Identification and expression analysis of cytokinin metabolic genes IPTs, CYP735A and CKXs in the biofuel plant Jatropha curcas

Introduction

Jatropha curcas is a multipurpose tree that belongs to the Euphorbiaceae family. It can endure drought and adapt to barren land in tropical and subtropical regions. Jatropha is considered a promising biofuel plant due to the high oil content in its seeds (Akashi, 2012; Francis, Edinger & Becker, 2005; Makkar & Becker, 2009). However, the seed yield is very low, potentially because of the relatively low number of total flowers and/or the ratio of female to male flowers in Jatropha (Kumar & Sharma, 2008; Kumar Tiwari, Kumar & Raheman, 2007; Rao et al., 2008). Recently, several studies have reported that exogenous cytokinin (CK) treatment can significantly increase the total number of flowers per inflorescence, the female-to-male flower ratio, and the seed yield (Fröschle, Horn & Spring, 2017; Pan & Xu, 2011).

Cytokinins are important hormones in plants and participate in many biological processes, such as apical dominance (Shimizu-Sato, Tanaka & Mori, 2009; Tanaka et al., 2006), root proliferation (Kudo, Kiba & Sakakibara, 2010; Werner et al., 2003), reproductive development (Ashikari et al., 2005), and senescence (Gan & Amasino, 1995). Endogenous CKs containing N⁶-(Δ²-isopentenyl)-adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), dihydrozeatin (DZ), and their conjugates are known as isoprenoid CKs (Mok & Mok, 2001). The major derivatives are generally iP- and tZ-type CKs (Sakakibara, 2006).

Cytokinin biosynthesis and degradation pathways have been well studied in the past decade (Fig. 1) (Galuszka et al., 2007; Kudo, Kiba & Sakakibara, 2010; Sakakibara, 2006). The first step of iP and tZ biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferases (IPTs). IPTs produce iP-ribotides from dimethylallyl diphosphate (DMAPP) and adenosine 5′-diphosphate (ADP) or adenosine 5′-triphosphate (ATP) (Ihara et al., 1984; Taya, Tanaka & Nishimura, 1978). iP-ribotides can then be hydroxylated to tZ-ribotides by cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A) (Takei, Yamaya & Sakakibara, 2004). These cytokinin ribotides are converted to free-base CKs by cytokinin-activating enzymes LONELY GUYs (LOGs) (Kurakawa et al., 2007; Kuroha et al., 2009; Tokunaga et al., 2012). In addition, cZ and tZ can be enzymatically interconverted by zeatin cis–trans isomerase (Bassil, Mok & Mok, 1993; Sakakibara, 2006). In Arabidopsis, IPT1 and IPT3–IPT8 are involved in iP and tZ biosynthesis (Kakimoto, 2001; Sun et al., 2003; Takei, Sakakibara & Sugiyama, 2001), while IPT2 and IPT9 are involved in cZ biosynthesis (Golovko et al., 2002). CYP735A1 is abundant in roots and flowers in Arabidopsis, while CYP735A2 specifically accumulates in roots (Takei, Yamaya & Sakakibara, 2004). CYP735As are required for shoot growth (Kiba et al., 2013). Cytokinin oxidase/dehydrogenase (CKX) catalyzes the irreversible degradation of CKs (Galuszka et al., 2001, 2007; Schmulling et al., 2003). CKXs play important roles in controlling CK levels in plant tissues. In Arabidopsis, CKX3 and CKX5 regulate the activity of reproductive meristems (Bartrina et al., 2011). In rice, OsCKX4 mediates crown root development by integrating cytokinin and auxin signaling (Gao et al., 2014).

Cytokinins play important roles in flower bud development and floral sex differentiation (Chandler, 2011; Gerashchenkov & Rozhnova, 2013; Yamasaki, Fujii & Takahashi, 2005). However, the roles of CK biosynthesis genes IPTs and CYP735A and catabolism gene CKXs in Jatropha are not clear. In this study, we isolated sequences of cytokinin metabolic genes, including six IPTs, one JcCYP735A, and seven JcCKXs, using the Jatropha Genome Database (Hirakawa et al., 2012; Sato et al., 2010; Wu et al., 2015). The 14 genes showed different expression patterns in different tissues of Jatropha. Some of them exhibited tissue-specific expression. JcIPT6 was only expressed in seeds a few days after pollination. JcCYP735A was highly expressed in roots and seeds. JcCKX4 was expressed mainly in seeds. In addition, CK types and contents were detected in flower buds and seeds. With flower bud development iP-type CKs increased, while tZ-type CKs decreased. With seed development, tZ-type CKs increased, while iP-type CKs decreased. The Jccyp735a mutants were obtained by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. Compared with the wild-type (WT) plants, the concentrations of tZ and tZ-riboside (tZR) decreased significantly in the Jccyp735a mutants, which showed severely retarded growth. These results will be helpful for future studies of the functions of these genes and for improving the biological characteristics of Jatropha.

Materials and Methods

Results

Cloning and identification of JcIPTs, JcCYP735A, and JcCKXs

Using all annotated Arabidopsis IPT, CYP735A, and CKX family members in the TAIR as query sequences to perform a BLAST analysis in GenBank and with our Jatropha transcriptome data (Chen et al., 2014; Pan et al., 2014), we identified and cloned IPT, CYP735A, and CKX orthologous sequences in Jatropha. The Jatropha IPT family included only six members, while there are nine members in Arabidopsis. These genes were named JcIPT1, JcIPT2, JcIPT3, JcIPT5, JcIPT6, and JcIPT9. The BLAST analysis identified only one member of the CYP735A family, JcCYP735A. The CKX gene family, encoding degradation enzymes, included the same seven members in Jatropha as in Arabidopsis. These genes were named JcCKX1, JcCKX2, JcCKX3, JcCKX4, JcCKX5, JcCKX6, and JcCKX7.

Sequence structure analysis showed that IPT, CYP735A, and CKX family members shared almost the same numbers of exons and introns between Jatropha and Arabidopsis and had similar exon lengths (Figs. 2 and 3). JcIPT6 has two more exons than IPT6 from Arabidopsis. However, the extra two exon sequences are short and are not part of the P-loop NTPase domain (Fig. 2A).

Phylogenetic analysis of JcIPTs, JcCYP735A, and JcCKXs

To analyse the phylogenetic relationships between orthologous genes, phylogenetic analysis were performed. IPT, CYP735A, and CKX family members from Arabidopsis thaliana, Ricinus communis, and Oryza sativa were compared with those from Jatropha. Orthologues of IPT1, 2, 3, 5, and 7 formed a clade, while IPT9 formed a single clade (Fig. 4A). JcCYP735A along with other dicotyledon CYP735As formed a clade, while CYP735A3 and 4 of O. sativa formed another clade (Fig. 4B). Orthologues of CKX1, 5, 6, and 7 formed a clade, while those of CKX2, 3, and 4 formed a separate clade (Fig. 5). These results showed that JcIPTs, JcCYP735A, and JcCKXs were most closely related to genes from R. communis, which also belongs to the Euphorbiaceae family.

Expression patterns of JcIPTs, JcCYP735A, and JcCKXs in different tissues

In order to gain more information of these gene family members in Jatropha, the temporal and spatial expression patterns of these genes were analyzed using qRT-PCR. JcIPT1 was mainly expressed in roots (Fig. 6A). JcIPT2 was mainly expressed in roots, shoot apical meristems, mature leaves, and flower buds (Fig. 6B). JcIPT3 showed much higher expression levels in stems and mature leaves than other tissues (Fig. 6C). JcIPT5 exhibited high expression levels in roots and mature leaves (Fig. 6D). JcIPT9 only showed high expression levels in mature leaves (Fig. 6F). The expression of JcIPT6 was not detected in most of the plant tissues indicated above. After analysing more tissues (Fig. S1), we found that JcIPT6 began to be expressed in seeds a few days after fertilization, with the strongest expression observed in seeds at 10 days after fertilization; the expression levels then decreased rapidly. In seeds at 20 days after fertilization, JcIPT6 expression decreased by a factor of 5 compared with seeds at 10 days after fertilization (Fig. 6E). The expression levels of JcCYP735A were higher in roots, flower buds, and seeds than other tissues (Fig. 7A). JcCKX1 was mainly expressed in flower buds, roots, and female flowers (Fig. 7B). JcCKX2 showed very strong expression in female flowers and seeds (Fig. 7C). JcCKX3 was highly expressed in male flowers (Fig. 7D). JcCKX4 exhibited high expression levels in mature leaves and female flowers, and extremely high expression in seeds (Fig. 7E). JcCKX5 was mainly expressed in stems, young leaves, and fruit (Fig. 7F). JcCKX6 was expressed in all tissues (Fig. 7G). JcCKX7 was mainly expressed in roots (Fig. 7H).

Jccyp735a mutants generated by CRISPR-Cas9 system showed retarded growth

To explore the biological function of JcCYP735A in Jatropha, we generated Jatropha transformants with JcCYP735A knocked out using the CRISPR-Cas9 system (Fig. 8). Three homozygous mutant lines, L1, L2, and L3, were obtained by DNA sequencing (Fig. 8A). Endogenous contents of CKs in the leaves of two lines of Jccyp735a mutants (L2 and L3) and WT plants were examined. The results showed that the concentrations of tZ and tZR, and cZ and cZR significantly decreased, whereas the concentrations of iP and iPR significantly increased in Jccyp735a mutants compared with those of the WT (Fig. 8B). Jccyp735a mutants showed severely retarded growth, and the mutant plants were only approximately a quarter the height of the WT plants (Figs. 8C and 8D).

Discussion

In our study, six IPT family members were identified in Jatropha. The number of IPT genes differs among plant species; for example, there are six IPTs in R. communis, while there are nine IPTs in both Arabidopsis (Kakimoto, 2001; Takei, Sakakibara & Sugiyama, 2001) and rice (Fig. 4A). JcIPTs have the same number of exons as those in Arabidopsis, except JcIPT6, which has two more exons than AtIPT6. The third exon of JcIPT6 has almost the same number of base pairs as the only exon in AtIPT6 (Fig. 2A). It appears that the other two small exons were lost during evolution. Expression pattern analysis revealed that different JcIPT members were expressed in different tissues in Jatropha (Fig. 6). JcIPT1 had an expression pattern similar to that of AtIPT1 and was mostly expressed in roots, shoot apical meristems, and seeds (Miyawaki, Matsumoto-Kitano & Kakimoto, 2004). JcIPT2 and JcIPT9 were assigned to the same cluster as their orthologues (Fig. 4A). In Arabidopsis, AtIPT2 and AtIPT9 are expressed ubiquitously, with stronger expression in proliferating tissues, including the root and shoot apical meristems and leaf primordia (Miyawaki, Matsumoto-Kitano & Kakimoto, 2004). Similarly, JcIPT2 and JcIPT9 were expressed ubiquitously in Jatropha. However, the strongest expression of both was in mature leaves instead of meristems and young leaves (Figs. 6B and 6F). Considering that AtIPT2 and AtIPT9 are tRNA-IPTs, which only catalyze cZ biosynthesis (Golovko et al., 2002), cZ might be produced mainly in mature leaves of Jatropha. AtIPT3 was found to be responsible for nitrate-dependent cytokinin biosynthesis and is predominantly expressed in the phloem (Miyawaki, Matsumoto-Kitano & Kakimoto, 2004; Takei et al., 2002). In our study, JcIPT3 was strongly expressed in stems and mature leaves, which contain abundant phloem (Fig. 6C). A study by Miyawaki, Matsumoto-Kitano & Kakimoto (2004) showed that GUS activity was not detected in Arabidopsis transformants carrying AtIPT6::GUS. RT-PCR analysis indicated that it was abundant in siliques. Analogously, we did not detect JcIPT6 in most tissues of Jatropha, including fruits. We found that JcIPT6 was mainly expressed in seeds at 10 days after pollination, and its expression decreased rapidly thereafter (Fig. 6E). It is possible that the previous study missed this critical phase in the seeds chosen for GUS staining (Miyawaki, Matsumoto-Kitano & Kakimoto, 2004). Our results suggested that different JcIPT family members play different roles in the development of Jatropha and that some JcIPT members could be used to cultivate high-yield varieties of Jatropha using transgenic technology.

Although none of the ATP/ADP JcIPTs were found to be highly expressed in flowers, iP, iPR, and tZ contents increased with the development of flower buds (Table 1). It is known that CKs can be transported through the plant vascular system (Hirose et al., 2007). JcCYP735A was found to be highly expressed in flower buds (Fig. 7A). iP-type CKs may be transported into flowers, and some of them may then be used to generate tZ-type CKs via JcCYP735A. In addition, it has been reported that the tZ concentration is up-regulated in the early development of tomato fruits (Matsuo et al., 2012). Our study showed that JcCYP735A was highly expressed and that the tZ concentration increased with seed development in Jatropha (Table 1). Thus, JcCYP735A might play important roles in seed development by controlling tZ biosynthesis. Similar to R. communis (Chan et al., 2010), only one CYP735A gene was found in Jatropha, although there are two CYP735A members in Arabidopsis and rice (Takei, Yamaya & Sakakibara, 2004; Tsai et al., 2012).

Unlike JcIPT or JcCYP735A family, the JcCKX family in Jatropha has the same number of members as that in Arabidopsis (Fig. 3). In addition, the JcCKX and AtCKX orthologues contain the same number of exons, with five exons in CKX1-6 and 4 exons in CKX7 (Fig. 3). Further expression analysis showed that JcCKX2 was mostly expressed in female flowers, whereas JcCKX3 was mostly expressed in male flowers (Figs. 7C and 7D). Both JcCKX2 and JcCKX4 were expressed strongly in seeds (Figs. 7C and 7E). These tissue-specific expression genes may be chosen to adjust the CK content in these tissues using transgenic methods. It has been reported that reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield (Ashikari et al., 2005). Decreased expression of CKX orthologues may also lead to increased yield in Jatropha. Moreover, overexpression of some CKX members can also improve resistance. Overexpression of CKX1 or CKX2 in Arabidopsis and other species causes elongation of the primary root and increases root branching (Galuszka et al., 2004; Mrízová et al., 2013; Pospisilova et al., 2016; Werner et al., 2001; Yang et al., 2003), while overexpression of AtCKX7 results in an opposite phenotype (Kollmer et al., 2014). Specific expression of JcCKX1 or JcCKX2 in roots might be used to transform a shallow root system into a deep root system to improve the growth and lodging resistance of Jatropha. Furthermore, root system development might enhance tolerance to drought stress. Remarkably, JcCKX4 expression was much higher in seeds than other tissues (Fig. 7E), suggesting that JcCKX4 may be a key enzyme regulating cytokinin levels to affect seed development.

In early flower bud development, the content of iP-type CKs increased significantly while that of tZ-type CKs decreased. This result indicated that iP-type CKs participate more in early flower bud development than tZ-type CKs. In tomato, iP-type CK contents decrease during fruit ripening (Matsuo et al., 2012). By contrast, in early seed development, the content of tZ-type CKs increased substantially, while that of iP-type CKs decreased. This result, which is in accordance with high expression of JcCYP735A in seeds, suggested that tZ-type CKs are dominant in early seed development. Many differences were observed in CK contents in different periods of Jatropha flower bud and seed development. Our results indicate that iP-type CKs can be used to improve the number of flowers in Jatropha, while tZ-type CKs can be used to enlarge seeds.

The single cyp735a1 or cyp735a2 mutant Arabidopsis showed slight decreases in tZ and tZR concentration compared with that of WT, while the double mutants showed great decreases (Kiba et al., 2013). In addition, cyp735a1 cyp735a2 double mutants exhibited retarded shoot growth (Kiba et al., 2013). Similarly, in this study, Jccyp735a mutants showed substantial decreases in tZ and tZR concentrations (Fig. 8B), which is consistent with that only a single member of JcCYP735A was found in Jatropha genome (Wu et al., 2015). We noticed, however, tZ and tZR did not completely disappear in Jccyp735a mutants (Fig. 8B), which may result from conversion of cZ and cZR. This notion is supported by the fact that the concentrations of cZ and cZR also significantly decreased in Jccyp735a mutants (Fig. 8B), and that cis–trans isomerase activity for interconversion between cZ-type and tZ-type CKs has been reported in several plant species (Bassil, Mok & Mok, 1993; Kudo et al., 2012; Suttle & Banowetz, 2000).

Conclusion

In this study, we isolated the members of the JcIPT, JcCYP735A, and JcCKX gene families and analyzed their temporal and spatial expression patterns. Different family members exhibited different expression patterns. Different types of CKs seemed to influence the development of flower buds and seeds, respectively. The analysis of the Jccyp735a mutants revealed that JcCYP735A plays an important role in tZ biosynthesis in Jatropha. These results will be helpful for further function studies of cytokinin metabolic genes and improving agronomic characteristics of Jatropha by genetic engineering of cytokinin metabolism.

Supplemental Information