Isolation and Functional Analysis of CmMAX1 from Chrysanthemum

A BSTRACT . More axillary buds 1 ( MAX1 ), initially identiﬁed in arabidopsis ( Arabidopsis thaliana ), is a key regulatory gene in strigolactone synthesis. CmMAX1 , an ortholog of MAX1 was cloned from chrysanthemum ( Chrysanthemum morifolium cv. Jinba). It had an open reading frame of 1611 bp and encoded 536 amino acid of P450 protein, with a conserved heme-binding motif of PFG · GPR · C · G, as well as PERF and KExxR motifs. The predicted amino acid sequence of CmMAX1 was most closely related to the MAX1 ortholog identiﬁed in lotus ( Nelumbo nucifera ), NnMAX1, with 55.33% amino acid sequence similarity. Expression analysis revealed there was no signiﬁcant difference of CmMAX1 expression among various tissues. Phosphorus (P) deﬁciency signiﬁcantly improved the expression levels of CmMAX1 . Strigolactone, auxin, and cytokinin negatively regulated the expression of CmMAX1 . Overexpression of CmMAX1 reduced the branch numbers of arabidopsis max1-1 . These results suggest that CmMAX1 may be a candidate gene for reducing the shoot branching of chrysanthemum.

Cytochrome P450s (CYPs), one of the biggest gene superfamilies in plant genomes (Nelson and Reichhart, 2011), participate in all major plant biosynthetic pathways, including biosynthesis of pigments, defense-related compounds, ultraviolet protectants, fatty acids, and hormones (Guttikonda et al., 2010). CYP711 was designated as a new clan of CYPs, with MAX1 identified as the first member (Booker et al., 2005).
MAX1 orthologs were discovered in a wide number of species, although absent from the moss Physcomitrella patens (Proust et al., 2011), which led to the hypothesis that MAX1 was incorporated into the MAX pathway after the divergence of the moss and tracheophyte lineages (Hepworth, 2012). Furthermore, several orthologs of MAX1 were present in monocots, such as five in rice (Umehara et al., 2010) and three in maize [Zea mays (Yoneyama et al., 2018)], and are generally present as a single copy in eudicots. Nevertheless, orthologs of MAX2, MAX3, and MAX4 were present in all plants searched (Hepworth, 2012), which made MAX1 more confusing.
Chrysanthemum is one of the most important flowers in the world; however, the reduction of branching is required in the cultivation of cut chrysanthemum to ensure the quality of the single flower. Existing cultivars generally produce lots of lateral branches, which increases cultivation costs by one third as a result of the manual pinching and bud picking. Therefore, new chrysanthemum cultivars with less or no branches are needed.
Previous complementation experiments showed that MAX1 was present and active in some species (Hepworth, 2012). Do they have conserved function in chrysanthemum? With the aim of answering this question and use of the gene for regulating shoot branching, we isolated an ortholog of MAX1 from chrysanthemum cultivar Jinba, analyzed its expression in different tissues and treatments, and investigated the function of CmMAX1 by complementation experiments. This study lays a foundation for further research into shoot branching in chrysanthemum, and also provides gene resources for breeding new cultivars with fewer branches.

Materials and Methods
PLANT MATERIALS AND GROWTH CONDITIONS. Chrysanthemum cultivar Jinba, arabidopsis Columbia wild type (WT), max1-1, and transgenic plants were all grown in a tissue culture room at 23 ± 2°C, with a photoperiod of 16/8 h (light/dark) and a light intensity of 100 mmolÁm -2 Ás -1 .
ISOLATION OF THE CMMAX1 GENE. Total RNA was extracted from stems of chrysanthemum plants using a Trizol reagent kit (Invitrogen, Shanghai, China) and was digested with RNasefree DNase (Takara, Dalian, China). First-strand complementary (cDNA) was synthesized using Superscript III reverse transcriptase (Invitrogen). Two sets of polymerase chain reaction (PCR) primers were designed for the cloning of CmMAX1 based on the conserved sequence of arabidopsis MAX1, Medicago MtMAX1, Populus PtMAX1, and rice OsMAX1. After obtaining a partial fragment by PCR using forward primer (5#-ATTTTCAGRTTTCABTGRAG-3#) and reverse primer (5#-AAGTCCATCTTVAGTTGHGTTGT-3#), the 3# and 5# ends of CmMAX1 were amplified using the 3# RACE System for Rapid Amplification of cDNA Ends (Invitrogen) and the 5#-full RACE core set (Takara), respectively. The CmMAX1 full-length cDNA was isolated by combining the three parts of the fragments. The products amplified using Primestar HS DNA Polymerase (Takara) were cloned into the PEASY-Blunt Simple Cloning Vector (Trans, Beijing, China) to determine the sequence of CmMAX1. The protein molecular formula was determined using the Protparam analysis tool on an ExPASy server (Gasteiger et al., 2005). Alignment of multiple sequences was carried out using DNAMAN software (version 5.2.2; Lynnon Biosoft, San Ramon, CA). Phylogenetic analysis was conducted using the MEGA 5.05 program (Tamura et al., 2011) by the maximum likelihood method. QUANTITATIVE R E A L -TIME PCR (QRT-PCR) A N D SEMIQUANTITATIVE REVERSE TRANSCRIPTION ANALYSIS. For the experiment of CmMAX1 expression in different tissues, roots, stems, leaves, axil, and apex were collected from 30-d-old chrysanthemum seedlings. The P deficiency experiment was conducted by treating the roots of the chrysanthemum seedlings with P-free nutrient solution for 12 h, then the roots were harvested for the further experiments. For the hormone treatment experiments, 5 mL of 2 mM GR24, 6-benzylamino adenine (6-BA; 50 mM), or indole-3-acetic acid (IAA; 100 mM) solutions were applied to node 4 (the fourth bud from the top downward) of 30-d-old chrysanthemum seedlings. After 2 h, the buds treated with different hormones were collected. RNA extraction, qRT-PCR, semiquantitative reverse transcription,   and analysis were performed as described by Dong et al. (2013).
STATISTICAL ANALYSIS. All data were repeated at least three times. Tukey's test at P < 0.05 was used to evaluate the degree of rescue of the max1 phenotype by the CmMAX1 gene.

CLONING OF CMMAX1 AND SEQUENCE ANALYSIS OF CMMAX1.
CmMAX1 cDNA included a complete ORF of 1611 bp, which encoded proteins of 536 amino acids. The deduced amino acid sequence contained a heme-binding motif (PFG · GPR · C · G), PERF, and a K-helix motif (KExxR), which were the most highly conserved regions of P450 (Poulos et al., 1987), with a calculated molecular mass of 61.29 kDa and an isoelectric point of 8.92. It was speculated that the molecular formula of the CmMAX1 protein was C 2822 H 4378 N 720 O 771 S 17 .

PHYLOGENETIC ANALYSIS OF C M M A X 1 H O M E O D O M A I N PROTEINS.
Sequence alignment of the deduced amino acid sequence of the chrysanthemum MAX1 ortholog with other known MAX1 proteins was performed (Fig. 1A). CmMAX1 shared 60.7%, 60.7%, and 57.9% similarity with PhMAX1, MtMAX1, and MAX1 proteins, respectively. To investigate the evolutionary relationships among the predicted MAX1 proteins, a phylogenetic tree was constructed by an alignment of MAX1 proteins from a taxonomically diverse set of species (Fig. 1B). The phylogenetic tree revealed that CmMAX1 was closest to NnMAX1.
TISSUE SPECIFICITY OF CMMAX1 EXPRESSION. To identify the tissues in which CmMAX1 is most highly expressed, the level of CmMAX1 transcripts in different tissues was examined. As shown in Fig. 2, the CmMAX1 transcript was detected in all tissues examined. The difference of CmMAX1 expression in roots, stems, leaves, and axil was not significant, and the expression level of CmMAX1 in apex was lowest. P DEFICIENCY IMPROVED EXPRESSIONS OF CMMAX1. To study the relationship between CmMAX1 expression and P deficiency, we analyzed the expression of CmMAX1 in chrysanthemum seedlings treated with a P deficiency. Our results showed that the expression level of CmMAX1 in P-deficient plants increased about six times compared with the control (Fig. 3).
EXPRESSION OF CMMAX1 WAS REPRESSED BY DIFFERENT PHYTOHORMONES. To study the regulation of CmMAX1 by strigolactone, auxin, and cytokinin, we detected the expression of CmMAX1 after 2 h of treatment with GR24, IAA, or 6-BA. The results showed that all three hormones repressed the expression of CmMAX1, and the expression level of CmMAX1 decreased to about 0.21, 0.27, and 0.32 of the control, respectively (Fig. 4).
FUNCTIONAL ANALYSIS OF CMMAX1. The CmMAX1 ORF driven by cauliflower mosaic virus 35S promoter (35S:: CmMAX1) was expressed in the arabidopsis max1-1 mutant background. Four independent lines-line 1, line 3, line 6, and line 7-were selected for further analysis. As expected, all four lines displayed high expression levels of the CmMAX1 gene. The number of rosette branches with a length of at least 5 mm was scored to quantify the degree of rescue of the max1-1 mutant phenotype by the CmMAX1 gene overexpression. CmMAX1 expression reduced the mean number of branches from 8.2 in max1-1 to 3.25, 3.05, 4.2, and 3.5, respectively (Tukey's test at P < 0.05; Fig. 5A and B), improving the height of plants from 202 mm in max1 to 266.8, 270.3, 255.5, and 263 mm (Tukey's test at P < 0.05; Fig. 5A and C).

Discussion
MAX1 encodes a cytochrome P450 family member that acts downstream of D27 to produce a carotenoid-derived branchinhibiting hormone (Waters et al., 2012). Mutation of MAX1 led to the bushy phenotype in arabidopsis (Stirnberg et al., 2002). However, MAX1 orthologs were absent from Physcomitrella, present in monocots and eudicots as multiple copies and as a single copy, respectively, which indicated that MAX1 had a different pattern compared with the other genes. Thus, studies on MAX1 have important implications for understanding the evolution of the MAX pathway. In addition, expression and functional analysis of MAX1 orthologs will provide valuable information for molecular breeding.
In this study, another ortholog of MAX1 was cloned from chrysanthemum cultivar Jinba, then submitted to Nelson's P450 databases, and named CYP711A34 (Nelson et al., 1993). We found only one copy existed in chrysanthemum, which was consistent with the hypothesis that MAX1 is generally present as a single copy in eudicots (Hepworth, 2012). Sequence analysis revealed that CmMAX1 had deduced an amino acid sequence that contains heme-binding motif PFG · GPR · C · G, the PERF motif, and a K-helix motif KExxR. All three motifs were known to be involved in hemebinding or thought to stabilize the heme-binding pocket (Paquette et al., 2009). A phylogenetic tree based on the amino acid sequences was constructed. It showed that all the CYP711 members originated from the same ancestral origin, but diverged subsequently at different phases of evolution. CmMAX1 was closely related to NnMAX1 from lotus. It shared 55.33% amino acid similarity with NnMAX1, demonstrating that CmMAX1 was definitely a member of CYP711 according to the theory that P450 proteins with more than 55% similarity were grouped into the same subfamily (Mizutani and Ohta, 2010).
During the transition phase from vegetative growth to reproductive growth in plants, the hormone levels will be altered, which might lead to changes in gene expression. Given this, we tested the expression of CmMAX1 during the vegetative growth stage. Expression analysis showed that CmMAX1 in detected tissues varied little. This result was different from the previous report in petunia, in which PhMAX1 was expressed highest in the low-stem samples (Drummond et al., 2012). Studies in multiple species have shown that the biosynthesis of strigolactones is increased during P deficiency (Wang et al., 2015;Xi et al., 2015;Yoneyama et al., 2007). P starvation upregulated the expression level of DgCCD7 and DgCCD8 in chrysanthemum (Xi et al., 2015). In our study, the expression levels of CmMAX1 increased significantly compared with normal condition, indicating P deficiency can induce the expression of CmMAX1.
Strigolactone, auxins, and cytokinin are the most important hormones regulating shoot branching. Strigolactone and cytokinin were thought to be the second messengers of auxin to inhibit or promote directly the growth of axillary buds (Domagalska and Leyser, 2011). CmMAX1 decreased significantly after 2 h of GR24 treatment, which indicated there was a feedback regulation in the synthesis pathway of strigolactone. The application of 6-BA caused a decrease in the expression of CmMAX1, suggesting that cytokinins may regulate the synthesis of strigolactone by inhibiting the expression of CmMAX1. Studies in arabidopsis showed that MAX3 and MAX4 transcripts are regulated positively by auxin (Hayward et al., 2009). However, IAA treatment led to the significant decrease of CmMAX1 expression (Fig. 4), suggesting the complex regulation of the strigolactone synthesis pathway by auxin.
The overexpression of CmMAX1 in arabidopsis not only reduced max1-1 branching, but also increased plant height (Fig. 3C). This result demonstrates that CmMAX1 is a functional gene. Overexpression of PgMAX1 (Picea glauca) in arabidopsis was found to be capable of complete rescue of max1-1, and the overexpression of SmMAX1 (Selaginella moellendorffii) obviously reduced branching, although SmMAX1 only shares 38.7% amino acid similarity with MAX1 (Hepworth, 2012). These results indicated the partial conservation of MAX1 function across a wide evolutionary range.
We obtained the full length of CmMAX1 cDNA from chrysanthemum cultivar Jinba, and detected the expression of tissues and different treatments. Moreover, we confirmed the function of the CmMAX1 gene in vivo by the complementation experiment. Our results presented here reveal that the MAX1 ortholog was present and active in chrysanthemum, and also that CmMAX1 might be a candidate gene for the regulation of shoot branching.