Targeted Editing of SlMAPK6 Using CRISPR/Cas9 Technology to Promote the Development of Axillary Buds in Tomato Plants

The mitogen-activated protein kinase (MAPK) cascade signaling system has been relatively conserved throughout the evolution of eukaryotes and is involved in the regulation of growth and development and metabolism. In this study, dwarf tomato plants were used as the research material. First, the tissue-specific expression of SlMAPK6 was measured in wild-type plants by quantitative RT-PCR. The results showed that SlMAPK6 was highly expressed in the tissues of the stems, leaves and flowers but was expressed at low levels in the tissues of the roots, sepals and fruits. Second, SlMAPK6-knockout lines CRISPR-3 and CRISPR-7 were obtained by CRISPR-Cas9 technology and Agrobacterium-mediated transformation. Compared with wild-type, the mutant lines CRISPR-3 and CRISPR-7 showed significant phenotypic characteristics, such as increased numbers of axillary buds and true leaves, thickened stems, and longer leaflets. In addition, to explore the molecular mechanism by which MAPK regulates axillary bud growth, we also showed that SlMAPK6 positively regulates the strigolactone synthesis genes SlCCD7 and SlCCD8 and the gibberellin (GA) synthesis genes GA20ox3 and GA3ox1 and negatively regulates the axillary bud development-related genes Ls, BL and BRC1b/TCP8 and the GA synthesis inhibitory gene GAI. Therefore, SlMAPK6 appears to regulate the synthesis of strigolactone and GA to induce the growth and development of tomato axillary buds.


Introduction
Plant morphogenesis has great influence on yield and quality, the number of side branches is very important index to plant architecture. The lateral branches develop from the axillary buds. Many factors, especially hormones, determine the development of axillary buds (Huh et al., 2013;Chen et al., 2016). Strigolactones and gibberellin (GA), two kinds of plant hormones, are reported to regulate shoot branching, stems elongation, plant growth and architecture (Yamaguchi et al., 2008;Vogel et al., 2010;Kohlen et al., 2012). In tomato, CAROTENOID CLEAVAGE DIOXYGENASE8 (SlCCD8) and CAROTENOID CLEAVAGE DIOXYGENASE7 (SlCCD7) are two key strigolactone biosynthesis-related genes. GA20oxs are essential enzymes for GA biosynthesis and control the GA content in different plant species. GA3oxs produce bioactive GAs, such as GA1, GA3, GA4 and GA7, in the final step (Yamaguchi et al., 2008).
The crosstalk between mitogen-activated protein kinases (MAPKs), and hormones, secondary messengers in plant signaling systems facilitates plant to adapt and survive in different environmental, including controlling plant architecture (Smekalova et al. 2014). MAPK cascade signaling system has been relatively conserved throughout the evolution of eukaryotes (Widmann et al., 1999;Group et al., 2002;Xu et al., 2015) and is involved in the regulation of the responses to various biotic and abiotic stresses, growth and development, and metabolism. MAPKs play an important role in the signaling pathway of eukaryotes. The MAPK signaling system is usually composed of tertiary protein kinases, including MAPK, MAPK kinase (MAPKK) and MAPKK kinase. MAPKs acts downstream of the tertiary protein kinase signaling system.
A total of 16 MAPKs have been identified in tomato, which can be further divided into four categories: A, B, C, and D (Kong et al., 2012). Among them, MAPK1, MAPK2 and MAPK3 belong to category A; these MAPKs have been the subject of many studies and are involved in the regulation of various stresses in plants (Stulemeijer et al., 2007;Zhou et al., 2014;Lv et al., 2017). The remaining 13 MAPKs belong to categories B, C, and D, which have been poorly studied. Among them, tomato MAPK6 (SlMAPK6) belongs to category B and has a loop containing a MEY motif. However, the function of SlMAPK6 is still unknown.
Genome-editing technology has been used in Arabidopsis, tomato, rice, apple, watermelon, potato, soybean, maize, wheat, sorghum and grape (Feng et al., 2014;Ito et al., 2015;Wang et al., 2016;Reem et al., 2019;Char et al., 2019a;Nishitani et al., 2016;Zhou et al., 2014;Tian et al., 2017;Char et al., 2019b;Di et al., 2019;Lee et al., 2019;Liang et al., 2019;Ren et al., 2019). This study aimed to investigate the function of the SlMAPK6 gene in the development of axillary buds in tomato plants; the CRISPR/Cas9 system was applied to generate SlMAPK6 gene knockout mutants. We investigated the development of axillary buds on CRISPR/Cas9-mediated slmapk3 mutants and wild-type (WT) plants as well as the possible by SlMAPK6-mediated regulatory mechanism that influences the development of axillary buds. This study provides insight into the regulatory mechanism of SlMAPK6-mediated axillary bud development in tomato plants.

Plant Materials and Growth Conditions
Dwarf tomato plants (Solanum lycopersicum) were used in this study as WT. The plants were grown in a pot (18 cm  22 cm) with matrix (Pere matrix Technology Development Co., LTD, Jiangsu of China), and all plants were growth in artificial climate chamber and watered every three days with Hongland nutrient solution. Transgenic cultures were grown under controlled conditions with a 16-h-day/8-h-night photoperiod, a temperature of 25 °C/18 °C (day/night), a relative humidity of 65-70%, and a light intensity of 250 µmol m -2 s -1 . The first generation (T0) of transgenic tomato originated from tissue culture, and the homozygous SlMAPK6 knockout lines were screened from the seedlings of the second generation (T1). The third generation (T2) of tomato homozygous lines were used in subsequent experiments.

Measurement of Plant Architecture Parameters
To study differences between the WT and SlMAPK6-knockout lines, we observed the plant morphological parameters in flower stage, including the stem diameter, number of true leaves, internode length, number of axillary buds and compound leaves from SlMAPK6-knockout lines and WT plants.

Construction of the SlMAPK6 Knockout Vector
Three target sequences of the SlMAPK6 gene from the CRISPR-GE web tool (http://skl.scau.edu.cn/) were used. The target sequences were introduced into three single-guide RNA (sgRNA) expression cassettes using overlapping PCR. These three sgRNA expression cassettes were then ligated into a pCAMBIA2301 vector. The SlMAPK6 knockout vector was constructed by the company of RexBiotech.
The diagram of the SlMAPK6 knockout vector was shown in Supplemental Figure S1.

Plant Transformation
Via the freeze-thaw method (Jyothishwaran et al., 2007), the confirmed pCAMBIA2301/2x35S:hSpCas9-Tnos/ AtU6-26::SlMAPK6 binary vector was transferred into Agrobacterium tumefaciens strain GV3101. Transgenic plants were subsequently obtained through the cotyledon transformation method of Agrobacterium mediation. Transgenic lines were screened on the basis of kanamycin resistance. After the regenerated buds rooted, the regenerated shoots were grown for one month and then planted in sterilized soil at 25±2 C with a 16-h/8-h (light/dark) photoperiod. jas.ccsenet.  Figure 4B) T ( Figure 3G).

Figure 5
Note. catabolism of GA. Our results are in accordance with those of a previous study of Martín-Trillo et al. (2010), where it was shown that the expression level of PRE1 was regulated by GA. Moreover, the expression levels of PRE1, PRE2 and PRE3 were significantly reduced in SlMAPK6-knockout lines (Martín-Trillo et al., 2010). However, our results suggest that SlMAPK6 positively regulates GA-related genes. Our results are in agreement with the development of tomato axillary buds being regulated by members of several transcription factor families, such as MYBs (e.g., the BL gene) (Schmitz et al., 2002), TCPs (e.g., the BRC1b gene) (Cubas et al., 1999;Martín-Trillo et al., 2010) and GRASs (e.g., the Ls gene) (Schumacher et al., 1999).
During the vegetative growth of the plants whose SlMAPK6 gene was knocked out, there were a greater number of compound leaves and more vigorous plants. These results showed an increase in the vegetative growth of the SlMAPK6-knockout lines. These results are in agreement with those of a previous study in which SlCCD7 and SlCCD8 were reported to inhibit the growth of branches of tomato plants through the methods of expressing antisense constructs or RNAi-mediated silencing (Kohlen et al., 2012;Vogel et al., 2010). The expression levels of the SlCCD7 and SlCCD8 genes were also examined in the SlMAPK6-knockout lines. It was observed that the expression levels of both genes were downregulated in SlMAPK6-knockout plants, indicating that with the abundant production of axillary buds, strigolactone biosynthesis might be decreased.
In this study, the increase in the number of axillary buds was examined due to the loss of function of SlMAPK6 in the knockout lines. The level of expression was observed in lateral buds for related developmental genes, such as Ls, BL and BRC1b, in both WT and knockout plants. The highly expressed BL genes in the SlMAPK6 knockout lines indicated that SlMAPK6 negatively regulates BL genes. Moreover, the BRC1b gene were highly upregulated in the SlMAPK6 knockout lines, which contradicts the increased axillary bud phenotype of the knockout plants. Shoot branching of angiosperms determines the aspects of plant morphological structure, such as visibility to pollinators, plant height and nutrient allocation. Thus, the vigorous vegetative growth of the SlMAPK6 knockout plant probably accounts for the low fruit set percentage of the transgenic lines.
These results were also in agreement with those of a previous study in which RNA interference was applied to the BL control of the development of lateral axes. After interference with BL in tomato, the number of lateral axes decreased because the initiation of lateral meristems was stopped (Schmitz et al., 2002). SlBRC1b was found to suppress the growth of shoot branches because increased branch outgrowth was shown in SlBRC1b-loss-of-function tomato plants (Martín-Trillo et al., 2011).

Conclusions
The function of SlMAPK6 was studied through CRISPR-Cas9 technology. First, it was observed that SlMAPK6 was ubiquitously expressed in the different tissues we examined, and SlMAPK6 expression levels were higher in the tissues of the leaves, stems and flowers than in the tissues of the roots, fruits and sepals. Second, the SlMAPK6-knockout lines CRISPR-3 and CRISPR-7 were obtained using CRISPR-Cas9 technology. It was found that, as a negative regulator, SlMAPK6 plays a key role in regulating the development of axillary buds and architecture of tomato plants. In addition, a potential molecular mechanism for increasing the number of axillary buds was studied in SlMAPK6-knockout plants. It was possible that SlMAPK6 negatively regulate strigolactone biosynthesis via SlCCD7 and SlCCD8 and positively regulated GA biosynthesis via PRE1, PRE2, PRE3, GA20ox3, GA3ox1 and GAI, which led to the phenotype of increased numbers of axillary buds.  Target Na   T1  T1  T1   T2  T2  T2   T3   T3  T3  T3 Note. The are marked

Copyright
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