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Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression in response to plant stresses. Given the importance regulatory roles of lncRNAs, providing methods for predicting the function of these molecules, especially in non-model plants, is strongly demanded by researchers. Here, we constructed a reference sequence for lncRNAs in P. vera (Pistacia vera L.) with 53220 transcripts. In total, we identified 1909 and 2802 salt responsive lncRNAs in Ghazvini, a salt tolerant cultivar, after 6 and 24 h salt treatment, respectively and 1820 lncRNAs in Sarakhs, a salt sensitive cultivar, after 6 h salt treatment. Functional analysis of these lncRNAs by several hybrid methods, revealed that salt responsive NAT-related lncRNAs associated with transcription factors, CERK1, LEA, Laccase genes and several genes involved in the hormone signaling pathways. Moreover, gene ontology (GO) enrichment analysis of salt responsive target genes related to top five selected lncRNAs showed their involvement in the regulation of ATPase, cation transporter, kinase and UDP-glycosyltransferases genes. Quantitative real-time PCR (qRT-PCR) experiment results of lncRNAs, pre-miRNAs and mature miRNAs were in accordance with our RNA-seq analysis. In the present study, a comparative analysis of differentially expressed lncRNAs and microRNA precursors between salt tolerant and sensitive pistachio cultivars provides valuable knowledge on gene expression regulation under salt stress condition.

Constitutively photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that has been studied extensively in the photomorphogenesis- and light-related processes in Arabidopsis. However, the possible role of COP1 in plant drought stress response remains unknown. Hence, in the present study, the stomatal behavior as one of the key elements in plant dehydration response was investigated in Arabidopsis cop1-4 and pea light-independent photomorphogenesis (lip1) mutants. We observed that water loss rate in the cop1-4 and lip1 detached leaves was significantly much faster than wild-type, resulting from failing to reduce the stomatal aperture by the mutants. But, interestingly, the cop1-4 and lip1 isolated leaves treated with abscisic acid (ABA) as well as cop1-4 and lip1 soil-grown under drought stress could close their stomata as wild-type. Hence, COP1 plays a fundamental role in the regulation of stomatal movements in response to dehydration and its function was conserved during evolution in both Arabidopsis and pea. Further evaluations showed the cop1-4 mutant was not significantly damaged from the oxidative stress derived from soil water limiting conditions when compared to wild-type. Similarly, the up-regulation level of several key stress-responsive genes was relatively lower in cop1-4 than wild-type. Therefore, COP1 might be considered as a potential key regulator of both short-and long-term dehydration response. Multiple stress-related cis-elements were also detected in the COP1 promoter region, which supported its up-regulation in response to drought, salt, and cold stresses. Besides, we figured out the constitutively open stomata of cop1-4 in darkness can be as a result of the reduced AtMYB61 expression.

The quality and quantity of RNA are critical for successful downstream transcriptome-based studies such as microarrays and RNA sequencing (RNA-Seq). RNA isolation from woody plants, such as Pistacia vera, with very high amounts of polyphenols and polysaccharides is an enormous challenge. Here, we describe a highly efficient protocol that overcomes the limitations posed by poor quality and low yield of isolated RNA from pistachio and various recalcitrant woody plants. The key factors that resulted in a yield of 150 μg of high quality RNA per 200 mg of plant tissue include the elimination of phenol from the extraction buffer, raising the concentration of β-mercaptoethanol, long time incubation at 65 °C, and nucleic acid precipitation with optimized volume of NaCl and isopropyl alcohol. Also, the A260/A280 and A260/A230 of extracted RNA were about 1.9–2.1and 2.2–2.3, respectively, revealing the high purity. Since the isolated RNA passed highly stringent quality control standards for sensitive reactions, including RNA sequencing and real-time PCR, it can be considered as a reliable and cost-effective method for RNA extraction from woody plants.