Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone

Abstract

Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d−1 for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including specific transcription factors (MYB and WRKY). This study demonstrates that a complex sequence of events takes place in the cells at intracellular and membrane level following O3 exposure and elucidates the effects of this oxidative stress on the transcriptional machinery of the non-model plant species V. lantana, with the final aim to provide the molecular supportive knowledge for the use of this plant as O3-bioindicator.

Transcriptome profiles of Quercus rubra responding to increased O3 stress

“…Higher levels of ROS in cells ultimately leads to programmed cell death [96]. Therefore, consistent with past studies [60,62], increased expression of HSP and amino acid glutathione, an important anti-oxidant, plus other ROS scavengers in plant tissues, as we observed, may provide detoxification methods that diminish O 3 induced damages [16,57,97].…”

“…To give a broad overview of annotations, GO term assignments were re-mapped to secondtier GO terms, yielding 70 total terms (Additional file 3: Figure S2), which included: 21,623 putative transcripts that were assigned to terms in the biological process ontology (BP), 20,073 putative transcripts that were assigned to terms in the cellular component ontology (CC), and 24,819 putative transcripts that were assigned to terms in the molecular function (MF) group. The most abundant GO terms for each category were classified as metabolic processes (16,696) and cellular processes (16,125) for BP, cell (14,036) and cell part (13,972) for CC, and binding (16,103) and catalytic activity (15,065) for MF categories. Based on the full set of retrieved GO terms, a total of 10,026 Enzyme Commission (EC) numbers were assigned to annotated putative transcripts, which were utilized to obtain Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assignments.…”

“…One of the key abiotic stressors implicated in oak decline is ozone (O 3 ), a compound that is formed by the interaction of sunlight and molecular oxygen and by the interactions of chemicals commonly found in industrial and automobile emissions such as nitrogen oxides and volatile organic compounds. Tree physiology is altered in the presence of O 3 as evidenced by elevated water use, increased respiration and transpiration, and modified carbon allocation, resulting in decreased tree vegetative growth and life span [16][17][18][19][20]. Forest productivity loss by exposure to O 3 in the eastern USA has been estimated between 1 and 10% [21].…”

The Biosynthesis of Phenolic Compounds Is an Integrated Defence Mechanism to Prevent Ozone Injury in Salvia officinalis

“…To date, we do not know how many PAL members are present in S. officinalis . In addition, an increased expression profile of genes encoding enzymes in the early part of the phenylpropanoid and flavonoid biosynthesis pathway (such as C4H ) [ 49 ] and in the early section of RA biosynthesis (such as TAT ) was observed throughout the whole experiment, suggesting that the mechanisms regulating proteins encoded by these genes were, at least in part, at transcriptional level, according to Gottardini et al [ 50 ]. Song and Li [ 51 ] suggested that four genes ( PAL , C4H , 4CL , and HPPR ) may serve as key genes in the regulation of rosmarinic acid accumulation in S. miltiorrhiza , while TAT may play an important role in regulating the content of salvianolic acid B.…”

Ozone Stress Responsive Gene Database (OSRGD ver. 1.1): a literature curated database for crop improvement

“…Transcriptome studies have also been done on Medicago truncatula and Pak choi using DNA microarray and RNA-seq respectively [Iyer et al, 2013; Zhang et al, 2017]. Apart from these crop plants, studies have also been performed for tree species such as Populus [Gupta et al, 2005; Street et al, 2011], Quercus [Natali et al, 2018; Soltani et al, 2020], Betula pendula [KONTUNEN-SOPPELA et al, 2010] and Viburnum lantana [Gottardini et al, 2016]. These studies have enriched the knowledge about genes which were differentially expressed under elevated ozone stress.…”