*Article*

## **Seed Transcriptome Annotation Reveals Enhanced Expression of Genes Related to ROS Homeostasis and Ethylene Metabolism at Alternating Temperatures in Wild Cardoon**

#### **Hector R. Huarte 1,**†**, Giuseppe. D. Puglia 2,\*,**†**, Andrey D. Prjibelski 3 and Salvatore A. Raccuia 2**


Received: 7 August 2020; Accepted: 13 September 2020; Published: 18 September 2020

**Abstract:** The association among environmental cues, ethylene response, ABA signaling, and reactive oxygen species (ROS) homeostasis in the process of seed dormancy release is nowadays well-established in many species. Alternating temperatures are recognized as one of the main environmental signals determining dormancy release, but their underlying mechanisms are scarcely known. Dry after-ripened wild cardoon achenes germinated poorly at a constant temperature of 20, 15, or 10 ◦C, whereas germination was stimulated by 80% at alternating temperatures of 20/10 ◦C. Using an RNA-Seq approach, we identified 23,640 and annotated 14,078 gene transcripts expressed in dry achenes and achenes exposed to constant or alternating temperatures. Transcriptional patterns identified in dry condition included seed reserve and response to dehydration stress genes (i.e., *HSPs*, peroxidases, and *LEAs*). At a constant temperature, we observed an upregulation of ABA biosynthesis genes (i.e., *NCED9*), ABA-responsive genes (i.e., *ABI5* and *TAP)*, as well as other genes previously related to physiological dormancy and inhibition of germination. However, the alternating temperatures were associated with the upregulation of ethylene metabolism (i.e., *ACO1*, *4,* and *ACS10*) and signaling (i.e., *EXPs*) genes and ROS homeostasis regulators genes (i.e., *RBOH* and *CAT*). Accordingly, the ethylene production was twice as high at alternating than at constant temperatures. The presence in the germination medium of ethylene or ROS synthesis and signaling inhibitors reduced significantly, but not completely, germination at 20/10 ◦C. Conversely, the presence of methyl viologen and salicylhydroxamic acid (SHAM), a peroxidase inhibitor, partially increased germination at constant temperature. Taken together, the present study provides the first insights into the gene expression patterns and physiological response associated with dormancy release at alternating temperatures in wild cardoon (*Cynara cardunculus* var. *sylvestris*).

**Keywords:** RNA-Seq; dormancy termination; gene expression; antioxidants; ethylene signaling; environmental signals
