The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants.

The mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack. A genome-wide analysis revealed the presence of at least 14 MAPKs, 6 MAPKKs, and 59 MAPKKKs in the cucumber genome. Phylogenetic analyses classified all the CsMAPK and CsMAPKK genes into four groups, whereas the CsMAPKKK genes were grouped into the MEKK, RAF, and ZIK subfamilies. The expansion of these three gene families was mainly contributed by segmental duplication events. Furthermore, the ratios of non-synonymous substitution rates (Ka) and synonymous substitution rates (Ks) implied that the duplicated gene pairs had experienced strong purifying selection. Real-time PCR analysis demonstrated that some MAPK, MAPKK and MAPKKK genes are preferentially expressed in specific organs or tissues. Moreover, the expression levels of most of these genes significantly changed under heat, cold, drought, and Pseudoperonospora cubensis treatments. Exposure to abscisic acid and jasmonic acid markedly affected the expression levels of these genes, thereby implying that they may play important roles in the plant hormone network. A comprehensive genome-wide analysis of gene structure, chromosomal distribution, and evolutionary relationship of MAPK cascade genes in cucumber are present here. Further expression analysis revealed that these genes were involved in important signaling pathways for biotic and abiotic stress responses in cucumber, as well as the response to plant hormones. Our first systematic description of the MAPK, MAPKK, and MAPKKK families in cucumber will help to elucidate their biological roles in plant.

Mitogen-activated protein kinase (MAPK) cascades have important functions in plant growth, development, and response to various stresses. The MAPKK and MAPKKK gene families in tomato have never been systematically analyzed. In this study, we performed a genome-wide analysis of the MAPKK and MAPKKK gene families in tomato and identified 5 MAPKK genes and 89 MAPKKK genes. Phylogenetic analyses of the MAPKK and MAPKKK gene families showed that all the MAPKK genes formed four groups (groups A, B, C, and D), whereas all the MAPKKK genes were classified into three subfamilies, namely, MEKK, RAF, and ZIK. Evolutionary analysis showed that whole genome or chromosomal segment duplications were the main factors responsible for the expansion of the MAPKK and MAPKKK gene families in tomato. Quantitative real-time RT-PCR analysis showed that the majority of MAPKK and MAPKKK genes were expressed in all tested organs with considerable differences in transcript levels indicating that they might be constitutively expressed. However, the expression level of most of these genes changed significantly under heat, cold, drought, salt, and Pseudomonas syringae treatment. Furthermore, their expression levels exhibited significant changes in response to salicylic acid and indole-3-acetic acid treatment, implying that these genes might have important roles in the plant hormone network. Our comparative analysis of the MAPKK and MAPKKK families would improve our understanding of the evolution and functional characterization of MAPK cascades in tomato.

Auxin response factors (ARFs) encode transcriptional factors that function in the regulation of plant development processes. A tomato ARF gene, SlARF5, was observed to be expressed at high levels in emasculated ovaries but maintained low expression levels in pollinated ovaries. The amiRNA SlARF5 lines exhibited ovary growth and formed seedless fruits following emasculation. These parthenocarpic fruits developed fewer locular tissues, and the fruit size and weight were decreased in transgenic lines compared to those of wild-type fruits. Gene expression analysis demonstrated that several genes involved in the auxin-signaling pathway were downregulated, whereas some genes involved in the gibberellin-signaling pathway were enhanced by the decreased SlARF5 mRNA levels in transgenic plants, indicating that SlARF5 may play an important role in regulating both the auxin- and gibberellin-signaling pathways during fruit set and development.

SMALL AUXIN UP-REGULATED RNAs (SAURs) are recognized as auxin-responsive genes involved in the regulation of abiotic stress adaptive growth. Among the growth-limiting factors, water-deficit condition significantly affects plant growth and development. The putative function of SAUR family member AtSAUR32 has the potential to diminish the negative impact of drought stress, but the exact function and mode of action remain unclear in Arabidopsis. In the current study, AtSAUR32 gene was cloned and functionally analyzed. AtSAUR32 localized to the plasma membrane and nucleus was dominantly expressed in roots and highly induced by abscisic acid and drought treatment at certain time points. The stomatal closure and seed germination of saur32 were less sensitive to ABA relative to AtSAUR32-overexpressed line (OE32-5) and wild type (WT). Moreover, the saur32 mutant under drought stress showed increased ion leakage while quantum yield of photosystem II (ΦPSII) and endogenous ABA accumulation were reduced, along with the expression pattern of ABA/stress-responsive genes compared with WT and the OE32-5 transgenic line. Additionally, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that AtSAUR32 interacted with clade-A PP2C proteins (AtHAI1 and AtAIP1) to regulate ABA sensitivity in Arabidopsis. Taken together, these results indicate that AtSAUR32 plays an important role in drought stress adaptation via mediating ABA signal transduction.