The basic leucine zipper (bZIP) family transcription factors play crucial roles in regulating plant development and stress response. In this study, we identified 62 ClabZIP genes from watermelon genome, which were unevenly distributed across the 11 chromosomes. These ClabZIP proteins could be classified into 13 groups based on the phylogenetic relationships, and members in the same group showed similar compositions of conserved motifs and gene structures. Transcriptome analysis revealed that a number of ClabZIP genes have important roles in the melatonin (MT) induction of cold tolerance. In addition, some ClabZIP genes were induced or repressed under red light (RL) or root-knot nematode infection according to the transcriptome data, and the expression patterns of several ClabZIP genes were further verified by quantitative real-time PCR, revealing their possible roles in RL induction of watermelon defense against nematode infection. Our results provide new insights into the functions of different ClabZIP genes in watermelon and their roles in response to cold stress and nematode infection.

Allene oxide synthase (AOS) and hydroperoxide lyase (HPL), members of the CYP74 gene family, are branches of the oxylipin pathway and play vital roles in plant responses to a number of stresses. In this study, four HPL genes and one AOS gene were identified in the watermelon genome, which were clustered into three subfamilies (CYP74A, CYP74B and CYP74C). Sequence analysis revealed that most HPL and AOS proteins from various plants contain representative domains, including Helix-I region, Helix-K region (ExxR) and Heme-binding domain. A number of development-, stress-, and hormone-related cis-elements were found in the promoter regions of the ClAOS and ClHPL genes, and the detected ClAOS and ClHPL genes were differentially expressed in different tissues and fruit development stages, as well as in response to various hormones. In addition, red light could enhance the expression of ClAOS in root-knot nematode-infected leaves and roots of watermelon, implying that ClAOS might play a primary role in red light-induced resistance against root-knot nematodes. These findings lay a foundation for understanding the specific function of CYP74 genes in watermelon.Agronomy 2019, 9, 872 2 of 17 isomerization or dehydration of fatty acid hydroperoxides and require neither molecular oxygen nor NAD(P)H-dependent cytochrome P450-reductase [1,2,6].CYP74 enzymes are broadly classified into four subfamilies (CYP74A, CYP74B, CYP74C, and CYP74D) based on their evolutionary heritage and relationship with the diversity of oxylipin structure [6]. AOS and DES members constitute the CYP74A and CYP74D subfamily, respectively, while CYP74B and CYP74C subfamilies contain enzymes with HPL activity [5,7]. In addition, AOS enzymes can be classified into three different types: The first two types can use either 13-hydroperoxide derivatives or 9-hydroperoxide derivatives as the substrate (13-AOS and 9-AOS, respectively), while the third type can use both of them (9/13-AOS) as the substrate [4,8]. Similarly, three types of HPL enzymes named as 13-HPL, 9-HPL, and 9/13-HPL were also reported in a number of plant species [9]. To date, AOS and HPL genes have been cloned and functionally characterized from a number of plant species, such as tomato (Solanum lycopersicum L. (Solanaceae)) [10,11], tobacco (Nicotiana attenuate Torr. ex S.Watson) [12], rice (Oryza sativa L.) [13,14], and barrel medic (Medicago truncatula Gaertn.) [15], whereas DES genes have been isolated in very few plants [16][17][18][19][20]. These findings reveal that plant CYP74 family genes are indispensable for oxylipin biosynthesis, and play important roles in plant growth and development, as well as in plant defense to various abiotic and biotic stresses.Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is an agricultural crop with high nutritional and economic values, but it is rather susceptible to various biotic and abiotic stresses during growth and development [21]. The root-knot nematodes (RKNs, Meloidogyne incognita) mainly infect the roots of host plants and increase sus...

The enzyme 12-oxo-phytodienoic acid reductase (OPR) is important in the jasmonic acid (JA) biosynthesis pathway and thus plays a vital role in plant defence. However, systematic and comprehensive analyses of OPR genes in watermelon and their roles in defence responses are extremely limited.• The physicochemical properties, phylogenetic tree, gene structure and cis-acting elements of watermelon OPR genes were analysed using bioinformatics, and qRT-PCR and RNA-Seq were applied to assay expression of OPR genes in watermelon.• A total of five OPR family genes were identified in watermelon, which were unevenly distributed across the four chromosomes. Phylogenetic analysis assigned OPR members from different plant species to five subfamilies (OPRI-OPRV). The motif compositions of OPR members were relatively conserved. Expression analysis using qRT-PCR revealed that ClOPR genes, except for ClOPR5, were highly expressed in the flower and fruit. RNA-seq analysis showed that the ClOPR genes had different expression patterns during flesh and rind development. Furthermore, the ClOPR genes, particularly ClOPR2 and ClOPR4, were significantly upregulated by exogenous JA, salicylic acid (SA) and ethylene (ET) treatments. In addition, red light induced expression of ClOPR2 and ClOPR4 in leaves and roots of root-knot nematode (RKN)-infected watermelon plants, suggesting their involvement in red light-induced defence against RKN.• These results provide a theoretical basis for elucidating the diverse functions of OPR family genes in watermelon.

Allene oxide cyclase (AOC, EC 5.3.99.6) catalyzes the most important step in the jasmonic acid (JA) biosynthetic pathway and mediates plant defense response to a wide range of biotic and abiotic stresses. In this study, two AOC genes were identified from watermelon. Sequence analysis revealed that each of ClAOC1 and ClAOC2 contained an allene oxide cyclase domain and comprised eight highly conserved β-strands, which are the typical characteristics of AOC proteins. Phylogenetic analysis showed that ClAOC1 and ClAOC2 were clustered together with AOCs from dicotyledon, with the closest relationships with JcAOC from Jatropha curcas and Ljaoc1 from Lotus japonicus. Different intron numbers were observed in ClAOC1 and ClAOC2, which may result in their functional divergence. qRT-PCR analysis revealed that ClAOC1 and ClAOC2 have specific and complex expression patterns in multiple organs and under hormone treatments. Both ClAOC1 and ClAOC2 displayed the highest transcriptional levels in stem apex and fruit and exhibited relatively lower expression in stem. JA, salicylic acid (SA), and ethylene (ET) could enhance the expression of ClAOC1 and ClAOC2, particularly that of ClAOC2. Red light could induce the expression of ClAOC2 in root-knot nematode infected leaf and root of watermelon, indicating that ClAOC2 might play a primary role in red light-induced resistance against root-knot nematodes through JA signal pathway. These findings provide important information for further research on AOC genes in watermelon.Agriculture 2019, 9, 225 2 of 12 AOC genes were found to have specific and complicated tissue expression patterns in different plants. In barley, HvAOC mRNA accumulation is abundant in root tip, scutellar node, and leaf base [13]. Soybean GmAOC1 and GmAOC2 also exhibited abundant mRNA transcripts in roots, while GmAOC3 and GmAOC4 showed relatively higher expression in leaves and stems, respectively [7]. Camptotheca acuminata CaAOC is constitutively expressed in various organs, with the highest expression level in leaves [14]. In Arabidopsis, AtAOC1, AtAOC2, and AtAOC3 promoters exhibit high activities in the leaves, while only AtAOC3 and AtAOC4 show promoter activity in roots [15]. These results suggest that AOC genes are involved in the regulation of multiple plant developmental processes.The expression of jasmonate biosynthetic pathway genes, including AOCs, is regulated by JA, and overexpression or suppression of these genes can greatly affect the JA levels in plants. For example, a severe deficiency of jasmonate was found in two AOC mutants (cpm2 and hebiba) of rice [12], and partial suppression of MtAOC1 in hairy roots of Medicago truncatula also resulted in lower JA levels in mycorrhizal roots [16]. In addition, elevated JA levels were found in transgenic plants overexpressing AOC genes from different plants, such as AaAOC from Artemisia annua [5], TaAOC1 from wheat [17], and GmAOC3 from soybean [18]. JA and its related compounds are involved in plant defense reactions against biotic and abiotic stresses, and th...

As a subfamily of basic helix-loop-helix (bHLH) transcription factors, phytochrome-interacting factors (PIFs) participate in regulating light-dependent growth and development of plants. However, limited information is available about PIFs in pepper. In the present study, we identified six pepper PIF genes using bioinformatics-based methods. Phylogenetic analysis revealed that the PIFs from pepper and some other plants could be divided into three distinct groups. Motif analysis revealed the presence of many conserved motifs, which is consistent with the classification of PIF proteins. Gene structure analysis suggested that the CaPIF genes have five to seven introns, exhibiting a relatively more stable intron number than other plants such as rice, maize, and tomato. Expression analysis showed that CaPIF8 was up-regulated by cold and salt treatments. CaPIF8-silenced pepper plants obtained by virus-induced gene silencing (VIGS) exhibited higher sensitivity to cold and salt stress, with an obvious increase in relative electrolyte leakage (REL) and variations in the expression of stress-related genes. Further stress tolerance assays revealed that CaPIF8 plays different regulatory roles in cold and salt stress response by promoting the expression of the CBF1 gene and ABA biosynthesis genes, respectively. Our results reveal the key roles of CaPIF8 in cold and salt tolerance of pepper, and lay a solid foundation for clarifying the biological roles of PIFs in pepper and other plants.