A stable and high efficient transform system was construction for B. juncea
B. juncea is a unique vegetable in China, normally planted along Yangtze River with latitude below 800. To accelerate breeding process and break breeding limitation, in vitro culture techniques were used for genetic research in B. juncea. Previous studies by Zhang and Chen demonstrated that using shoots as explants resulted in a high induction proportion of callus, adventitious buds and roots (Chen et al. 2005; Zhang et al. 2012). Additionally, Chen found that the use of NAA and 2, 4-D was necessary for plants regeneration from cotyledon protoplasts (Chen et al. 2005). In this study, four different cultivars of B. juncea ("A168", "ZL", "4–16" and "yonganxiaoye") were used to induce callus from both cotyledon and epicotyl. The results showed that only "yonganxiaoye" cotyledon had a high callus induction ratio of 83% when using a 6-BA concentration of 2.0 mg/L and NAA concentration of 0.1 mg/L (Table 1). The induced callus appeared soft green and compact texture, which facilitated the induction of adventitious buds and further growth into complete B. juncea plants (Table S1, Fig. 1). Furthermore, it was found that cotyledon with petiole as explants resulted in easier induction of adventitious buds, with a success rate of 95.6% (Shen et al. 2012; Zhang et al. 2012). In our study, callus was induced from petiole showed a green and compact appearance, which made it easier to induce adventitious buds (Table 1, Fig. 1). Based on the developed regeneration system, we further optimized the concentration and time of agrobacterium infection and successfully constructed an efficient transformation system for B. juncea "Yonganxiaoye", with a transgenic rate of 10.3% (Fig. 2). These findings provide valuable insights into the in vitro culture techniques and transform system for B. juncea, which can contribute to future studies on the function study in B. juncea.
Total Chl content was decreased in BjuLKP2 OE line and leading to plant yellowing
LKP2 as plant specific blue light photoreceptor, was reported functions in circadian clock and delay flowering time (Baudry et al. 2010; Takase et al. 2011). To identify BjuLKP2 function in B. juncea development, agrobacterium-mediated BjuLKP2 OE lines were obtained based on constructed agrobacterium-mediated genetic transform system (Fig. S1, Fig. 3). Two lines, BjuLKP2 OE20 and OE26 were obtained, which showed more than 4-fold and 14-fold up-regulation of BjuLKP2 expression, respectively (Fig. S1, Fig. 3d). Phenotypic observations revealed that both BjuLKP2 OE20 and OE26 displayed yellowing cotyledons, shoot tips and leaf (Fig. 3a, 3b). This suggests that overexpression of BjuLKP2 have an impact on Chl synthesis. Previous study found that leaves color becoming yellow directly related to Chl content (Xiao et al. 2013). In our study, Chl a, Chl b, carotenoid and total Chl content were all reduced in both BjuLKP2 OE20 and OE26, which consistence with yellowing leaves (Fig. 3e).
Chl is the primary photoreceptor responsible for capturing light energy in plants. The synthesis of Chl is a complex process that involves the participation of more than 27 genes and 17 enzymes (Zhao et al. 2020). Among these genes, HEMA1, HEMA3 and HEML2 are involved in the first stage of Chl synthesis. HEMA1 has been shown to positively regulate Chl biosynthesis, while HEMA3 is expressed at a low level (Fang et al., 2016; Tsang et al. 2003; Matsumoto et al. 2004; Tan et al. 2008). Overexpression of HEMA1 has been found to increase the content of protochlorophyllide, a precursor of Chl, in yellowing plants (Schmied et al. 2011). HEML2 functions as a positive regulator of Chl synthesis, reducing its expression through antisense technology results in a decrease in Chl levels in plants (Mock and Grimm, 1997; Mohanty et al. 2006). In this study, the expression of BjuHEMA1 and BjuHEML2 were up-regulated in BjuLKP2 OE line (Fig. 4a). Surprisingly, despite the up-regulation of these genes, the total Chl content was reduced in BjuLKP2 OE26 line (Fig. 3e, Fig. 4a). This suggests that BjuLKP2 may have additional functions beyond positively regulating the first stage of Chl synthesis genes. Further investigations are required to elucidate the specific role of BjuLKP2 in Chl synthesis and understand how its overexpression leads to a reduction in total Chl content. It is possible that BjuLKP2 may influence other stages or regulatory steps in the Chl synthesis pathway, or it may interact with other factors that affect Chl levels in plants.
In the second stage of Chl biosynthesis, ten genes (HEMB1, HEMB2, HEMC, HEMD, HEME1, HEME2, HEMF1, HEMF2, HEMG1 and HEMG2) are involved. The expression levels of HEMB1 and HEMB2 are highly dependent on leaf developmental age. However, in the BjuLKP2 OE line, the expression levels of BjuHEMB1 and BjuHEMB2 were almost unchanged compared to the wild type (Fig. 4b). This suggests that BjuLKP2 is not involved in the regulation of HEMB1 and HEMB2 expression in Chl synthesis process. HEMC encodes PBGD, a chloroplastic soluble protein whose expression level depends on leaf developmental stage and light (He et al. 1994; Mock and Grimm, 1997; Smith, 1998; Ayliffe et al. 2009). In the BjuLKP2 OE line, the expression of BjuHEMC was significantly up-regulated, indicating that BjuLKP2 positively regulates BjuHEMC expression (Fig. 4b). HEMD encodes UROS, which catalyzes the conversion of hydroxymethylbilane to Urogen III. HEMD functions in plant phototoxicity defense but does not regulate total Chl content (Mock and Grimm, 1997; Ishikawa et al. 2001). In this study, the expression level of BjuHEMD was almost the same in both wild type and BjuLKP2 OE line and neither displayed any phototoxicity phenotype (Fig. 3e, Fig. 4b). This suggests that BjuLKP2 is not involved in the regulation of HEMD expression. HEMEs encode UROD, which is responsible for the decarboxylation of Urogen III to Coprogen III (Shalygo et al. 1998; Ishikawa et al. 2001). qRT-PCR assays showed that both BjuHEME1 and BjuHEME2 were up-regulated in BjuLKP2 OE line (Fig. 4b), indicating that BjuLKP2 positively regulates the expression of BjuHEMEs. HEMFs are involved in leaf development, and mutants of HEMFs exhibit developmentally regulated and light-dependent lesions on leaves (Narita et al. 1996). In BjuLKP2 OE line, BjuHEMF1 was up-regulated while BjuHEMF2 was down-regulated. However, no lesions were observed on the leaves of BjuLKP2 OE line (Fig. 3b, Fig. 4b). This suggests that the up-regulation of BjuHEMF1 may offset the lesions caused by the down-regulation of BjuHEMF2 in BjuLKP2 OE line. HEMGs are highly expressed in leaves (Rissler et al. 2002). In this study, only BjuHEMG2 was significantly up-regulated, indicating that BjuLKP2 positively regulates its expression (Fig. 4b).
In the third stage of Chl biosynthesis, twelve genes (CHLH, CHLI1, CHLI2, CHLD, CHLM, CRD1, DVR, PORA, PORB, PORC, CHLG and CAO) are involved. Mgch is composed of three subunits (CHLI, CHLD and CHLH) encoded by CHLH, CHLI1, CHLI2 and CHLD (Kruse et al. 1997; Hiriart et al. 2002; Matsumoto et al. 2004; Pontier et al. 2007). In Arabidopsis, mutations in the CHLH subunit result in albino formation, while mutations in the CHLI subunit lead to a light yellow-green phenotype (Mochizuki et al. 2001). In the BjuLKP2 OE line, only BjuCHLI1 was down-regulated, while the other three Mgch subunits were up-regulated (Fig. 3e, Fig. 4c). This suggests that BjuCHLI1 is responsible for the yellowing leaf phenotype in BjuLKP2 OE line, while the other three subunits are not affected. CHLM functions in positive regulation of Chl protein complexes (Bang et al. 2008). In this study, BjuCHLM was significantly down-regulated in BjuLKP2 OE line, indicating that BjuLKP2 negatively regulates chlorophyll protein complexes (Fig. 4c). CRD1 is involved in Chl synthesis and mutations lead to an elevated Chl a/b ratio and a pale green phenotype (Frick et al. 2003; Hansson and Jensen, 2009). Although BjuDVR was significantly up-regulated in BjuLKP2 OE line, it also displayed yellowing leaf phenotype (Fig. 3). It means that the reduction of Chl a/b ratio is responsible for BjuLKP2 OE line yellowing phenotype not BjuDVR expression changes (Fig. 4c). PORA, PORB and PORC encode POR, and double mutants of porbporc result in a decrease in Chl a content (Nagata et al. 2005). In our study, BjuPORA, BjuPORB and BjuPORC were up-regulated in BjuLKP2 OE line, and Chl a content was reduced (Fig. 4c). This suggests that BjuPORB and BjuPORC function to increase Chl a content, while BjuPORA functions to decrease Chl a content. CHLG functions in the positive regulation of ALA synthesis and antisense CHLG gene expression reduces ALA synthesizing capacity (Tanaka et al. 2001). In our study, BjuCHLG was up-regulated in BjuLKP2 OE line, indicating that BjuLKP2 positively regulates CHLG expression (Fig. 5c). CAO functions to increase Chl b content, and qRT-PCR assays found that BjuCAO was up-regulated in BjuLKP2 OE line, resulting in an increased Chl b content (Fig. 3e, Fig. 4c). This suggests that BjuLKP2 is involved in the regulation of Chl b content through BjuCAO.
BjuLKP2 functions in oxidative damage
In plants, changes in the expression of Chl biosynthesis genes can lead to alterations in pigment proportions in chloroplasts, which can subsequently result in oxidative damage. Ultimately, this can manifest as leaf color mutations (Chen et al. 2005). To investigate whether the yellowing leaf phenotype in BjuLKP2 OE line is caused by oxidative damage, we assessed the activity of antioxidant enzymes (APX, CAT, POD and SOD) in both wild type and BjuLKP2 OE line (Fig. 5). The results showed that APX, CAT and SOD activities were decreased in BjuLKP2 OE line, while POD activity was up-regulated, indicating a decrease in the antioxidant capacity of BjuLKP2 OE line (Fig. 5a-d). Further analysis of photosynthetic characteristics revealed a reduction in the potential photosynthetic ability of BjuLKP2 OE line, which is consistent with the yellowing phenotype observed (Fig. 5e). Based on these findings, we speculate that overexpression of BjuLKP2 leads to changes in the expression of Chl biosynthesis genes, resulting in a decrease in antioxidant capacity and ultimately leading to leaf color mutations. In A. thaliana, AtLKP2 was found to be expressed in seedlings, sepals and young siliques (Schmied et al. 2011). In our study, GUS assays found that BjuLKP2 was also expressed in seedlings, sepals and young siliques, except in seeds (Fig. 6). It was reported that the chloroplast number and grana lamella reduction will affects Chl content and light energy conversion and utilization, which results in leaves yellowing (Xiao et al. 2013; Yang et al. 2014). In this study, granum and grana lamella decreased in both sponge and palisade tissues, which consistent with yellowing leaves in BjuLKP2 OE line (Fig. 7).