A long non-coding RNA PelncRNA1 is involved in Phyllostachys edulis response to UV-B stress

Plant material and UV-B treatment

The moso bamboo seeds were collected from a single mother bamboo. Seedlings with five mature leaves and consistent growth status were cultivated in a dark incubator (25 °C) for three days before treatment to eliminate the influence of UV in visible light (Biever, Brinkman & Gardner, 2014). For UV-B stress, UV-B treatments were applied for 2, 4, 6, and 8 h (Hunter et al., 2018; Makarevitch et al., 2015; Weber et al., 2020). The seedlings not subjected to the above stress treatments served as a control group (CK). All the samples were harvested directly into liquid nitrogen and stored at −80 °C until used for RNA extraction.

Screening and identification of lncRNA

The annotation information of lncRNA was acquired from the whole transcriptome sequencing of moso bamboo (Ding et al., 2022). Based on this database, we screened lncRNA, and their target genes, which were differentially expressed under UV-B treatment. There are four software used to determine the coding of transcripts and identify typical lncRNAs, including CNCI (Coding On-Coding Index) (Sun et al., 2013), CPC2 (Coding Potential Calculator) (Kong et al., 2007), PLEK (predictor of long non-coding RNAs, and messenger RNAs based on an improved k-mer scheme), Pfam (PfamScan) (Finn et al., 2016).

Target gene prediction and validation of lncRNA

There are two methods for predicting the target genes of lncRNAs. First, based on the position of genes near lncRNAs, we identified genes within 100 kb of the lncRNA as their cis-target genes with Perl scripts (Jia et al., 2010). The second method uses the online software LncTar (http://www.cuilab.cn/lnctar) to determine the normalized free energy according to how the bases of lncRNAs and mRNAs are paired (Li et al., 2015). Genes below the normalized free energy threshold were identified as trans-target genes of lncRNA.

Based on the transcriptome data, the differentially expressed target genes that corresponded to the relevant lncRNA transcription change trend were identified as the focus. QRT-PCR was used to confirm the relevance between PelncRNA1 and its target gene expression trends, and weakly correlated target genes were eliminated.

PelncRNA1 cloning, vector construction

Two specific primers (Table S1) were designed to amplify PelncRNA1. Full-length PelncRNA1 was cloned by PCR using KOD One™ PCR Master Mix (TOYOBO, KMM-101S, Shanghai, China). The positive PCR product was ligated into the transient over-expression vector pUBQ10 and the stable over-expression vector pER8, which were named pUBQ10-lncRNA and pER8-lncRNA respectively, and confirmed by Sanger sequencing. In the pUBQ10-lncRNA and pER8-lncRNA vectors, PelncRNA1 was promoted by the 35S promoter.

Protoplast isolation, polyethylene glycol (PEG) transfection, UV-B treatment

The protoplast isolation and PEG-mediated method were conducted as previously described (Hisamoto & Kobayashi, 2010; Yoo, Cho & Sheen, 2007), with some modifications. Briefly, 21-day-old moso bamboo leaf sheaths grown in soil were cut into small pieces using a razor blade and incubated for 4 h in an enzyme solution. After centrifugation, 2 − 3 × 10⁴ protoplasts were resuspended in a 200 µL MMG solution (4 mM MES-KOH [pH 5.7], 0.4 M mannitol, and 15 mM MgCl₂). A total of 10 µg of pUBQ10-lncRNA were mixed well with 100 µL of protoplasts and a PEG solution (40% PEG4000, 0.8 M mannitol, and 1 M CaCl₂). After 4 min of incubation, W5 solution (4 mM MES-KOH [pH 5.7], 0.5M mannitol, and 20 mM KCl) was added to the sample. The protoplasts were incubated and harvested.

Transfection was observed using a confocal laser scanning microscope (Zeiss, LSM510, Germany) after 12 to 16 h of incubation. Then, WT and transgenic protoplasts were exposed to UV-B radiation for 30 s. The protoplast viability would decrease if radiation exceeded 30 s (Fanguo & Guangmin, 2001; Gu, Lu & Yuan, 2004). For each treatment, three replicates were used for PelncRNA1 and its target gene expression analyses.

Genetic transformation and UV-B treated transgenic Arabidopsis

The constructs of pER8-lncRNA were transferred into Agrobacterium tumefaciens strain GV3101 and transformed into wide-type (WT) Arabidopsis (Col-0 ecotype) using the floral dip method (Zhang et al., 2006). The T0 transgenic plants were raised to adulthood. On half MS medium with 25 mg/L hygromycin, T1 seeds were harvested and germinated. Using PelncRNA1-specific primers, PCR was used to examine the segregation patterns in T1 progenies. T2 seeds from independent T1 lines showing 3:1 Mendelian segregation ratios were harvested separately and germinated on a hygromycin-containing rooting medium. T2 seedlings with absent segregation patterns were considered homozygous lines, and six were selected and used for subsequent UV-B stress experiments.

The 4-week-old transgenic and WT Arabidopsis were exposed to UV-B radiation for 1 and 2 h (Han & Han, 2015; You et al., 2011) to test their susceptibility to UV-B stress. Each treatment had three replicates, with natural daylight as the control group. The leaves of the samples were collected and immediately frozen in liquid nitrogen and stored at −80 °C until total RNA extraction was required.

RNA extraction and qRT-PCR

Total RNAs were extracted with an RNA extraction kit (TIANGEN, DP441, Beijing, China). Reverse transcription was performed with the Hifair^® II 1st Strand cDNA Synthesis SuperMix for qPCR (YEASEN, 11123ES, Shanghai, China). Real-time quantitative PCR was performed on a standard protocol (CFX 96TM Touch Deep Well, BIO-RAD) using NTB and ACTIN2 as a control. Primers for qPCR (Table S1) were designed with Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/). The data was analyzed using the 2^−ΔΔCT method (Livak & Schmittgen, 2001).

Phenotypic identification of transgenic Arabidopsis under UV stress

The malondialdehyde (MDA) and chlorophyll content of plant leaves were determined using the thiobarbituric acid colorimetric method and the SPAD-502plus chlorophyll meter (Minolta, Tokyo, Japan) after 0 h, 1 h, and 2 h of UV-B treatment, respectively. The single photon avalanche diode (SPAD) value is proportional to the relative content of chlorophyll (Ruiz-Espinoza et al., 2010). Three replicates were set up for each treatment, and the experimental results were calculated using IBM SPSS Statistics and Excel.

Identification and analysis of PelncRNA1 and its target gene

We screened the differentially expressed PelncRNA1 and its target genes for differential expression in response to UV-B stress. It was identified as a long intergenic non-coding RNA (lincRNA) located in an intergenic region on chromosome 13 of the moso bamboo genome. It consists of one exon, and the full length of the transcript is 373 bp (Table S2). Then, CNCI, CPC2, PLEK, and Pfam were used to evaluate the authenticity of PelncRNA1. PelncRNA1 cannot encode proteins, according to results from CNCI and Pfam software. PelncRNA1 was also unlikely to encode proteins by CPC2 (Coding Probability: 0.0234985, Fickett Score: 0.40197) and PLEK (score −2.343590, RNA with a score less than 0 is considered non-coding RNA). Therefore, PelncRNA1 is considered to be a typical non-coding RNA.

Next, the online software LncTar was used to calculate the normalized free energy of PelncRNA1 and mRNA pairing sites. Genes below normalized free energy were identified as candidate target genes of PelncRNA1 (Table S3). Furthermore, transcriptome data were used to filter the expression patterns of candidate genes that were consistent with or inverse to the expression modes of PelncRNA1. Finally, the differentially expressed candidate genes were chosen as the predicted target genes (Table S4), including PH02Gene33364 (Probable WRKY transcription factor 50), PH02Gene38550 (Wall-associated receptor kinase 3), PH02Gene43330 (Transcription factor BHLH148), PH02Gene19065 (CBL-interacting protein kinase 2), PH02Gene05460 (Purine permease 3), PH02Gene26812 (Berberine bridge enzyme-like 18), PH02Gene35897 (Chorismate synthase 2, chloroplastic), PH02Gene50461 (Hydroquinone glucosyltransferase). Details of the homologous genes of these target genes in Arabidopsis are listed in Table S4.

Expression pattern of PelncRNA1 and target genes in moso bamboo seedlings under UV-B stress

To study the expression pattern of PelncRNA1 and its predicted target genes under UV-B treatment, we investigated the expression level of PelncRNA1 and its predicted target genes under UV-B treatment at different times. The results showed that the PelncRNA1 expression level increased during UV-B treatment (2 h, 4 h, 6 h, 8h). Moreover, the expression pattern of eight predicted target genes revealed a positive correlation with the expression level changes of the corresponding PelncRNA1. PelncRNA1 was more strongly associated with its eight predicted target genes after UV-B treatment (Fig. 1).

Over-expression of PelncRNA1 in moso bamboo protoplasts

To verify the authenticity of the expression pattern of PelncRNA1 and its target genes under UV-B treatment, we constructed a pUBQ10-lncRNA vector (Fig. S2A) and transfected it into moso bamboo protoplasts by the PEG-mediated method (Fig. S2C). Compared with WT moso bamboo protoplasts, the relative expression level of PelncRNA1 was significantly increased in protoplasts transfected with pUBQ10-lncRNA after UV-B treatment. Similarly, the relative expression level of target genes of PelncRNA1 was also significantly increased (Fig. 2). The above results indicate that PelncRNA1 and its target genes can respond to UV-B, and their expression patterns are positively correlated.

Over-expression of PelncRNA1 in Arabidopsis

We determined the non-homology of PelncRNA1 by BLASTN analysis that no sequence similar to PelncRNA1 was found in the other species, such as Arabidopsis, Oryza sativa, Nicotiana tabacum, Glycine max, and Populus poplars. This indicates that PelncRNA1 is a moso bamboo-specific lncRNA. The full-length PelncRNA1 transcript was cloned to construct a stable over-expression vector pER8-lncRNA (Fig. S2B). Ten independent transgenic lines were obtained, and six over-expression (OE-) lines were selected for further study. Using WT Arabidopsis as a negative control, PCR detection was performed using specific primers (Table S1). The results showed that six lines of transgenic Arabidopsis had successfully over-expressed PelncRNA1 (Fig. S1).

WT Arabidopsis and transgenic Arabidopsis T2 plants (OE-PelncRNA1) were treated with UV-B for 1 h and 2 h. In OE-PelncRNA1 plants, the expression level of PelncRNA1 was significantly up-regulated after UV-B stress treatment (Fig. 3A). Under untreated conditions, the expression levels of these target genes in OE-PelncRNA1 plants were similar to those in WT Arabidopsis. However, the expression of these genes increased in both WT and OE-PelncRNA1 plants after UV-B stress treatment (Figs. 3B–3I). The expression levels of target genes in OE-PelncRNA1 plants were significantly higher than that of WT after UV-B treatment, especially AT5G43650 (Fig. 3D). These data indicated that PelncRNA1 could regulate plant tolerance to UV-B stress by regulating the expression of homologous target genes.

Additionally, phenotypic observation found no abnormal phenotype of OE-PelncRNA1 under natural light compared to WT (Fig. 4A). After one hour of UV-B treatment, OE-PelncRNA1 leaves did not wilt or lose water, but WT leaves started to wilt lightly (Fig. 4B). After 2 h of treatment, the leaves of OE-PelncRNA1 were only slightly wilted, whereas the WT leaves were severely wilted and significantly damaged by UV-B radiation (Fig. 4C). This result demonstrates that PelncRNA1 increases the resistance of transgenic Arabidopsis to UV stress.

We also examined the malondialdehyde (MDA) and chlorophyll content of transgenic Arabidopsis with OE-PelncRNA1 under UV-B stress. Under natural light conditions, the contents of MDA and chlorophyll in OE-PelncRNA1 and WT lines were similar. However, after 1 h UV-B treatment, the MDA content of OE- PelcRNA1 was not significantly different but significantly increased in WT. MDA was increased in both OE-PelncRNA1 and WT plants after 2 h treatment, but the elevation in WT plants was much larger than in OE-PelncRNA1 plants (Fig. 5A). The content of MDA concentration can represent the degree of cell membrane lipid peroxidation and is a crucial indication for detecting plant stress (Tsikas, 2017). The result implies that OE-PelncRNA1 plants had lower membrane lipid peroxidation under UV-B stress than WT plants.

After UV-B treatment, both OE-PelncRNA1 and WT plants’ SPAD values (single photon avalanche diode) decreased, and the SPAD value of WT plants decreased much more than the OE-PelncRNA1 plants (Fig. 5B). Since the SPAD value was proportional to chlorophyll content, this experiment demonstrated that the UV-B treatment on chlorophyll in OE-PelncRNA1 plants was less adverse than in WT plants. The physiological and phenotypic observations confirmed that OE-PelncRNA1 plants were more tolerant to UV-B stress than control plants.