RNA Sequencing indicates gene expression changes in Silene sendtneri seeds after seed priming with silicic acid

To improve our understanding of the molecular mechanisms underlaying seed priming, RNA transcriptome analysis was performed using primed and non-primed seeds of Silene sendtneri . Seed priming was performed by submergence in 1% silicic acid for 24h at 4°C, followed by rinsing with sterilised water and desiccation to original moisture content. Silene sendtneri is a species with no sequenced genome and annotation of de novo assembly of transcriptome was done against several species. Gene ontology (GO) analysis indicated that genes related to heavy metal transporters and heat shock proteins are differentially expressed after priming with silicic acid. Within these gene categories, genes such as heavy metal-associated isoprenylated plant protein 26-like (log2fold -8.79) were downregulated, while others such as heavy metal ATPase 5 (log2fold 6.46), heat shock factor protein HSF30-like isoform X1 (log2fold 5.98) were upregulated.


Introduction
Silene sendtneri is an endemic plant species with high capacity to accumulate cadmium and can be classified as a Cd hyperaccumulator . Heavy metal presence in the environment, especially in agricultural land, can lead to harmful effects on human health and different strategies are employed to alleviate such effects through soil remediation. Phytoremediation is one of the approaches, but recently other options are being investigated, such as changes in root absorption rate through exclusion of heavy metals. Seed priming is known as an effective method to improve plant traits including their tolerance to different biotic and abiotic stressors, but the mechanisms of this "primed" memory is still not well understood (Mladenov et al., 2020). Germination as a process, represents probably the most critical stage of plants life and consists out of three phases; phase 1water absorptionimbibition; phase 2activation of metabolic processes; phase 3growth processesradicle protrusion. Seed priming exploits the processes of seed germination specifically events of phase II of germination influencing metabolic processes and gene expression through exposure to short stress (priming agent). Desiccation of the seeds postpriming must be performed before the commence of radicle growth and protrusion to preserve germination capacity of the seeds creating "primed" memory. Through desiccation process seed "memorises" the priming-induced changes enabling primed seeds to perform better under stress conditions. One of most prominent effects of seed priming is synchronisation of germination but it can affect the whole plant life cycle (Srivastava et al., 2021). From plant memory side, the most prominent events commence during signalling events in the phase II of germination process under priming conditions. Triggered signalling pathways and consequent genome-level changes are integrated into "primed" memory. Most investigated priming agents are selenium, salicylic acid, polyethylene glycol (PEG), CaCl 2 and thiourea (Srivastava et al., 2021). The priming process triggers changes at mRNA level as well as protein level in correspondence to specific priming agent. Several priming agents have been associated to upregulation of genes such as genes encoding different antioxidants (Paul et al., 2022), while others have been associated with downregulation of genes, such as PEG where downregulation of genes encoding antioxidants are identified in rice (Lei et al., 2021). The objective of presented study was to investigate molecular mechanisms underlying seed priming memory initiated by seed priming using silicic acid through transcriptome analysis of of primed/non-primed seeds. The study contributes to further elucidation of primed memory and identification of candidate genes responsible for primed memory and enhanced performance of primed plants.

Plant material
Seeds of Silene sendtneri were collected from natural growing population at locality Pjeskovita ravan (43.9128° N, 18.4628° E). Seeds were separated from fruits; dried and voucher specimen was deposited at Laboratory for Plant physiology, Faculty of Science, University of Sarajevo. Priming of seeds was performed using 1% silicic acid for 24 h followed by rinsing in sterile distilled water and desiccation to original water content at room temperature. Primed seeds were stored at 4 °C till use.

RNA isolation and transcriptome sequencing
Total RNA was extracted from 20 uniformed healthy primed or non-primed seeds using a RNAprep Pure Plant Plus Kit (Tiangen) according to the manufacturer instructions. The concentration of total RNA was measured using Qubit and appropriate Qubit kit. The purity and integrity of RNA was checked by Agilend Bioanalyser 2100 system. The cDNA library construction and RNA sequencing were performed in NovaGene (UK). cDNA libraries were constructed using Ilumina TruSeqTM RNA Sample Preparation Kit following manufacturers instruction. All samples were sequenced using Ilumina system HiSeq2500 following standard procedure.

Read mapping, Gene Annotation and Analysis of Gene Expression Level
Sequencing raw reads were processed by filtering out reads with adaptor contamination, reads when uncertain nucleotides constitute more than 10 percent of either read (N > 10%), reads when low quality nucleotides (Base Quality less than 5) constitute more than 50 percent of the read.

Identification of Differentially Expressed Genes (DEGs)
After GO annotation, the successfully annotated genes were grouped into three main GO domains: Biological Process (BP), Cellular Component (CC), Molecular Function (MF). (Result Directory: Result/3.Annotation/GO classification). Genes with fold change (FC) of expression levels >8 and q-value (adjusted P-value) <0.01 were considered as significantly expressed genes (DEGs) (Wang et al., 2010).

Results and Discussion
Seed priming is correlated to significant improvements of stress tolerance in plants, but mechanisms by which primed memory is established are still elusive (Mladenov et al., 2021). In our previous experiments we have described how silicic acid seed priming can contribute to better cadmium tolerance in Silene sendtneri . Through transcriptome analysis of current study, we aimed to investigate the molecular mechanisms in primed state that subsequently influences seedling performance under stress conditions in plants developing from primed seed. Collected data reflect the RNA-seq data of primed state in SiA primed seed of Silene sendneri in correlation to non-primed seeds.

Transcriptome sequencing
After removing the adaptor sequences, low quality, and short reads, 27899385 and 2548320 clean reads were obtained for non-primed and SiA primed seeds, respectively. The Q30 percentage exceeded 90% and CG content (guanine-cytosine) was over 45% which suggests highly accurate and reliable sequencing (Table 1). Transcriptome assembly was performed using methods for species with no reference genome (Grabherr et al., 2011).

Analysis of new genes and differentially expressed genes (DEGs)
In the present study we used high-throughput RNA-sequencing (RNA-seq) to identify differentially expressed genes (DEGs) in the primed seeds with 1% silicic acid in relation to non-primed seeds. A total of 76 944 and 65 218 gene transcripts were assembled for non-primed and SiA-primed seeds, respectively. By comparing the genes that show differential expression in SiA-primed seeds compared to nonprimed seeds we found a total of 221 DEGs using log2FC≥4 and q value ≤ 0.01. The heatmap carried out on the 221 common genes, show clearly it is possible split the DEGs analysed in two different cluster based on their different expression in SiA-primed seeds and nonprimed seeds, respectively. Venn diagram was constructed to determine the number of DEGs unique and in common between each of the two comparisons ( Figure 1). A total of 221 differentially expressed genes (DEGs) were analysed. Among them, 117 genes unique to SiAprimed seeds and 102 to non-primed seeds respectively. Only two DEGs results in common among the two different experimental conditions analysed. DEGs in common were also analysed with MultiExperiment viewer (MeV).

Functional annotations
All DEGs identified in SiA-primed seeds were functionally analysed using gene ontology (GO) classification, COG (Cluster of Orthologous Group), KEGG (KyotoEncyclopedia of Genes and Genomes), Swiss-Prot and NR (non-redundant) databases using BLAST (Basic Local Alignment Search Tool) software ( For the GO classification analysis of DEGs, in the summary graph Figure 2A we can identify different subcategories with indicated significant change in expression, such as ribosome related genes, protein-containing complexes, processes related to rRNA binding, signal transduction and cytoplasm and cytosol. Furthermore, DEGs were assigned to three main categories (biological processes, molecular function, and cellular compartment) with different sub-categories where several DEGs were assigned to more than one sub-category. In category of biological processes ( Figure 2B) subcategory translation and signal transduction were two categories with significantly affected differential gene expression. In category cellular compartment subcategories ribosome, cytoplasm, protein containing complex, intracellular, cell, and cytosol included statistically significant differentially   expressed genes after silicic acid priming compared to non-primed seeds ( Figure 2C). The category molecular function included three subcategories with statistically different gene expression: structural constituent of ribosome, structural molecule activity and rRNA binding ( Figure 2D). These results indicate that primary effect of seed priming using silicic acid results in different gene expression related to ribosome activity and other processes correlated to identified nine subcategories with statistically different gene expression. According to the KEGG mapping, genes related to ribosome structure and function have significantly changed expression patterns in seeds primed with SiA ( Figure 3).
An analysis using KEGG database on biological pathways showed statistically significant change of gene expression levels in genes involved in ribosome function (Figure 3). There are several upregulated genes such as genes L3, L18, S13 and L13 (Figure 4). Upregulation of L3 could be related with early development considering that seed priming can stimulate early germination and faster seedling emergence. L3 if silenced can result in decrease of pre-rRNA affecting biogenesis, and decreased L3 levels can delay development (Popescu et al., 2004). L18 are involved in virus infections as identified in infections with cauliflower mosaic virus (CaMV) (Leh et al., 2000).   On the other hand, multiple genes related to ribosomal function show downregulation patterns in SiA primed seeds, such as S4, S1, L5 and L5e (Figure 4). Ribosomal protein L5 is also known to bind specifically to 5S rRNA and is involved in its nucleocytoplasmic transport (Rosorius et al., 2000), and the nuclear re-entry of 5S rRNA is mediated exclusively by the ribosomal protein L5 (Murdoch et al., 1996). Downregulation of this gene can affect rRNA transport. S4 protein also plays a role for the cytoplasm-nuclear translocation (Yi et al., 2002).

Identification of candidate genes involved in primed memory after SiA priming
Seed priming is a known mechanism for enhancement of plant stress response and tolerance. Analysis of annotated genes revealed several stress related genes up-and downregulated after SiA priming (Supplementary table 1). Presented study gives information on genes identified for the first time in Silene sendtneri such as heavy metal-associated isoprenylated plant protein and some predicted genes such as probable metal-nicotianamine transporter YSL6 which are connected to plants ability to detoxify toxic metals (Divol et al., 2013). These genes could be also responsible for the Cd accumulation abilities of S. sentneri. SiA treatment downregulated large number of heavy metals related as well as some other stress response genes such as heat shock protein genes. In the group of upregulated genes, members of pumilio (Pum)/Puf family RNA binding proteins genes, were upregulated after SiA priming. Members of this gene family are mostly related to plants response to stress, biotic as well as abiotic stress. In biotic stress expression of Pum genes is related to plants immunity (Huh, 2021) and synthesis of antibacterial peptides (Gerber et al., 2006). Expression of Pum genes is also changed under salt, cold or drought stress and upregulated expression of these genes can enhance heat stress tolerance (Nyiko et al., 2019). Considering DEGs identified after SiA seed priming we can postulate that SiA seed priming could be suitable for enhancement of plant tolerance toward biotic and abiotic stress, but it could reduce plant's ability to tolerate, accumulate and detoxify heavy metals. Further research is necessary to verify gene expression patterns in developing plant under stress.

Conclusions
Seed priming is a widely used technique but molecular mechanisms underlaying behind primed memory are still unclear. Silicic acid is a priming agent with known beneficial effects on plants tolerance of different stresses through induction of primed state in primed seeds. Large number of genes are affected by seed priming with SiA mostly affecting ribosome function and expression of genes correlated with plants response to biotic and abiotic stress. Based on obtained results from RNA-Seq selected subset of differentially expressed genes will be further validated by quantitative real-time polymerase chain reaction (RT-qPCR).