MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes.

Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach to their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, the electrophoretic mobility shift assay, and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.

Mapping and functional verification of leaf yellowing genes in watermelon during whole growth period.

Increasing light energy utilization efficiency is an effective way to increase yield and improve quality of watermelon. Leaf is the main place for photosynthesis, and the color of leaf is directly related to the change of photosynthesis. In addition, leaf yellowing can be used as a marker trait to play an important role in watermelon hybrid breeding and improve seed breeding. It can not only be used to eliminate hybrids at seedling stage, but also be used to determine seed purity. In this study, transcriptome analysis was first carried out using the whole growth period leaf yellowing watermelon mutant w-yl and inbred line ZK, and identified 2,471 differentially expressed genes (DEGs) in the comparison group w-yl-vs-ZK. Among the top 20 terms of the gene ontology (GO) enrichment pathway, 17 terms were related to photosynthesis. KEGG pathway enrichment analysis showed that the most abundant pathway was photosynthesis-antenna proteins. The F2 population was constructed by conventional hybridization with the inbred line ZK. Genetic analysis showed that leaf yellowing of the mutant was controlled by a single recessive gene. The leaf yellowing gene of watermelon located between Ind14,179,011 and InD16,396,362 on chromosome 2 by using indel-specific PCR markers, with a region of 2.217 Mb. In the interval, it was found that five genes may have gene fragment deletion in w-yl, among which Cla97C02G036010, Cla97C02G036030, Cla97C02G036040, Cla97C02G036050 were the whole fragment loss, and Cla97C02G0360 was the C-terminal partial base loss. Gene function verification results showed that Cla97C02G036040, Cla97C02G036050 and Cla97C02G036060 may be the key factors leading to yellowing of w-yl leaves.

Transcriptomic and Metabolomic Analysis of the Effects of Exogenous Trehalose on Salt Tolerance in Watermelon (Citrullus lanatus).

Trehalose can effectively protect the biomolecular structure, maintain the balance of cell metabolism, and improve the tolerance to various abiotic stresses in plants. However, the molecular mechanism underlying the improvement in salt tolerance by exogenous trehalose in watermelon (Citrullus lanatus) seedlings is still unclear. To understand these molecular mechanisms, in this study, watermelon seedlings under salt stress were treated with various concentrations of exogenous trehalose. An amount of 20 mM exogenous trehalose significantly improved the physiological status; increased the activities of enzymes such as POD, SOD, and CAT; and increased the K+/Na+ ratio in watermelon seedlings under salt stress. RNA-seq and metabolomic analysis were performed to identify the specifically expressed genes and metabolites after trehalose treatment. Watermelon seedlings were divided into salt stress (CK2), control (CK1) and trehalose treatment (T) groups as per the treatment. Overall, 421 shared differentially expressed genes (DEGs) were identified in the two comparison groups, namely CK2-CK1 and T-CK2. Functional annotation and enrichment analysis revealed that the DEGs were mainly involved in MAPK signaling pathway for plant hormone signal transduction and phenylpropanoid biosynthesis. Furthermore, 129 shared differential expressed metabolites (DEMs) were identified in the two comparison groups using liquid chromatography-mass spectrometry, which were mainly involved in the metabolic pathway and phenylpropanoid biosynthesis. The combined transcriptomic and metabolomic analyses revealed that genes involved in phenylpropanoid biosynthesis, plant hormone signal transduction, and carbohydrate biosynthesis pathways, especially bHLH family transcription factors, played an important role in improving salt tolerance of watermelon seedlings after exogenous trehalose treatment.

Comparative Transcriptome Analysis Reveals Differential Gene Expression in Resistant and Susceptible Watermelon Varieties in Response to Meloidogyne incognita.

M. incognita is a major parasitic plant disease in watermelon production, causing serious economic losses. Although there are many studies on root-knot nematode, the resistance mechanism is still unclear. In this study, in order to fully understand the mechanism of watermelon resistance to root-knot nematode, the relatively strongly resistant 'Hongzi watermelon' variety and the susceptible 'M16' watermelon variety were used as materials, combined with RNA sequencing (RNA-seq), to analyze the expression abundance of resistant and susceptible varieties at 0, 2, 8 and 15 days post-infection (DPI) by M. incognita. The number of differentially expressed genes (DEGs) in the four comparison groups (A0_B0, A1_B1, A2_B2 and A3_B3) was 3645, 2306, 4449 and 2362, respectively, and there were 835 shared DEGs among them. GO annotation and KEGG pathway enrichment analysis showed that 835 DEGs were mainly involved in phenylpropane biosynthesis and carbon metabolism. Furthermore, lignin-biosynthesis-related genes (4CL (4-coumaric acid-CoA ligase), C3H (coumaric acid 3-hydroxylase), CSE (caffeoyl shikimate esterase), COMT (caffeic acid-O-methyltransferase), CCR (cinnamyl CoA reductase) and PRX (peroxidase)), defense-related proteins (UDP-glucoronosyl/UDP-glucosyl transferase, UGT84A13; salicylic acid binding protein, SABP2) and some transcription factors (TFs) were highlighted, which may be potential candidate genes for further analysis in the infection process of M. incognita. These results suggest that watermelon can achieve resistance to M. incognita by increasing the content of lignin and phenols in root or improving ROS level. These RNA-seq data provide new knowledge for future functional studies and will be helpful to further elucidate the molecular mechanism of resistance to M. incognita in watermelon.

Comparative Transcriptome Profiling Provides Insights into Plant Salt Tolerance in Watermelon (Citrullus lanatus).

Salt stress seriously reduced the yield and quality of watermelon and restricted the sustainable development of the watermelon industry. However, the molecular mechanism of watermelon in response to salt stress is still unclear. In this study, 150 mmol·L-1 NaCl was used to deal with the seedlings of salt-tolerant and salt-sensitive watermelon varieties. Physiological characteristics showed that salt stress significantly reduced the biomass of watermelon seedlings and the accumulation of K+ in roots and leaves and significantly increased the content of Na+, Cl-, and malondialdehyde (MDA). Compared with the salt-sensitive variety, the salt-tolerant variety had higher K+ accumulation, lower Cl-, Cl- accumulation, and MDA content in roots and leaves. Then, RNA-seq was performed on roots and leaves in normal culture and under 150 mmol·L-1 NaCl treatment. A total of 21,069 genes were identified by RNA-seq analysis, of which 1412 were genes encoding transcription factors (TFs). In the comparison groups of roots and leaves, 122 and 123 shared differentially expressed genes (DEGs) were obtained, respectively. Gene ontology (GO) annotation and KEGG enrichment results showed that there were many identical GO terms and KEGG pathways in roots and leaves, especially the pathways that related to sugar or energy (ATP or NADP+/NADPH). In addition, some DEGs related to salt tolerance were identified, such as plant hormone indole-3-acetic acid (IAA) and gibberellin (GA) signal transduction pathway-related genes, K+/Na+/Ca2+-related genes, lignin biosynthesis-related genes, etc. At the same time, we also identified some TFs related to salt tolerance, such as AP2-EREBP, bZIP, bHLH, MYB, NAC, OFP, TCP, and WRKY and found that these TFs had high correlation coefficients with salt tolerance-related genes, indicating that they might have a potential regulatory relationship. Interestingly, one TCP TF (Cla97C09G174040) co-exists both in roots and leaves, and it is speculated that it may be regulated by miR319 to improve the salt tolerance of watermelon.

Genome-wide identification and characterization of AINTEGUMENTA-LIKE (AIL) family genes in apple (Malus domestica Borkh.).

AINTEGUMENTA-LIKE (AIL) genes play a key role in various growth and developmental processes in plants. Thus far, the genome-wide identification of AIL genes has been reported for some plant species. However, genome-wide identification of AIL genes has not been conducted in apple (Malus domestica Borkh.). The current study focused on a comprehensive analysis of the AIL genes in the apple genome (i.e., MdAIL genes). In total, 27 MdAIL genes in the apple genome were identified and then divided into four groups according to phylogenetic analysis. The chromosomal locations, gene and protein structures, and physicochemical characteristics of MdAIL genes were analyzed. Synteny analysis revealed that segmental duplication events played a major role in the expansion of the AIL gene family in apple. The analysis of cis-regulatory elements in MdAIL promoter regions indicated that most of the MdAIL genes are involved in embryo development and seed germination. Moreover, the analysis of tissue-specific expression patterns and transcript levels in adventitious bud regeneration indicated that MdAIL genes play an extensive regulatory role in apple growth and development, especially in the regulation of germination and adventitious bud regeneration from in vitro leaves of apple. In conclusion, this is the first genome-wide analysis of the AIL genes in apple. The current results may help in better understanding the evolution and function of MdAIL genes and thus facilitate further research on plant growth and development.

Genome-Wide Identification and Characterization of the Trehalose-6-phosphate Synthetase (TPS) Gene Family in Watermelon (Citrullus lanatus) and Their Transcriptional Responses to Salt Stress.

With the increase in watermelon cultivation area, there is an urgent need to explore enzymatic and genetic resources for the sustainable development of watermelon, especially under salt stress. Among the various compounds known, trehalose plays an important role in regulating abiotic stress tolerances in diverse organisms, including plants. Therefore, the present study comprehensively analyzed the trehalose-6-phosphate synthase (TPS) gene family in watermelon. The study analyzed the functional classification, evolutionary characteristics, and expression patterns of the watermelon TPS genes family. Seven ClTPSs were identified and classified into two distinct classes according to gene structure and phylogeny. Evolutionary analysis suggested the role of purifying selection in the evolution of the TPS family members. Further, cis-acting elements related to plant hormones and abiotic stress were identified in the promoter region of the TPS genes. The tissue-specific expression analysis showed that ClTPS genes were widely expressed in roots, stems, leaves, flowers, and fruits, while ClTPS3 was significantly induced under salt stress. The overexpression of ClTPS3 in Arabidopsis thaliana significantly improved salt tolerance. Finally, the STRING functional protein association networks suggested that the transcription factor ClMYB and ClbHLH regulate ClTPS3. Thus, the study indicates the critical role of ClTPS3 in watermelon response to salt stress.

Major Latex Protein MdMLP423 Negatively Regulates Defense against Fungal Infections in Apple.

Major latex proteins (MLPs) play critical roles in plants defense and stress responses. However, the roles of MLPs from apple (Malus × domestica) have not been clearly identified. In this study, we focused on the biological role of MdMLP423, which had been previously characterized as a potential pathogenesis-related gene. Phylogenetic analysis and conserved domain analysis indicated that MdMLP423 is a protein with a 'Gly-rich loop' (GXGGXG) domain belonging to the Bet v_1 subfamily. Gene expression profiles showed that MdMLP423 is mainly expressed in flowers. In addition, the expression of MdMLP423 was significantly inhibited by Botryosphaeria berengeriana f. sp. piricola (BB) and Alternaria alternata apple pathotype (AAAP) infections. Apple calli overexpressing MdMLP423 had lower expression of resistance-related genes, and were more sensitive to infection with BB and AAAP compared with non-transgenic calli. RNA-seq analysis of MdMLP423-overexpressing calli and non-transgenic calli indicated that MdMLP423 regulated the expression of a number of differentially expressed genes (DEGs) and transcription factors, including genes involved in phytohormone signaling pathways, cell wall reinforcement, and genes encoding the defense-related proteins, AP2-EREBP, WRKY, MYB, NAC, Zinc finger protein, and ABI3. Taken together, our results demonstrate that MdMLP423 negatively regulates apple resistance to BB and AAAP infections by inhibiting the expression of defense- and stress-related genes and transcription factors.

Genome-wide identification and expression analysis of major latex protein (MLP) family genes in the apple (Malus domestica Borkh.) genome.

Major latex protein/ripening-related proteins (MLP/RRP) subfamily are a class of proteins that play crucial roles in response to defense and stress response. However, their biological function is still not clear, the identification and characterization will provide essential information for understanding their roles. Here, we carried out a genome-wide evolutionary characteristics and gene expression analysis of the MLP family in apple (Malus domestica, Borkh.). A total of 36 MdMLP genes were screened in apple genome. They were uneven located on 5 chromosomes, where were mainly arranged in tandem clusters, and the phylogenetic analysis put forward further views on the evolutionary relationship and putative functions among the genes. The conserved motifs showed that the MLP proteins which contained motif 1 had the potential function, and tissue-specific expression analysis showed that apple MLP members had diverse biological roles. Furthermore, the results showed seven of the MdMLPs that harbored cis-acting regulatory elements in response to defense and stress, and our expression data proved that they were involved in biotic stresses. The present study provides new views to the evolution and regulation of MdMLP genes, which represent objectives of future research and incorporate in resistance-related molecular breeding projects.

An Integrated Transcriptome and Proteome Analysis Reveals New Insights into Russeting of Bagging and Non-Bagging "Golden Delicious" Apple.

Apple skin russeting naturally occurs in many varieties, particularly in "Golden Delicious" and its pedigree, and is regarded as a non-invasive physiological disorder partly caused by excessive deposition of lignin. However, the understanding of its molecular mechanism is still limited. In this study, we used iTRAQ (isobaric tags for relative and absolute quantitation) and RNA-seq to detect the changes in the expression levels of genes and proteins in three developmental stages of russeting formation, in russeted (non-bagging) and non-russeted (bagging) skin of "Golden Delicious" apple. 2856 differentially expressed genes and 942 differentially expressed proteins in the comparison groups were detected at the transcript level and protein level, respectively. A correlation analysis of the transcriptomics and proteomics data revealed that four genes (MD03G1059200, MD08G1009200, MD17G1092400, and MD17G1225100) involved in lignin biosynthesis are significant changed during apple russeting formation. Additionally, 92 transcription factors, including 4 LIM transcription factors, may be involved in apple russeting formation. Among them, one LIM transcription factor (MD15G1068200) was capable of binding to the PAL-box like (CCACTTGAGTAC) element, which indicated it was potentially involved in lignin biosynthesis. This study will provide further views on the molecular mechanisms controlling apple russeting formation.

A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour.

A complete and accurate genome sequence provides a fundamental tool for functional genomics and DNA-informed breeding. Here, we assemble a high-quality genome (contig N50 of 6.99 Mb) of the apple anther-derived homozygous line HFTH1, including 22 telomere sequences, using a combination of PacBio single-molecule real-time (SMRT) sequencing, chromosome conformation capture (Hi-C) sequencing, and optical mapping. In comparison to the Golden Delicious reference genome, we identify 18,047 deletions, 12,101 insertions and 14 large inversions. We reveal that these extensive genomic variations are largely attributable to activity of transposable elements. Interestingly, we find that a long terminal repeat (LTR) retrotransposon insertion upstream of MdMYB1, a core transcriptional activator of anthocyanin biosynthesis, is associated with red-skinned phenotype. This finding provides insights into the molecular mechanisms underlying red fruit coloration, and highlights the utility of this high-quality genome assembly in deciphering agriculturally important trait in apple.