Study of the role of Zinc Finger transcription factor of Arabidopsis thaliana 6 (ZAT6) in forage turnip (Brassica rapa L.) salinity tolerance

Soil and water salinity has significantly reduced crop yields and threatened human food security worldwide. Understanding the molecular basis of how crop plants receive and respond to salinity and identifying the main components of their stress tolerance are essential for developing tolerant plants through genetic manipulation. Today, functional genomics methods such as transcriptomics, proteomics, and metabolomics have revolutionised the study of plant stress response and facilitated the identification of important pathways and components governing stress tolerance. For example, the production and analysis of EST sequences, RNA microarray, and RNA-sequencing technology (RNA-seq) have made it possible to study and identify gene transcripts in genome-wide stress response networks. Accordingly, this study aimed to analyse the transcript of forage turnip (Brassica rapa L.) under salinity stress, determine the functional orientation of the genome, and identify the important components and genes involved in salinity tolerance.

Two Expressed Sequenced Tags (ESTs) libraries of forage turnip under non-stress and salinity stress conditions were analysed using bioinformatic and statistical methods. Non-stress and salinity stress libraries had 8408 and 6894 sequences, respectively. After sequences trimming, the genome functional orientation of forage turnip in response to salinity stress was determined based on Fisher's exact test using the DAVID tool. Differentially expressed genes (DEGs) were assigned using the Audio and Claverie statistical test (AC test) implemented in the IDEG6 tool, at a significance level of 5%. Based on Fisher's exact test, a hierarchical gene network among DEGs was predicted. Then, gene network topology analysis was performed using the NetworkAnalyzer plugin in Cytoscape 4.3. One gene was identified as an important gene (Hub gene) based on gene network topology analysis. In a greenhouse experiment using a tolerant genotype and a susceptible genotype of forage turnip, the expression profile of the identified important gene along with some traits related to the plant antioxidant system, including anthocyanin content, antioxidant enzymes activity, and malondialdehyde content, were evaluated in response to different salinity time points. Finally, the relationship among the changes in gene expression, evaluated traits, and salinity tolerance was determined using Correspondence Analysis (CA).

Pre-processing of 8408 EST sequences of the non-stress library resulted in 8,403 high-quality sequences. Also, out of 6894 EST sequences from the salinity stress library, 6894 high-quality sequences were obtained. BlastX against Arabidopsis proteins showed that 8075 (96.1%) of the non-stress EST sequences had at least one specific hit. 6597 (95.7%) of the EST sequences of the salinity stress library also had at least one specific hit in BlastX. Based on Fisher's exact test, 15 functional groups were significantly (FDR≤0.01) more active in salinity stress conditions than in non-stress conditions. These results clearly showed that under salinity conditions, the genome functional activity of forage turnip was oriented towards response to stresses, especially oxidative stresses, response to internal inductions such as plant hormones, control of metabolic activities, and photosynthetic reactions. Audic and Claverie’s statistical test showed 344 genes were differentially expressed in response to salinity stress; among them, 242 genes were upregulated, and 102 genes were downregulated. The predicted gene network indicated a complex relationship among DEGs, regulatory molecules (especially melatonin and plant hormones), and downstream responsive pathways. Among identified DEGs, the gene encoding transcription factor ZAT6 was assigned as an important gene in the salinity response gene network. The expression profile of the ZAT6 gene, quantity of anthocyanin, the activity of antioxidant enzymes and content of malondialdehyde were significantly different between the two studied genotypes. ZAT6 was significantly more expressed in the stress-tolerant genotype than the stress-susceptible genotype at all time points. In addition, the antioxidant system of the tolerant genotype was more potent than the susceptible genotype. Also, results revealed a significant relationship between the expression profile of ZAT6 and evaluated traits in the context of salinity tolerance. Based on the results, changes in ZAT6 gene expression are directly or indirectly involved in regulating forage turnip plant responses to salinity stress, especially through the control of evaluated traits.

Transcriptome study clarified some of the molecular bases of the forage turnip response to salinity stress. Accordingly, it seems that the gene encoding the ZAT6 transcription factor plays an important role(s) in the salinity stress tolerance of forage turnip. There was a significant relationship between high expression levels of this gene and enhanced antioxidant activities, which could confirm the hypothesis. However, further studies are needed to assign detailed functions of ZAT6, particularly the association of this gene with the regulatory pathway of melatonin as a major regulatory molecule in plants. This can be an effective starting point for further studies.