesis, pollen formation, and other biological processes. Therefore, low or excessive copper causes damage to plants. Mulberry is an essential perennial economic tree. At present, research on the abiotic stress responses in mulberry is mainly focused on the identification of resistant germplasm resources and cloning of resistant genes. In contrast, studies on the resistance function of microRNAs and the regulatory gene responses to stress are rare. In this study, small RNA libraries (control and copper stressed) were constructed from mulberry leaf RNA. High-throughput sequencing and screening were employed, a total of 65 known miRNAs and 78 predicted novel mature miRNAs were identified, among which 40 miRNAs were differentially expressed under copper stress. Subsequently, expression patterns were verified for 14 miRNAs by real-time fluorescence quantitative PCR (qPCR). The target genes of miRNAs were validated by 5' RLM-RACE. Our results provide the bases for further study on the molecular mechanism of copper stress regulation in mulberry.