Genome-wide identification of MAXs genes for strigolactones synthesis/signaling in solanaceous plants and analysis of their potential functions in tobacco

Identification of MAX genes

Genomic information, chromosome locations, protein sequences, and coding sequences (CDSs) of Nicotiana tabacum TN90, Nicotiana tabacum BX, Nicotiana tabacum K326, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana benthamiana, Solanum lycopersicoides, Solanum tuberosum, Petunia hybrida, Capsicum annuum, Arabidopsis thaliana, and Oryza sativa were downloaded from The Sol Genomics Network (https://solgenomics.net). Two methods were used to identify members of the MAX gene family. First, the conserved domains of MAX1–MAX4 were downloaded from the Pfam database (http://pfam.xfam.org/; MAX1: PF00067, MAX2: PF18511, MAX3 and MAX4: PF03055) (Czarnecki et al., 2014). These were used as template sequences to identify candidate family members using HMM3.0 (http://hmmer.org/download.html) with an E-value cutoff of 1.0. S, the amino acid sequences of the Arabidopsis MAX1–MAX4 members (AT2G26170.1, AT2G42620.1, AT2G44990.1, AT4G32810.1) were compared within the whole genome using the BLASTP method (threshold E≤1e−10) to obtain candidate members. Candidate family members were manually filtered using SMART (http://smart.embl-heidelberg.de) (PFAM domains) and NCBI (https://www.ncbi.nlm.nih.gov) to obtain the final set of MAX gene family members.

Evolutionary analysis and classification of MAX genes

The protein sequences of the identified MAX gene family members (Table S1) were compared with known Arabidopsis and rice MAX protein sequences using ClustalW (Larkin et al., 2007). Multiple sequence alignments were displayed with Jalview (Waterhouse et al., 2009). An unrooted phylogenetic tree was constructed using MEGA7.0 (Sudhir, Glen & Koichiro, 2016) with the maximum likelihood method based on the poisson correction model and 1,000 bootstrap replicates, the gap opening penalty was 10.

Sequence and structural characterization of MAX genes

Physicochemical properties such as amino acid number, molecular weight (MW), and theoretical isoelectric point (pI) of MAXs were calculated with ExPASy (https://www.expasy.org/vg/index/Protein). Subcellular localizations were predicted with CELLO v.2.5 (https://mybiosoftware.com/cello-v-2-5-subcellular-localization-predictor.html). Protein sequences of the MAX family were analyzed using MEME (https://meme-suite.org/meme/), and the Gene Structure Display Server (GSDS, v2.0, http://gsds.gao-lab.org/) was used to map MAX gene structure.

Analysis of cis-acting elements of MAX promoters

For each tobacco MAX gene, the 3-kb region upstream of the transcription start site was extracted with TBtools (Chen et al., 2020) and analyzed with the PLACE database (https://www.dna.affrc.go.jp/PLACE/?action=newplace) and the PlantPAN3.0 database (http://plantpan.itps.ncku.edu.tw/promoter.php). This was done to identify potential transcription factor binding sites and cis-acting elements associated with growth, development, hormone responses, and stress responses.

Re-analyzing RNAseq dataset of MAX

The transcriptome data of root, leaf, flower and fruit (six developmental stages) of tomato MAX genes (The Tomato Genome Consortium, 2012), and the transcriptome data of roots (0, 0.5, 1, 3, 5, 8 and 24 h after topping) of tobacco Yunyan 87 (Qin et al., 2020) MAX genes were analyzed separately. The heatmap was constructed by TBtools.

Total RNA extraction and fluorescence quantitative PCR (qPCR)

Nicotiana tabacum L. cv. ‘honghuadajinyuan’ was grown in the greenhouse. The seeds were obtained from the Tobacco Research Institute (TRI) of the Chinese Academy of Agricultural Sciences (CAAS). Total RNA was extracted from the roots, stems, leaves, flowers, apical buds, and axillary buds during the vigorous growth period using the GeneJET™ Plant RNA Purification Mini Kit (MBI Fermentas, Burlington, Canada). Samples were run on 1% agarose gels and purity was assessed with a NanoDrop2000 spectrophotometer. Total RNA was reverse transcribed using the RevertAid™ First-Strand cDNA Synthesis Kit (MBI Fermentas, Burlington, Canada). Quantitative reverse transcription (qRT)-PCR and qPCR were performed with the resulting cDNA and TB Green™ Premix Ex Taq™ II (TliRNaseH Plus) (TaKaRa, Japan) with primers specific to the candidate genes (Table 1). Actin gene was selected as the internal reference gene for normalization. Each sample was analyzed with three replicates. The relative expression levels were calculated using the 2 ^{$-\mathrm{\Delta }\mathrm{\Delta }\mathrm{C}\mathrm{t}$} method (Livak & Schmittgen, 2002).

Hormone, topping, and stress treatments

Hormone treatments were applied to N. tabacum L. cv. ‘honghuadajinyuan’ during the vigorous growth period. These treatments comprised 0.1 mmol/l abscisic acid (ABA) (Wang & Qiang, 2012), 0.0001 mol/l indole-3-acetic acid (IAA) (Vehn & Sauer, 2017), 300 mM sucrose, 50 µM of the synthetic strigolactone analog GR24 (Fan et al., 2021), and 200 µM 6-benzylaminopurine (6-BA) (Wang et al., 2017) according to published references. The axillary buds of the first leaf were also sampled before topping and 1, 3, and 5 d after topping according to published references (Wang et al., 2018). Plants were treated with simulated drought stress (260 µmol L-1 mannitol) or low temperature stress (4 °C) then sampled at 1, 2, 4, and 8 d (Zhang et al., 2015) or at 1 and 2 d (Gu et al., 2021), respectively. There were three biological replicates for each treatment. The results were analyzed with TBtools and Sigmaplot 10.0 software.

Subcellular localization analysis of representative MAX proteins in tobacco

CDSs of NtMAX2, NtMAX3, and NtMAX4 were each cloned with the stop codon removed, then inserted into the PYG57 vector using the restriction enzymes SacI and SpeI. The recombinant vectors and empty plasmid were each transformed into Agrobacterium tumefaciens EHA105. Four-day-old leaves of Nicotiana benthamiana were inoculated with transformed Agrobacterium EHA105 via needle prick. Plants were then incubated continuously at 28 °C with a 16/8 h light/dark cycle. Leaves were photographed using a Leica TCS SP8 laser confocal microscope (Leica, Mannheim, Germany).

Identification of MAX genes

A total of 74 non-redundant MAX family proteins were identified in tobacco, tomato, potato, petunia, and pepper. The gene ID, protein length, molecular weight, isoelectric point, and subcellular localization of 89 MAX family members (including in Arabidopsis and rice) are shown in Table 2. PI values of MAX1 homologs were all in the alkaline range, whereas PI values of the MAX2 and MAX4 homologs were all in the acidic range. It is noteworthy that for the selected solanaceous crops, members of the MAX1, MAX2, and MAX3/MAX4 sub-families were mainly localized in the mitochondria, nucleus, and cytoplasm, respectively. This implies that different sub-types of MAX proteins may perform different functions. Table 3 shows the number of MAX sub-family members predicted in different species, as well as the ranges of PI and MW values and protein lengths. The results showed that the MAX family was not large in either monocotyledons or dicotyledons; Nicotiana sylvestris contained the largest MAX4 sub-family, with 16 members, but whether these 16 homologous genes have functional redundancy needs further experimental verification.The distribution of genes within each sub-family was similar between species.

Phylogenetic and structural analysis of MAX genes

To better understand the evolutionary relationships between MAX family members, an unrooted phylogenetic tree was constructed using the full-length sequences of 87 MAX1–MAX4 proteins (Fig. 1); two sequences (mRNA_108065 and mRNA_110488) were removed during construction of the evolutionary tree due to their relatively long distances from other sequences. Based on maximum likelihood phylogenetic analysis, the MAX1–MAX4 sub-families contained 16, 26, 13, and 32 members, respectively. The sequences of the MAX family members are listed in Table S1. The exon–intron structures and conserved motifs of the MAX genes were analyzed (Fig. 2). There were at least two exons in all members of the MAX1, MAX3, and MAX4 sub-families, with maximum exon numbers of five, seven, and nine, respectively. In contrast, members of the MAX2 family contained only one to two exons each, except for PGSC0003DMT400057424, which contained six. MEME analysis of MAX protein sequences revealed the existence of sub-family-specific motifs. For example, motifs 2, 6, 7, 9, and 10 were uniquely found in MAX2 proteins, and motif 4 was specific to MAX3 and MAX4 genes. In contrast, motif 5 was common to the MAX1–MAX4 sub-families. The results showed that each sub-family had a unique and consistent gene structure with conserved motifs. Alignment of representative MAX1–MAX4 proteins showed that they all contained typical conserved domains (Fig. 3 and Fig. S1).

Analysis of cis-acting elements and transcription factor binding sites in the promoter region of the tobacco MAX family

To investigate the potential biological functions of MAX genes in tobacco, the 3-kb promoter sequences of MAX1–MAX4 gene family members were analyzed using the PlantCARE database. Cis-acting elements associated with meristem development and responses to hormones, sucrose, light, and stress were screened (Table S2). We found that ASF1MOTIFCAMV, CPBCSPOR, ERELEE4, GAREAT, MYBGAHV, and TATCCAOSAMY (which are closely associated with responses to auxin, cytokinin, ethylene, gibberellin (GA), and sucrose) are widely distributed in the tobacco MAX promoters, consistent with previous observations. It has been demonstrated that strigolactone can inhibit plant branching by inducing cytokinin and auxin to promote axillary bud dormancy, and can inhibit stem elongation by acting on photoreceptors and auxin (Yao, Li & Xie, 2018; Luo et al., 2019; Jia et al., 2014; Xiong, Wang & Li, 2014). Recent discoveries indicate that SL biosynthesis is regulated by GAs (Marek, 2017) and that SLs can induce ethylene to elongate root hairs (Kapulnik et al., 2011). Sucrose can promote shoot branching by suppressing the inhibitory effect of SL (Patil et al., 2021).

In recent years, it has been found that SLs play key roles in response to abiotic stresses such as drought, high salt, and nutrient deficiencies in plants (Visentin et al., 2016). In tomato, strigolactone biosynthesis is induced by heat and cold stresses (Chi et al., 2021). This is consistent with our finding that a large number of cis-elements associated with cold and other stress responses exist in MAX gene promoters. Many cis-acting elements related to pathogenesis and wounding induction were also identified, suggesting that SL may have functions in disease resistance, which is in line with previous research (Torres-Vera et al., 2014). These results imply that the cis-elements in the promoter sequences of MAX1/3/4, which are involved in SL biosynthesis, and MAX2, which is involved in SL signal sensing and transduction, are together able to ensure appropriate SL functioning with respect to plant branching and responding to cues such as light and hormones.

Previous transcriptome analysis revealed that genes responsive to SL include TCP, NAC, WRKY, and MYB transcription factors (Tang & Chu, 2020; Wang et al., 2021). We analyzed and counted potential transcription factor binding sites in MAX family members of three tobacco cultivars (N. tabacum K326, KN90, and BX), two ancestral species of cultivated tobacco (Nicotiana sylvestris and Nicotiana tomentosiformis), and triploid Nicotiana benthamiana (Table 4). We found that the number of transcription factor binding sites varied widely between tobacco species with different ploidy levels. This was especially true of AP2, MYB, TCP, and WRKY transcription factors in MAX1 homologs; AP2, bHLH, bZIP, and TCP in MAX2 homologs; AP2, MYB, and TCP in MAX3 homologs; and AP2, bHLH, bZIP, and MYB in MAX4 homologs. Prior studies have noted the importance of AP2 transcription factors in responding to drought, low temperature, and high salt in plants (Xie et al., 2019). Based on these data, we can infer that MAX family members play important roles in abiotic stress responses.

Tissue-specific MAX expression analysis in tobacco

To analyze the roles of MAX genes in the growth and development of tobacco, tissue-specific expression analysis was performed for MAX1–MAX4 genes in apical buds, axillary buds, leaves, stems, flowers, and roots of cultivated tobacco (Fig. 4A). All MAX genes were highly expressed in the flowers, with MAX1 and MAX3 specifically expressed in flowers and MAX4 specifically expressed in apical buds and flowers. Expression trends were similar for MAX1/3/4 sub-family members, whereas MAX2 members were expressed in all tissues, which is consistent with Arabidopsis (Stirnberg, Furner & Leyser, 2007). The tomato transcriptome data indicated that MAX1 and MAX4 have the highest expression in tomato roots, while MAX2 has the highest expression in fruits, which indicates that tomato MAX2 may play vital roles in fruit ripening, however MAX3 has very low expression in different tissues (Fig. 4E). All the raw data for fluorescent quantitative PCR is listed in Table S3.

MAX gene expression analysis in response to topping, stress, and hormone treatments

An important physiological role of strigolactone is to regulate branching by interacting with other plant hormones. Tobacco production in the field requires topping treatments, which lead to axillary buds due to the loss of apical dominance. To further analyze the role of MAX genes in the responses of tobacco to topping, stress, and hormone treatments, expression levels of MAX1–MAX4 were analyzed at 1, 3, and 5 d after topping or hormone application, at 1, 2, 4, and 8 d after drought treatment, and at 1 and 2 d after cold treatment. The expression level of MAX2 and MAX3 after topping rises and then falls, in contrast to the expression level of MAX1 and MAX4 after topping, which falls and then rises (Fig. 4B). These results differ from the expression pattern of Yunyan 87, in which within 24 h after topping, MAX1 and MAX2 rise first and then fall, while the expression levels of MAX3 and MAX4 falls and then rise (Fig. 4E), indicating that MAX genes differentially respond to topping in different tobacco varieties.

MAX family members were more sensitive to drought than to cold treatment in cultivated tobacco, with a significant increase in expression of MAX1/3/4 after drought treatment (Figs. 4C and 4D). Despite the low expression, MAX2 expression level changed after drought treatment, a finding that is similar to the results of previous studies, suggesting that the max2 mutant in Arabidopsis was sensitive to drought stress (Bu et al., 2014). However, the sensitivity of max2 was significantly lower under drought treatment compared to max3 and max4 mutant plants (Chien et al., 2014), and the sensitivity of MAX2 to drought was further reduced in tobacco compared to Arabidopsis. Unraveling the molecular evolutionary mechanisms behind this interesting phenomenon will be a future focus of our research. The data suggest that SLs may have varying regulatory mechanisms in response to drought stress in different species, nonetheless, a number of questions remain to be answered regarding the diversity and function of SLs.

Detailed analysis was conducted to measure the expression levels of MAX1–MAX4 genes in response to ABA, 6-BA, IAA, sucrose, GR24, and topping treatments (Figs. 5A–5D). From these data, we reached several general conclusions. First, MAX1–MAX4 genes in cultivated tobacco all responded positively to GR24 and topping treatments. Second, MAX1, MAX3, and MAX4, which are involved in SL biosynthesis, did not have consistent responses to each treatment; only MAX4 responded to sucrose, whereas both MAX3 and MAX4 responded to IAA, and MAX2–MAX4 responded to both 6-BA and drought treatment (Fig. 5E).

Subcellular localization of MAX proteins in tobacco

The fusion vectors PYG57::NtMAX2-GFP, PYG57::NtMAX3-GFP, and PYG57::NtMAX4-GFP were constructed and co-cultured in tobacco leaves using an Agrobacterium tumefaciens EHA105-mediated transformation method. The subcellular localization of each target protein was indicated by green fluorescence of the fusion protein (Fig. 6). There was no green fluorescence in the plasma membrane, cytoplasm, or nucleus of leaves injected with the control vector. MAX2 was localized to the cytoplasm, which was inconsistent with the result predicted by CELLO. MAX3 and MAX4 were also localized to the cytoplasm, which was consistent with the predicted results.