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The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.

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Calcium-dependent protein kinases (CDPKs) are encoded by a large gene family and play important roles against biotic and abiotic stresses and in plant growth and development. To date, little is known about the CDPK genes in strawberry (Fragaria x ananassa). In this study, analysis of Fragaria x ananassa CDPK gene family was performed, including gene structures, phylogeny, interactome and expression profiles. Nine new CDPK genes in Fragaria x ananassa were identified based on RNA-seq data. These identified strawberry FaCDPK genes were classified into four main groups, based on the phylogenetic analysis and structural features. FaCDPK genes were differentially expressed during fruit development and ripening, as well as in response to abiotic stress (salt and drought), and hormone (abscisic acid) treatment. In addition, the interaction network analysis pointed out proteins involved in the ABA-dependent response to plant stress via Ca 2+ signaling, especially RBOHs. To our knowledge, this is the first report on CDPK families in Fragaria x ananassa, and it will provide valuable information for development of biofortified fruits and stress tolerant plants. Plants are constantly exposed to stress conditions, such as drought, low or high temperature, and high salinity 1. Plants adapt to these conditions by capturing external signals and modulating their responses using complex mechanisms. These mechanisms involve the perception of the stimulus and subsequent signal transduction, which lead to the activation of various chemical, molecular, biochemical, and physiologic changes, improving plant plasticity 2. The calcium ion (Ca 2+) plays central roles in the regulation of different physiological processes in plants as an important secondary messenger. Transient changes in the cytoplasmic concentration of Ca 2+ are detected by several types of sensor proteins that initiate rapid signal transduction processes by triggering cascades of phosphorylation events. Several major classes of Ca 2+ binding proteins have been characterized in plants, including Ca 2+-dependent protein kinases (CDPK), calmodulin (CaM), CAM-like proteins (CML), and calcineurin B-like proteins (CBL) 3-6. Among these, CDPK constitutes a large calcium-sensing family only found in plants, protists, oomycetes, and green algae, and not in animals and fungi 7. CDPK structures include four domains: N-terminal, self-regulatory/autoinhibitory, serine/threonine kinase, and finally calmodulin-like regulatory domains (CaM-LD) 8. The CaM-LD domain contains three or four EFhand Ca 2+-binding motifs that recognize distinct Ca 2+ signatures with variable affinities. The C-terminal lobe of the CaM-LD binds Ca 2+ with high affinity. At low Ca 2+ levels, the structure is stabilized by the interaction of this lobe with the auto-inhibitory region of the protein. Otherwise, a conformational change induced by the binding of Ca 2+ to the low-affinity N-terminal lobe of CaM-LD results in the release of the auto-inhibition 9,10. CDPKs are involved in stress signaling, hormone respon...