Genome-wide identification and characterization of lncRNAs responding to iron deficiency in Oryza sativa


 Background: Iron (Fe) plays a vital role in various cellular processes in plants, including chlorophyll biosynthesis, photosynthesis and respiration. Fe deficiency directly affects crop growth and development, ultimately resulting in reduced crop yield and quality. Long non-coding RNAs (lncRNAs) have recently been demonstrated to play critical regulatory roles in a multitude of pathways across numerous species. However, systematic screening of lncRNAs responding to Fe deficiency in plants has not been reported. In this work, genome-wide identification and characterization of lncRNAs responsive to Fe deficiency were performed by strand-specific RNA sequencing in rice. Results: In total, 6477 lncRNAs were identified. In Fe-deficient conditions, 47 lncRNAs were up-regulated and 33 lncRNAs were down-regulated in shoots, while 89 lncRNAs were up-regulated and 32 lncRNAs were down-regulated in roots compared to normal conditions. The lncRNAs that were differentially expressed under Fe-sufficient and -deficient conditions appear to be clustered in the genome. Among five monocotyledon species, 88 lncRNAs were conserved, but only 3 lncRNAs responded to Fe deficiency in shoots. Two lncRNAs were identified as putative target mimics of miRNAs that respond to Fe deficiency. Conclusions: Our results provide evidence that lncRNAs are involved in Fe-deficiency signaling pathway in plants, providing a new pathway for further investigation into Fe-regulatory and response mechanisms.

Background Iron (Fe) is an essential micronutrient for plants, but is often limited due to low availability in the soil [1]. To overcome Fe deficiency, plants have evolved two strategies to optimize Fe acquisition and uptake, i.e., the reduction strategy (Strategy I) for non-gramineous plants and the chelation strategy (Strategy II) for gramineous plants [2,3]. Rice, which is adapted to growing in the paddy field where the reduced form of Fe is available, utilizes both strategy I and strategy II Fe-uptake systems [4][5][6]. A large number of genes are known for their involvement in Fe uptake and homeostasis, including those that encode transcription factors for regulating expression of downstream Fe-responsive genes, enzymes for synthesis of phytosiderophores (MAs), and transporters of MA-Fe(III) or Fe(II) in rice [2, 4, 3 7-14]. However, regulation of Fe-responses by long noncoding RNAs (lncRNAs) has not been reported.
LncRNAs are transcripts of more than 200 nucleotides in length but without coding potential [15] that have recently gained widespread attention. LncRNAs plays roles in numerous crucial biological processes across many species by regulating expression of mRNAs at epigenetic, transcriptional, post-transcriptional, translational and post-translational levels [16][17][18]. LncRNAs are classified as sense, antisense, intronic, and intergenic according to their position in relation to neighboring coding genes [16,19]. In plants, lncRNAs were reported to be involved in development and stress responses [20,21]. For instance, the lncRNAs COLDAIR (Cold-Assisted Intronic noncoding RNA) and COOLAIR (Cold-Induced Long Antisense Intragenic RNA) are both located in the FLC gene, which regulates flowering time. COLDAIR and COOLAIR regulate expression of FLC at the epigenetic level by interacting with an evolutionarily conserved repressive complex PRC2 (Polycomb Repressive Complex 2) [22 -24]. Another lncRNA, the 1,236-nucleotide long LDMAR (Long-day-specific male-fertilityassociated RNA), regulates photoperiod-sensitive male sterility (PSMS) in rice [25]. A number of lncRNAs have been reported to regulate phosphate homeostasis. IPS1 (Induced by phosphate starvation 1) reduces phosphorus acquisition by inhibiting the activity of miR399, through the target mimicry mechanism [26]. The cis-natural antisense RNA (cis-NATPHO1;2), transcribed from OsPHO1;2, was found to be a translational enhancer of its sense gene (OsPHO1;2) [27]. In yeast, prt (pho1repressing transcript), generated from the promoter region of the pho1 gene, regulates expression of pho1 responding to different phosphate levels [28]. These studies show a complicated network involving lncRNAs that regulate phosphate homeostasis. In contrast, there are no reported lncRNAs responding to Fe deficiency. In this study, the transcriptome of rice was surveyed to systematically identify and characterize any lncRNAs that respond to Fe deficiency.

Genome-wide identification of lncRNAs
To systematically identify and characterize lncRNAs in rice, ssRNA sequencing was performed on shoot and root samples from rice seedlings grown in Fe-sufficient and -deficient conditions. After 10 days of Fe-deficient growth, rice plants showed significant chlorosis and lower chlorophyll content in 4 the young leaves ( Fig. 1A and 1B). The expression of typic Fe-deficiency responsive genes, such as the iron-related bHLH transcription factor 2 (IRO2), nicotianamine synthases 1 and 2 (NAS1/2), Fe(III)-DMA transporters (YSL15/16) and Iron-Regulated Transporter 1 (IRT1), were significantly increased ( Fig. 1C), indicating that the rice seedlings is under iron deficiency condition and at the sampling time.
The pipeline for lncRNA identification and characterization is shown in Figure S1 (see methods). Using this pipeline, the approximately 700 million 150-bp pair-end reads were assembled into 31947 transcripts using Cufflinks. The Coding Potential Calculator (CPC) was used to evaluate the proteincoding potential of the transcripts to distinguish protein coding transcripts and lncRNAs. Transcripts more than 200 bp in length with CPC scores < 0 were defined as lncRNAs, the remaining transcripts were classified as protein-coding transcripts (mRNAs). Using this method, 25470 mRNAs and 6477 lncRNAs were identified.

Fe-deficiency responsive lncRNAs and mRNAs in rice shoot and root
To identify the lncRNAs and mRNAs that are differentially expressed in response to Fe deficiency, the normalized expression levels (in fragments per kilobase of exon per million fragments mapped, FPKM) of lncRNAs or mRNAs were compared between the Fe-deficient and Fe-sufficient treatments ( Figure   S2). In shoots, 80 differently expressed lncRNAs were identified (Log2 (fold change) ≥ 1 or ≤-1, probability > 0.8). Among them, 47 lncRNAs were up-regulated and 33 lncRNAs were down-regulated  Table S2B). In addition, 394 and 841 mRNAs were differentially expressed in either roots or shoots due to Fe deficiency, respectively (Log2 (fold change) ≥ 1 or ≤-1, probability > 0.8). In shoots, 240 mRNAs were up-regulated and 154 were downregulated ( Figure S2C and S3B; and Table S2C), while in roots, 536 mRNAs were up-regulated and 305 mRNAs were down-regulated ( Figure S2D and S3B; Table S2D).
The differentially expressed lncRNAs and mRNAs were used to generate a heat map (Fig. 2). Classes I and III contained lncRNA and mRNA transcripts that were expressed significantly higher in Fesufficient than in Fe-deficient conditions in either roots (Class I) or shoots (Class III), respectively. In contrast, transcripts in Classes Ⅱ and Ⅳ had higher expression in roots or shoots under Fe-deficient conditions, respectively. Transcripts in Class Ⅴ were more highly expressed in both shoots and roots under Fe-deficient conditions. Among the five groups, Class Ⅱ, the transcripts induced under Fedeficient roots, contained the largest number of both lncRNAs ( Fig. 2A) and mRNAs (Fig. 2B). In total, 171 lncRNAs and 1001 mRNAs were differentially expressed under different Fe supply conditions ( Fig. 2; Table S3).
Verification of lncRNAs responding to Fe deficiency using quantitative RT-PCR Quantitative RT-PCR (qRT-PCR) was performed to verify the accuracy of the RNA-seq data for the lncRNAs. Nine intergenic lncRNAs were picked for the verification. Expression of the Class IV lncRNAs XLOC_006153 and XLOC_028199 were induced in shoots but not detected in roots regardless of the Fe supply status. The Class II lncRNAs XLOC_052823 and XLOC_007199 were up-regulated by Fe deficiency in the roots. The remaining 5 lncRNAs belonged to Class Ⅴb, which were induced upon Fe deficiency in both shoots and roots (Fig. 3). Thus, qRT-PCR results were consistent with the RNA-Seq results.

Distribution of lncRNAs and mRNAs in the rice genome
Based on their relative position to protein-coding genes, lncRNAs can be classified into three types.
Intergenic lncRNAs have no overlap with any protein-coding sequences, while sense lncRNAs and antisense lncRNAs overlap with one or more exons of another transcript on the same or opposite strand, respectively [20]. Among the 6477 lncRNAs identified in this work, 3730 (57%) were intergenic lncRNAs, 1696 were cis-lncRNAs, and 1051 were antisense lncRNAs ( Figure S4).
Recent studies have shown that lncRNAs regulate the expression of genes via either cis-or transacting modes based on their genomic proximity to protein-coding genes [29]. To determine the modes of action of the lncRNAs in response to Fe deficiency, the genomic locations of the Fe-related lncRNAs and mRNAs mapped to each chromosome of the rice genome (Fig. 4). No significant genomic proximities were found between the Fe-responsive mRNAs and lncRNAs. Fe-related lncRNAs showed a higher degree of clustering in the genome than did the protein coding transcripts.

The conservation of lncRNAs
To investigate the conservation of the lncRNAs in the Oryza genus, putative lncRNAs were aligned Most of the lncRNAs were highly conserved among the Oryza species. In total, 1662 lncRNAs were detected in more than 7 species, while 860 lncRNAs were conserved in all 8 species (Fig. 5A).
The 860 conserved lncRNAs were aligned with lncRNAs from the monocots Zea mays, Hordeum vulgare, Sorghum bicolor, and Triticum aestivum. Among these 5 grass species, 88 of the lncRNAs were conserved (Fig. 5B; Table S4). However, only 3 of these conserved lncRNAs showed significant response to Fe deficiency (Fig. 5C). The results demonstrated that the lncRNAs were weakly conserved between Oryza and other gramineous plants.
Prediction of potential miRNA precursors and miRNA target mimics miRNAs regulate key aspects of development, cell signaling, and responses to various biotic and abiotic stresses via binding to specific complementary transcripts, including protein coding or noncoding sequences, resulting in the degradation or translational repression of the target. LncRNA have been shown to function as precursors of miRNA in many studies [18,30]. In this study, 23 lncRNAs were identified to be precursors of 29 known miRNAs in rice (Table S5). TCONS_00034827 was predicted to be the precursor of four miRNAs (osa-miR1428b, osa-miR1428c, osa-miR1428d, osa-miR1428e). TCONS_00000537 was predicted to generate two miRNAs (osa-miR156b and osa-miR156c). The results indicated the lncRNAs are quite complex and versatile and that at least some Fe-responsive lncRNAs may function through miRNAs.
In addition to generating miRNAs, lncRNAs are also targets of miRNAs. In this case, lncRNAs function as target mimicry with the sequestered transcript known as an endogenous target mimic (eTM) to inhibit miRNA activity [26]. In order to further verify whether the target mimicry mechanism is involved in Fe regulation in rice, any potential interactions between the Fe-responsive lncRNAs and known Fe-related microRNAs were investigated. Two endogenous target mimics (eTMs), osa-eTM159 and osa-eTM408, were identified. The lncRNAs, XLOC_012715 and XLOC_054182, were predicted to bind the miR159 and miR408, respectively (Fig. 6). The results demonstrated that target mimicry might be a part of the regulation of Fe uptake. 7

Discussion And Conclusions
LncRNAs play important roles in a wide range of biological processes, including development, stress responses, and plant nutrition. In this work, lncRNAs that respond to Fe deficiency in rice roots and shoots were identified. Results generated in this study promote our understanding of how rice plants respond to Fe deficiency.
LncRNAs arise from intergenic, intronic, or coding regions in the sense and antisense directions with lower expression level than mRNAs. Thus, identification of lncRNAs requires the use of an ssRNA-seq strategy. In this study, 6477 lncRNAs were identified and characterized. Differentially expressed lncRNAs and mRNAs were identified by comparing the expression level between + Fe and -Fe conditions. The expression patterns divided them into five classes (Fig. 2). Class Ⅱ, which comprised the molecules up-regulated in the rice root, had the greatest number of transcripts. Among the differentially expressed lncRNAs and mRNAs, we found that the number of lncRNAs and mRNAs responding to Fe deficiency had a similar trend, with more lncRNAs and mRNAs up-regulated under Fe deficiency in both shoots and roots. Moreover, a greater number of lncRNAs and mRNAs were detected in roots in Fe-deficient condition. LncRNA orthologs in different species of Oryza and five grass plants were also found, despite limited sequence similarity. Just 3 lncRNAs that were involved in response to Fe limitation were identified as conserved across the five gramineous plants (Fig. 5).
LncRNAs have been shown to regulate phosphate homeostasis in plants by a novel mechanism called target mimicry. Two endogenous lncRNA target mimics (eTMs) were identified in rice, namely osa-eTM159 and osa-eTM408, which target the miRNAs osa-miR159 and osa-miR408, both of which are known to be related to Fe deficiency (Fig. 6). We speculated that these two lncRNAs might function as target mimics to regulate Fe homeostasis in rice.
Our study provides an insight into the potential functions and regulatory interactions of mRNA and lncRNA molecules when rice plants are grown in Fe-deficient conditions. We believe our study would be an initial and reference for understanding the function of the lncRNAs in regulating iron homeostasis in Oryza sativa.

Methods
Plant growth condition 8 Oryza sativa L. cv. Nipponbare (Nip) was used in this study as the wild type (WT) [14]. The WT seeds were germinated in the dark for 3 days, and then placed on a net floating on a solution with or without iron (1.43 mM NH 4  Strand-specific RNA library construction and sequencing Shoots and roots were separately collected from seedlings grown hydroponically for 10 days after germination with or without Fe, and frozen in the liquid nitrogen immediately. Three biological replicates were used for each sample. Total RNA was extracted from these tissues using TRIzol mRNA transcripts (CPC scores > 0) were also identified from the transcriptome in this work. 9 Validation of several lncRNAs using RT-qPCR Tissues were collected from shoots and roots grown with or without iron for 10 days. Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from total RNA using a cDNA Synthesis Kit (TIANGEN), and RT-qPCR was performed on a LightCycler480 machine (Roche) with SYBR Green Supermix (CWBIO). ACTIN mRNA was used as the internal control for sample normalization. Means ± SD were calculated by three biological repeats. The RT-qPCR primers (synthesized by TSINGKE) are shown in Table S1.

Conserved lncRNAs in eight species of Oryza
To analyze the sequence conservation of lncRNAs, all the lncRNAs identified in this work were used as the query dataset and searched against the lncRNA transcripts collected in CANTATAdb    Figure S4. Location, of lncRNAs relative to the nearest protein-coding genes. Table S1. Primers used in this study for validation of lncRNAs.    18 Figure 3 qRT-PCR validation of lncRNAs responding to Fe deficiency. Nine lncRNAs were chosen. Actin was used as a reference gene, Means ± SD were determined from three biological repeats.

Figure 4
Distribution of differentially expressed mRNAs and lncRNAs on the 12 rice chromosomes.
The transcripts that were differentially expressed in shoots (A) and roots (B) are mapped to the chromosomes separately for clarity. Different colors represent different densities of mRNAs on the chromosome, with red color denoting a high density and blue a low density.
Each triangle represents one lncRNA identified in this study. 20