Genome-wide identification and expression analysis reveals spinach brassinosteroid-signaling kinase (BSK) gene family functions in temperature stress response

doi:10.21203/rs.3.rs-1507914/v1

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Genome-wide identification and expression analysis reveals spinach brassinosteroid-signaling kinase (BSK) gene family functions in temperature stress response

https://doi.org/10.21203/rs.3.rs-1507914/v1

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Background: Brassinosteroid-signaling kinase (BSK) is a critical family of receptor-like cytoplasmic kinase for BR signal transduction, which plays important roles in the development, immunity-/ abiotic stress- response, and hormone regulation of plants. Spinach (Spinacia oleracea) is cold tolerant but heat sensitive green leafy vegetable. A comprehensive study on the mechanisms of BSK family members and BSK- mediated stress response in spinach have not been performed.

Results: We identified and cloned seven SoBSKs in spinach. Phylogenetic and collinearity analyses suggested that SoBSKs had close relationship with dicotyledonous sugar beet (Beta vulgaris) rather than monocotyledons. The gene structure, and conserved protein domain/ motif analyses indicated that most SoBSKs were relative conserved, while SoBSK6 could be a truncated member. The predicted functional post-translation modification (PTM) sites in SoBSKs suggested their probable roles in signal transduction, redox regulation, and protein turnover of SoBSKs, and the N-terminal myristoylation site in SoBSKs was critical for their localization to cell periphery. Cis-acting elements for light, drought, temperature (heat and cold), and hormone response distributed wildly in the promoters of SoBSKs, implying the pivotal roles of SoBSKs in response to diverse abiotic stress and phytohormone stimuli. Most SoBSKs were highly expressed in leaves, except of SoBSK7 in roots. Many SoBSKs were differentially regulated by heat stress, cold stress, as well exogenous BL and ABA treatments. The bsk134678 mutant seedlings exhibited more heat tolerance than wild-type and SoBSK1-overexpressed seedlings.

Conclusions: A comprehensive genome- wide analysis of SoBSK in spinach presented a global identification and functional prediction of SoBSKs. Seven SoBSKs had relative conserved gene structure and protein function domains. Except for SoBSK6, all the other SoBSKs had the similar motifs and conserved modification sites. Most SoBSKs participated in the temperature and hormone (BR and ABA) responses. These findings paved the way for further functional analyses on BSK- mediated mechanism in spinach development and stress responses.

Spinach

BSK family

Expression pattern

BR signaling

Heat stress

Spinach (Spinacia oleracea) is an important and nutritious green leafy vegetable in China, which belongs to the Amaranthaceae family [1]. Generally, spinach is cold- tolerant but heat- sensitive vegetable, which limits its distribution and production in tropics [2]. The genomic analyses on spinach presented a high-quality chromosome- scale reference genome, and the transcriptome sequencing and population genomic investigations discovered the genome architecture, evolution, and domestication, which provided valuable information for understanding the genetic basis of important agronomic traits and breeding [1, 3]. In addition, high- throughput heat- responsive proteomics on heat- tolerant variety Sp75 and heat- sensitive variety Sp73 of spinach indicated that the plant variety- specific reactive oxygen species (ROS) scavenging, and heat signal perception and transduction were pivotal for the heat resistance [4, 5]. However, the finetuned heat- responsive mechanism in spinach keeps unknown due to the lack of efficient genetic transformation system [6].

Brassinosteroids (BRs), a group of steroid hormones, regulate diverse physiological processes upon plant development and stress response [7]. The BR signal transduction in Arabidopsis has been well addressed [8]. BR signal is precepted by leucine-rich repeat receptor-like kinase BR INSENSITIVE1 (BRI1) and coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) at the cell surface [9, 10]. The activate BRI1 by sequential transphosphorylation from BAK1 can phosphorylate BR SIGNALING KINASE (BSK) and subsequently enhance BRI1 SUPPRESSOR 1 (BSU1) activity [11, 12], and then the activated BSU1 inhibits BRASSINOSTEROID INSENSITIVE2 (BIN2) through dephosphorylation BIN2, promoting the unphosphorylated BRASSINAZOLE RESISTANT1/bri1 EMS SUPPRESSOR1 (BZR1/BES1) transcription factors to accumulate and enter the nucleus for regulating BR-targeted gene expressions [13, 14].

BSK is a critical family of receptor-like cytoplasmic kinase (RLCK) in the initial steps, activating downstream phosphatase BSU1 for BR signal transduction [12]. Three Arabidopsis BSKs (i.e. BSK1, BSK2, and BSK3) have been identified as BR- responsive proteins [11]. The function of some members among 12 Arabidopsis AtBSKs and five rice OsBSKs has been well investigated, which has conserved kinase catalytic domain at the N-terminus and tetratricopeptide repeats (TPRs) domain at the C-terminus [11, 15-17]. Besides, myristoylation site at the N-terminus for plasma membrane (PM)-localization, critical functional serine (S) and lysine (K) sites (e.g., S²³⁰ and K¹⁰⁴ in AtBSK1), as well as several sumolyation and ubiquitination sites were also conserved in most AtBSKs [17].

BSKs were involved in BR signal transduction but also other diverse signaling pathways [18, 19]. For plant growth and development, several BSKs (i.e. BSK3, BSK4, BSK6, BSK7, and BSK8) were partially redundant in BR signaling [20]. However, BSK3 was the only reported member for BR- mediated plant root growth [21] and foraging under low nitrogen [22]. On the contrary, BSK12 (also called SHORT SUSPENSOR, SSP) activated the YODA mitogen-activated protein kinase (MAPK) pathway, but not BR signaling, upon embryogenesis of Arabidopsis [23]. For abiotic stress responses, BSK5 inhibited the expression of abscisic acid (ABA) deficient3 (ABA3) and 9-cis-expoxycarotenoid dioxygenase (NCED3) for ABA biosynthesis in Arabidopsis under salinity stress [24]. Besides, BSK5 promoted BES1-PHYTOCHROME INTERACTING FACTOR4 (PIF4)/PIF5 module- mediated auxin signaling for plant hypocotyl elongation under shade (low ratio of red to far-red light), but this enhancement was blocked by NaCl- induced ABA signaling [25].

During plant immunity response process, BSK1 positively regulated pathogen- associated molecular pattern (PAMP)- trigged immunity (PTI) (i.e. edr- mediated resistance) by physically associating with the PAMP receptor FLAGELLIN SENSING2 (FLS2) in response to FLS2- mediated ROS bursting [26, 27]. BSK1-FLS2 module did not activate MAPK signaling, but BSK1 directly phosphorylated S²⁸⁹ in mitogen-activated protein kinase kinase kinase5 (MAPKKK5) to form immune complex for triggering the MAPK cascade [28]. During the resistance to bacterial pathogens, the immune signals can be transmitted from RECEPTOR-LIKE KINASE 902 (RLK902) to BSK1 through the phosphorylation of BSK1 at S²³⁰ [29], which was also the key phosphorylation site for BR signal transduction [11]. Besides, BSK5 can interact with 11 immunity- associate receptor like kinases (e.g., STRUBBELIG-RECEPTOR FAMILY (SRF)6/ 7, FERONIA, WALL-ASSOCIATED RECEPTOR KINASE-LIKE (WAKL)8/ 14/ 18, BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1), LYSIN-MOTIF RECEPTOR KINASE5 (LYK5), PEP1 RECEPTOR1 (PEPR1), ERECTA, and SUPPRESSOR OF BIR1-1 (SOBIR1)) for promotion of multiple PAMPs/ damage- associated molecular patterns (DAMPs)- induced PTI processes [18, 30]. In addition, BSK8 was associated with FLS2 [31] and phosphorylated on flg22 treatment [32], although its molecular mechanism is still unclear. On the other hand, in the process of effector- triggered immunity (ETI) initiation, PM- localized BSK1 and BSK8 were identified as the component of Ribosomal protein S2 (RPS2) protein complex [31]. RPS2 is a well- studied resistance proteins for the perception of pathogen. Therefore, BSKs played key roles in pathogen perception upon PTI.

Interestingly, the dynamic spatiotemporal reorganization of BSK1 within PM was a critical node for the finetuned signal- specific activation of the balance between growth and immunity [33]. BSK1 multimerization and dissociation from the complexes of FLS2-BSK1 or BRI1-BSK1 were induced by the flg22 or exogenous BR treatment, respectively. Moreover, the flg22- triggered BSK1 translocated from membrane rafts to non- membrane- raft regions for immunity response, whereas BR- induced BSK1 remained in membrane rafts for growth regulation [33].

Besides of Arabidopsis, several BSKs from other plants were reported to function in diverse biological processes, such as the sprouting of potato (Solanum tuberosum) [34], drought response of maize (Zea mays) [35], wild barley (Hordeum spontaneum) [36] and Kentucky bluegrass (Poa pratensis) [37], cold tolerance of Populus tomentosa [38], as well as immune response of rice (Oryza sativa) [15] and tomato (Solanum lycopersicum) [39]. However, the BSK- regulated mechanisms in these courses need to be further studied.

In this study, seven members of the spinach BSK gene family were identified by screening the spinach whole- genome database [1, 3]. The phylogenetic trees were constructed along with their homologous genes from sugar beet (Beta vulgaris), Arabidopsis, rice, maize, as well as common tobacco (Nicotiana tabacum) and woodland tobacco (Nicotiana sylvestris) for evaluating the molecular evolution relationship and classification. The chromosomal distribution, gene structure, conserved function domains/ sites, and cellular localization of SoBSKs were analyzed. Moreover, we examined the expression patterns of SoBSKs in different organs from heat- tolerant variety Sp75 and heat- sensitive variety Sp73, and upon exposure to temperature stresses and exogenous hormone treatments. Furthermore, diverse PTMs were found in several SoBSKs, implying the potential function of SoBSKs. All these results lay a solid foundation for understanding the SoBSK- mediated molecular mechanisms in spinach development and stress response.

2.1 Phylogenetic relationship of BSKs from spinach and other plant species

To evaluate the molecular evolution relationship of BSKs from spinach with other plant species, an un- rooted neighbor- joining (NJ) phylogenetic tree was constructed using MEGA-X with the maximum likelihood method (Fig. 1). The protein sequences of seven BSKs from spinach, seven from sugar beet, 12 from Arabidopsis, 18 from common tobacco, 12 from woodland tobacco, five from rice, and 11 from maize were clustered in the phylogenetic tree (Fig. 1 and Additional file 1). Among them, 12 Arabidopsis AtBSKs [11, 17-32, 40, 41], five rice OsBSKs [15, 16], two woodland tobacco NsBSKs (i.e. NsBSK1 and NsBSK3) [42], a common tobacco NtBSK2 [42], and nine maize ZmBSKs [35] have been reported previously, and all the other sequences of BSKs were obtained by searching against the genomics database using BLASTp. Totally, 72 proteins were grouped into seven classes (Class I to Class VII), and seven SoBSKs distributed into four classes (Class I, Class II, Class V, and Class VII) (Fig. 1). In Class I, SoBSK1 and SoBSK7 were clustered into two sub-branches with their homologous sugar beet BvBSK1 and BvBSK7, respectively, and the BSKs from monocotyledonous rice and maize were grouped into another sub-branches. In Class II, SoBSK2, BvBSK2, and AtBSK2 were grouped into one subgroup, and the BSKs from rice (OsBSK2) and maize (ZmBSK2 and ZmBSK3) were grouped into another subgroups. In Class V, SoBSK3 was grouped into a subgroup with BvBSK3 and AtBSK5. Three NsBSKs and six NtBSKs were grouped into another three subgroups. Class VII included four main subgroups, Subgroup I (contained AtBSK8 and AtBSK7), Subgroup II (i.e. SoBSK5, SoBSK6, BvBSK5, and BvBSK6), Subgroup III (i.e. SoBSK4, BvBSK4, and two AtBSKs), and Subgroup IV contained two NsBSK2 and five NtBSKs. Besides, Class III, Class IV and Class VI did not contain any SoBSKs. Herein, AtBSK6, AtBSK9, and AtBSK10 were clustered into Class IV. Two OsBSK3 and five ZmBSKs were clustered into Class III (i.e. OsBSK4, ZmBSK8, and ZmBSK9) and Class VI (i.e. OsBSK3, ZmBSK5, ZmBSK6-1, and ZmBSK6-2). The phylogenetic analysis indicated that seven SoBSKs had close homology with BvBSKs from sugar beet, and all these reported BSKs were highly conserved among monocotyledons and dicotyledons.

2.2 Chromosomal distribution analysis of SoBSKs

The physical map positions of seven SoBSKs on chromosome were analyzed using MapGene2Chrom web v2 (http://mg2c.iask.in/mg2c_v2.0/) against spinach genome. Seven SoBSKs were distributed nonrandomly and unevenly across four out of six spinach chromosomes, except for chromosome 1 and 3 (Fig. 2A). SoBSK4, SoBSK7, and SoBSK1 were localized on chromosome 2, 4, and 5, respectively, while chromosome 6 harbored SoBSK2, SoBSK5, and SoBSK6. In addition, SoBSK3 was mapped onto an unanchored scaffold (Scf_01469) according to the draft spinach Sp75 genome, which was assembled with low coverage rate (47%) [1].

2.3 Collinearity analysis of BSKs between spinach and other plants

The collinearity relationship was constructed between spinach and three plant species (i.e. Arabidopsis, sugar beet, and maize) (Fig. 2B). Collinearity analysis showed that SoBSK genes had homologous genes in Arabidopsis, sugar beet, and maize, of which Arabidopsis had six homologous gene pairs located in chromosome 1, 4, and 5, followed by sugar beet (five gene pairs in chromosome 1, 7, and 8, as well as an unanchored scaffold chr5_nu.sca001), and maize (only one gene pair in chromosome 10) (Fig. 2B and Additional file 2). Interestingly, SoBSK4 (LOC110789384) had homologous gene pairs with all three plant species, especially three gene pairs in Arabidopsis, including AtBSK3 (AT4G00710), AtBSK4 (AT1G01740), and AtBSK7 (AT1G63500). SoBSK6 (LOC110778653) and SoBSK7 (LOC110788203) had homologous gene pairs with both sugar beet and Arabidopsis. In addition, both SoBSK4 and SoBSK6 had close relationship with AtBSK7. SoBSK6 was also homologous with AtBSK8 (AT5G41260). These results indicated that SoBSKs had close phylogenetic relationship with dicotyledons rather than monocotyledons.

2.4 Gene structure and protein function domain analysis of SoBSKs

Phylogenetic analysis indicated that SoBSKs were grouped into three groups according to their amino acid sequences. SoBSK5, SoBSK6, and SoBSK4 were included in Group I, SoBSK1, SoBSK7, and SoBSK2 were classed into Group II, while SoBSK3 was a distinct Group III (Fig. 3A).

To predict the evolutionary feature and functional diversification, exon-intron organization of seven SoBSKs was analyzed according to their gene sequences in SpinachBase (http://www.spinachbase.org/) using TBtools. SoBSK4 contained eleven introns, SoBSK2, SoBSK3, and SoBSK5 had nine introns, SoBSK1 and SoBSK7 had eight introns, while SoBSK6 had four introns (Fig. 3B). The diversity of various introns and exons implied the possible alternative splicing in SoBSKs was critical for spinach development and stress response, which were reported in Arabidopsis BSKs [17]. In addition, SoBSK4 and SoBSK6 only had 3’ UTR, SoBSK5 only had 5’UTR, while the remaining four SoBSKs had both 5’UTR and 3’UTR (Fig. 3B).

The conserved motifs were detected using MEME online search software (https://meme-suite.org/meme/tools/meme) (Fig. 3C and Additional file 3). Thirteen conserved motifs were found in the six SoBSKs, except for SoBSK6 which only had seven motifs (Motif 1, 2, 4, 5, 8, 9, and 11) (Fig. 3C). Among 13 motifs, seven motifs (Motif 1, 3, 5, 6, 10, 11, and 12) were localized in the kinase domain, while three motifs (Motif 2, 4, and 9) were distributed in the TPR domain. Importantly, more than half amino acid sites (26 out of 50) in Motif 1 were highly conserved, among of which contained four conserved S sites (S22, S26, S36, and S43) (Fig. 3D). Besides, the 2^nd serine in Motif 1 also conserved in SoBSKs, except for SoBSK6 (Fig. 3D and G). The Arabidopsis homologous site of the 2^nd serine in Motif 1 (S²³⁰ in AtBSK1 and S²¹⁰ in AtBSK3) had been reported to a phosphorylated site by AtBRI1 in the BR signaling pathway [11, 16]. Moreover, the glycine (G) 16, G28, and G37 in Motif 1 were also conserved in all SoBSKs (Fig. 3D and G). The homologous sites in AtBSK3 of G16 and G28 (G²²⁶ and G²³⁸ in AtBSK3) can interfere other BSKs in BR signaling and protein stability of AtBSK3, respectively [21]. Motif 6 has 26 conserved amino acids out of 50 amino acids, including two lysines (K10 and K29 in Motif 6), and K29 was the key site for ATP binding and BSK kinase activity (Fig. 3E and G) [21]. In addition, the 2^nd glycine in Motif 13 was highly conserved myristoylation site for membrane localization of BSKs (Fig. 3F and G) [16, 18].

2.5 Analyses of conserved function domain and modification sites in SoBSKs

A common feature of BSKs from Arabidopsis and rice is the presence of a N-terminal kinase domain and two or three C-terminal TPR domains, which can interact directly with each other for autoregulating its kinase activity [11, 16]. In spinach, we also found the conserved N-terminal kinase domain and C-terminal TPR domains in seven SoBSKs using the NCBI web CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) with the default parameters (Fig. 3G). SoBSK2 and SoBSK3 contained two C-terminal TPR domains, but all the other SoBSKs had three C-terminal TPR domains (Fig. 3G).

We performed the prediction of some conserved amino acid sites for protein PTM. The sequence alignment with these reported functional sites in Arabidopsis [11, 18, 21, 26, 27, 29, 41] and rice [16], the GPS-SNO 1.0 [43] with default parameters, and UbiComb (http://nsclbio.jbnu.ac.kr/tools/UbiComb/) with a threshold value of 0.5 [44] were used for the predictions of phosphorylation, myristoylation, S-nitrosylation, and ubiquitination, respectively (Fig. 3G). Except for SoBSK6, all the other SoBSKs had a serine site and a lysine site in the kinase domain (Fig. 3G), which were the homologous sites with the conserved phosphorylated site by BRI1 and ATP- binding site for kinase activity determination in Arabidopsis, respectively [11, 26]. Similarly, most SoBSKs, except for SoBSK6, had a glycine site for myristoylation modulating its plasma membrane localization, and more than one cystines for S-nitrosylation in response to nitric oxide signal [43] (Fig. 3G). In addition, SoBSK1 and SoBSK5 had a predicted ubiquitination site (K¹⁰⁰ in SoBSK1 and K¹⁰⁴ in SoBSK5) in the kinase domain (Fig. 3G), which were supposed to be a critical site for BSK degradation through ubiquitin-proteasome system. This indicated that these conserved PTM sites determined subcellular localization, redox regulation, and protein turnover of SoBSKs.

2.6 Analysis of cis-acting elements in SoBSK promoters

In order to explore the potential function of SoBSKs, cis-acting elements in seven SoBSK promoter sequences of 2,000 bp upstream from the translation start site of individual gene were predicted using the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and the prevalence distribution of cis-acting elements for stress- and hormone- responses were schematically depicted (Fig. 4A). A total of 294 cis-acting elements were found in SoBSKs involved in diverse abiotic stresses, such as light (80 elements), drought (74), heat (29), anaerobism (22), wound (11), cold (2), and phytohormone (76) (Fig. 4B and Additional file 4). Among them, the elements in response to drought (i.e. DRE, MYB, MRS, MYC, and MBS), light (e.g., Box 4, G-box, and GT1-motif), heat (i.e. CCAAT-box, STRE, and AT-rich), and phytohormone accounted for the main parts in each SoBSK, and some of these elements were conserved across seven SoBSKs, which implied that SoBSKs could play important roles in response to drought, light, heat, and various hormones. Importantly, 76 hormone-responsive elements included 28 for ABA (i.e. ABRE), 23 for methyl jasmonate (MeJA) (i.e. TGACG-motif, CGTCA-motif, and JERE), nine for salicylic acid (SA) (i.e. TCA-element), eight for ethylene (i.e. ERE), three for gibberellin acid (GA) (i.e. P-box and TATC-box), and five for auxin (i.e. TGA-element and AuxRR-core) (Fig. 4B). Collectively, these conserved elements in the promoter region suggested that SoBSKs were pivotal for these abiotic stresses and phytohormone stimuli.

2.7 Localization of SoBSK1 and SoBSK6 proteins

The subcellular localization of SoBSKs was performed using tobacco leaves. SoBSK1 and SoBSK6 were fused respectively in frame to the 3’-terminus of the GFP reporter gene under the control of CaMV 35S promoter. The recombinant SoBSK1-GFP, SoBSK6-GFP, and GFP alone were transiently expressed in the epidermal cells of tobacco leaves. The 35S::GFP signals were distributed in the cytosol, nucleus, and PM in tobacco epidermal cells, while the signal from SoBSK1-GFP was concentrated in the PM (Fig. 5). This suggested that the SoBSK1-GFP recombinant protein localized on the cell membrane. Interestingly, unlike SoBSK1-GFP, SoBSK6-GFP recombinant protein localized not only in the cell membrane, but also in the cytoplasm and nucleus, because SoBSK6 was lack of the N-terminal myristoylation site for potential membrane localization [45]. This indicated that the truncated N-terminus without myristoylation site in SoBSK6 led to the missing of exclusive localization to cell periphery.

2.8 Organ-specific expression analysis of SoBSKs

To better understand organ- specific expression of seven SoBSK genes, total RNA from roots, stems, and three pairs of alternate leaves (i.e. the 1^st, 2^nd, and 3^rdleaves) from heat- sensitive variety Sp73 and heat- tolerant variety Sp75 were prepared. The transcription level of each SoBSK gene was evaluated for three replicates using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. In heat- sensitive variety Sp73, SoBSK6 in stems, SoBSK3 in the 1^st leaves, SoBSK4 in the 2^nd leaves, and SoBSK2 in the 3^rd leaves had the highest expression levels (Fig. 6). However, in heat- tolerant variety Sp75, SoBSK5 in stems, the 1^st and 2^nd leaves, as well as SoBSK4 in the 3^rdleaves exhibited the highest expressions (Fig. 6). On the other hand, for each gene in variety Sp73, SoBSK1, SoBSK2, and SoBSK4 in the 3^rd leaves, SoBSK3 in the 1^st leaves, SoBSK5 and SoBSK7 in the roots, as well as SoBSK6 in the stems exhibited the highest expression (Fig. 6). While, in variety Sp75, it was SoBSK1 and SoBSK4 in the 3^rd leaves, SoBSK2, SoBSK3, SoBSK5, and SoBSK6 in the 2^nd leaves, as well as SoBSK7 in the roots that had the highest expression (Fig. 6). These suggested that, except of SoBSK7 in roots, the other SoBSKs mainly functioned in leaves rather than stems and roots in different varieties of spinach (Fig. 6).

2.9 ABA- and brassinolide (BL)- responses of SoBSKs in leaves

BSKs are critical components in BR signaling pathways [12]. We found multiple SoBSKs had ABRE cis-acting elements in their promoters for ABA signaling (Fig. 4). This implied that the cross-talk of ABA and BR signaling would happen in regulating plant growth, development, and stress response [46, 47]. Therefore, we evaluated the expression profiles of seven SoBSKs in leaves under exogenous BL and ABA treatments using RT-qPCR analysis (Fig. 7).

For BL treatment, all the SoBSKs in variety Sp73 were induced more than 1.5- fold at 2 and 4 h. SoBSK1, SoBSK2, SoBSK3, SoBSK4, SoBSK5, and SoBSK7 reached their highest expression levels at 4 h, while SoBSK6 maintained a higher level with more than 2- fold during whole process (Fig. 7). Except of SoBSK1 and SoBSK3, the other SoBSKs in variety Sp73 were increased at 24 h (Fig. 7). However, in variety Sp75, SoBSK2, SoBSK3, and SoBSK5 were apparently induced at 2, 4, and 12 h, but reduced at 24 h (Fig. 7). Besides, SoBSK1 and SoBSK4 were increased, but SoBSK6 and SoBSK7 were decreased in variety Sp75 under most conditions (Fig. 7). These indicated that, although the seven SoBSKs were all involved in BL- response, most SoBSKs, except of SoBSK6 and SoBSK7, were BL- induced in both Sp73 and Sp75 at the early stages (2 and 4 h) of BL treatment, and most SoBSKs exhibited significantly different even opposite responses in Sp73 and Sp75 under 24 h of BL treatment (Fig. 7). Interestingly, no SoBSKs were obviously ABA- induced in Sp73, and most of them were reduced in leaves under various exogenous ABA treatment conditions (Fig. 7). On the contrary, in Sp75, most SoBSKs in leaves were ABA- induced at 2 and 4 h, but ABA- reduced at 24h (Fig. 7). All these suggested that heat- sensitive variety Sp73 and heat- tolerant variety Sp75 probably employed different BSK- mediated mechanisms in response to exogenous BR and ABA treatments.

2.10 Temperature stress- response of SoBSKs in leaves

Temperature extremes inhibit seed germination and reduce plant growth and reproduction [48]. We have predicted that 29 heat- responsive cis-acting elements and two cold- responsive cis-acting elements existed in SoBSKs promoters (Fig. 4). To evaluate the function of these SoBSKs in temperature response, the expression profiles of SoBSK genes in the heat- sensitive variety Sp73 and heat- tolerant variety Sp75 under heat and cold stresses were validated using RT-qPCR analysis. Most SoBSKs were significantly altered in both varieties under certain temperature stress condition when compared with the normal condition (0 h) (Fig. 8). Importantly, the opposite temperature- responsive expression patterns of most SoBSKs were observed in varieties of Sp73 and Sp75. Five SoBSKs, except of SoBSK1 and SoBSK6, were reduced in Sp73, but induced in Sp75 at 4 and 12 h of cold and heat stresses (Fig. 8). Under heat conditions, SoBSK2 and SoBSK4 were obviously decreased in Sp73, but SoBSK5 and SoBSK7 were increased at 12 and 24 h. However, five SoBSKs (i.e. SoBSK2, SoBSK3, SoBSK4, SoBSK5, and SoBSK7) were heat- induced in Sp75 under each time point, which was represented by the significantly heat- induced SoBSK2 (Fig. 8). The similar expression patterns were found under cold conditions. Five SoBSKs (i.e. SoBSK2, SoBSK3, SoBSK4, SoBSK5, and SoBSK7) were cold- increased in Sp75, but cold- decreased in Sp73, while two SoBSKs (i.e. SoBSK1 and SoBSK6) were slightly induced in Sp73, but reduced in Sp75 (Fig. 8).

2.11 Heat- responsive genes in BR- and ABA- signaling in leaves

The expression levels of six BR signaling- related genes and 16 ABA signaling- related genes were detected in leaves from variety Sp73 and variety Sp75 under heat treatments for 0, 2, 4, 12, and 24 h using RNA-sequencing assay (Fig. 9). Two genes (BRL2 and BSK2) for BR signal perception were slightly heat- induced in variety Sp75 at 2 and 4 h, but significantly decreased in variety Sp73 at 12 and 24 h. However, the BKI1 for inhibition of BR binding with BRI1 was heat- reduced in Sp73 and Sp75 at 12 and 24 h (Fig. 9). Besides, three genes encoding transcription factors in the BR- mediated signaling pathway, including BZR1, BEH2, and BEH4, were induced in variety Sp75 under heat treatment (Fig. 9). This implied that BR signaling was probably employed in variety Sp75 for facilitating its heat tolerance.

The other 16 heat-altered genes were involved in ABA biosynthesis, transport, signal transduction, and transcriptional regulation (Fig. 9). Five genes for ABA biosynthesis, including two ZEPs, ABA4, NCED1, and NCED5, were reduced in variety Sp73, and four of them were also reduced in variety Sp75, except of NCED1 induced at 2 and 24 h (Fig. 9). Besides, ABCC2 for ABA transport from cytoplasm to vacuole was induced, but ABA efflux transporter ABCG25 and influx transporter ABCG22 for long-distance ABA transport were reduced in both Sp73 and Sp75 (Fig. 9). These indicated that the biosynthesis and transport of ABA were heat- disturbed in spinach, while some specific pathways probably were enhanced in heat- tolerant variety Sp75. Significantly, the expressions of four ABA receptor encoding genes (i.e. PYR1, PYL4, PYL5, and PYL9) were changed under heat treatment. Among them, PYL5 was significantly heat- induced in variety Sp75 and Sp73, and PYL4 was induced in Sp75 at 2 and 4 h (Fig. 9). Interestingly, PP2C24 and PP2C51, the negative regulation factor of ABA signaling, were obviously reduced in Sp75 under heat stress. Besides, the expressions of transcription factor ABI5 and AREB3 were altered, and AREB3 was increased in both Sp73 and Sp75 under heat treatments (Fig. 9). All these implied that variety Sp75 exhibited more enhanced ABA signaling to regulate heat- responsive process when compared with variety Sp73.

2.12 Heat-responsive phenotypes of SoBSK1-overexpressing Arabidopsis plants

The SoBSK1 expression pattern was distinct with other SoBSKs in heat- tolerant variety Sp75 in response to heat treatment (Fig. 8). To investigate the function of SoBSK1 on heat tolerance, we constructed overexpressing SoBSK1 transgenic Arabidopsis plants under the control of the strong constitutive CaMV 35S promoter, and compared the survival rates of wild-type (WT), bsk134678 mutant, and SoBSK1- overexpressed seedlings under heat treatment (Fig. 10). Three- day- old WT, bsk134678 mutant, and SoBSK1- overexpressed seedlings grew under 22 ℃ for five days as control, or treated under 43 ℃ for 4 h, and following under 22 ℃ for five days as heat treatments (Fig. 10A and B). There were no differences among WT, bsk134678 mutant, and SoBSK1- overexpressed seedlings under control condition, but they exhibited obvious different phenotypes under heat treatments (Fig. 10A). The bsk134678 mutant seedlings showed higher survival rate (92%), which was about 3.7- fold and 4.7- fold higher than WT and SoBSK1- overexpressed seedlings, respectively (Fig. 10A and C). While WT and SoBSK1- overexpressed seedlings exhibited similar phenotype, and no significantly different survival rate was observed (Fig. 10C). These implied that SoBSK1 probably regulated plant heat tolerance as a negative factor and the members of BSK family had function redundance.

3.1 The SoBSKs had close homology with sugar beet BvBSKs

The BSKs belonged to the receptor-like cytoplasmic kinase superfamily RLCK-XII, which were firstly identified in A. thaliana [11]. Recently, the genome- wide identification of 143 BSKs from 17 representative plant species has been reported [17]. Besides, the expressional patterns of some members of BSKs were investigated in wild barley [36], Kentucky bluegrass [37], and P. tomentosa [38] in response to drought and cold, respectively. However, only a few of BSKs were cloned and characterized in plants, such as AtBSK1, AtBSK2, AtBSK3, and AtBSK5 in Arabidopsis [11, 18, 21-30, 33, 40], OsBSK1-2 and OsBSK3 in rice [15, 16], NsBSK1 and NsBSK3 in woodland tobacco [42], NtBSK2 in common tobacco [42], and ZmBSK1 in maize [35]. The phylogenetic and functional analyses of BSKs from green vegetables, such as in spinach, have not been conducted so far.

In this study, seven SoBSK genes were identified in the genome and cloned from spinach variety Sp75. These SoBSKs, together with other 65 BSKs from Arabidopsis, sugar beet, common tobacco, woodland tobacco, rice, and maize, were grouped into seven classes in the NJ phylogenetic tree (Fig. 1). Our results indicated that seven SoBSKs were closely homologous with BvBSKs from sugar beet, which was consistent with the results of whole- genome comparative analysis [1]. Sugar beet was another Caryophyllales species, which genome was relative smaller (760 Mb) and closely related to spinach [1]. Moreover, the predicted number of protein-coding genes (25,495), the average length of the coding sequences (1,157 bp), the number of average exons (5.3), and the number of transcription factors (1,202) in spinach genome were all similar with those in sugar beet genome [1]. Therefore, BSK was a well representative gene family revealing the close relationship between spinach and sugar beet.

In addition, our results indicated that the BSKs from dicotyledons (i.e. spinach, sugar beet, Arabidopsis, common tobacco, and woodland tobacco) and monocotyledons (i.e. rice and maize) were classed into separated subgroups in each class (Fig. 1), implying the lineage- specific evolution of BSK genes after the divergence of dicots and monocots. This was also found in the analysis of the genome- wide identification of 143 BSKs from 17 plant species, although spinach, common tobacco, woodland tobacco, and sugar beet were not included in [17]. Interestingly, the expansion of BSK gene family attributed to the whole genome duplication (WGD) in some of the previously reported 17 plant species, such as two- fold expansion in Arabidopsis (12 BSKs), soybean (Glycine max) (13 BSKs), and Populus trichocarpa (14 BSKs), as well as three- fold expansion in Brassica rapa (21 BSKs) [17], but the WGD events were not observed in spinach (7 BSKs) [1].

3.2 SoBSKs had conserved TPR domain and kinase domain

Seven SoBSKs had conserved TPR domain at the C-terminus, including two TPRs in SoBSK2 and SoBSK3, and three TPRs in the other five SoBSKs (Fig. 3G). The conserved TPRs were also existed in the BSKs from Arabidopsis [17] and rice [16]. TPR domain functioned to facilitate the specific interaction between partners acting as scaffolds for the assembly of multiprotein complexes [21, 49]. The TPR in rice OsBSK3 was proved to an autoregulatory domain as a “phospho-switch” for modulating BSK kinase activity [16]. The TPR bound to the kinase domain of BSK3 for the prevention of BSK3 from binding to and activating BSU1 without BR signals, while the phosphorylated kinase domain of BSK3 by BR- activated BRI1 was free from the binding with the TPR domain, and then facilitated the binding affinity of BSKs for downstream phosphatase BSU1 to promote BR signaling pathway [16]. It has been found that TPR domain was critical for several BSK’s functions in embryogenesis and defense responses [16, 26, 50]. In Arabidopsis, the missense mutation of the strictly conserved Arginine (R) site R⁴⁴³ (R443Q) in the TPR domain led to the loss-of-function of AtBSK1, conferring the defective defense responses [26]. Moreover, the TPR deletion of SSP/ BSK12 completely inactivated its function, resulting in the defective suspensor development upon Arabidopsis embryogenesis [50]. In rice, the TPR domain negatively regulated OsBSK3 activity in BR signaling [16]. On the contrary, the truncated AtBSK3 protein without TPR motifs (BSK3^TPR-Δ) was less efficient to rescue the Arabidopsis root growth defect and BR resistance phenotype of bsk3-1 when compared with BSK3 protein, although it can fully complement the bsk3-1 mutant phenotypes. This indicated that TPR domain was not essential, but required for the full function of AtBSK3 in BR signaling [21]. Additionally, TPR deletion impaired the interactions of AtBSK3-AtBSK3 and AtBSK3-AtBSK1, implying its regulation to BSK homodimer and/ or heterodimer formation [21]. Interestingly, all the seven SoBSKs had the conserved TPR domain and R residue inside (Fig 3G), which functions remained to be elucidated.

All SoBSKs contained conserved phosphorylation site of serine in the kinase domain, except of SoBSK6 (Fig 3G). Their homologous serine sites in AtBSKs and OsBSKs were also conserved and critical for their kinase functions [11, 16, 26, 41]. The S²³⁰ in AtBSK1, S²¹⁰ in AtBSK3, S²⁰⁹ and T²¹⁰in AtBSK5, S²¹⁵ in OsBSK3, as well as the adjacent S, T and Tyrosine (Y) residues in AtBSK6 and AtBSK8 have been reported to be phosphorylated by BRI1, RLK902, PEPR1, and ELONGATION FACTOR-TU RECEPTOR (EFR) and functioned in the BR signaling pathway upon plant immunity, respectively [11, 16, 18, 29]. Therefore, these conserved phosphorylation site in SoBSKs would be pivotal for their functions in spinach development and stress response. Additionally, in the kinase domain, a lysine in six SoBSKs except of SoBSK6 were conserved, and two lysines (K¹⁰⁰ in SoBSK1 and K¹⁰⁴ in SoBSK5) were predicted to be ubiquitinated (Fig 3G). The conserved lysine in six SoBSKs were homologous with K¹⁰⁴ in AtBSK1 [26], K⁸⁶ in AtBSK3 and AtBSK5 [18, 21], as well as K⁸⁷ in AtBSK8 [51], which have been proved to the key sites of ATP binding for BSK kinase activity. However, the regulation role of this ubiquitination site in kinase domain for balancing the BSK degradation and its kinase activity need to be investigated. Additionally, the conserved glycine sites in all SoBSKs (Fig 3G) were homologous with G²³⁸ and G²²⁶, which were found to interfere the protein stability of AtBSK3 and other BSKs in BR signaling, respectively [21].

3.3 SoBSKs played important roles in spinach development and heat response

BSKs represent a critical kinase family that play partially redundant or overlapping roles in BR signaling and/ or other kinase signaling pathways during plant development and stress response [11, 20]. In spinach, different SoBSK members displayed a constitutive and unique expression pattern in roots, stems, and leaves from variety Sp73 and variety Sp75, and most of them had higher expression in developing leaves (i.e. the 3^rd and 2^nd leaves) than that in the mature leaves (the 1^stleaves) (Fig. 6), which implied SoBSKs were important for spinach growth and development. The similar expression patterns of BSKs were also found in Arabidopsis. Twelve AtBSKs were expressed in multiple tissues and organs (i.e. roots, flowers, rosette leaves, mature pollen, and seeds) during various developmental stages [17]. Phenotype analyses of various single, double, and multiple mutations of each AtBSK indicated that BSKs were partially redundant positive regulators of BR signaling in Arabidopsis [20]. Besides, the complex genetic interactions between BSKs (e.g., homodimers and/ or heterodimers) and their downstream proteins were supposed to the possible reasons of distinct functional characteristics for BSKs in diverse physiological and developmental processes [20].

In our results, most SoBSKs were BL- induced in leaves from both heat- tolerant variety Sp73 and heat- sensitive Sp75 (Fig. 7), which indicated that most SoBSKs were involved in the BR signaling pathway, and 2 to 4 h of BL treatments would be the critical timepoint for BR signal perception and original transduction in two spinach varieties. Importantly, several SoBSKs (i.e. SoBSK2, SoBSK5, SoBSK6, and SoBSK7) were significantly ABA- induced in leaves from heat- tolerant variety Sp75, but ABA- reduced in heat- sensitive variety Sp73 under certain timepoints (Fig. 7). Moreover, SoBSK1 and SoBSK3 were highly heat- induced in variety Sp75 when compared with that in variety Sp73 (Fig. 7). This implied that heat- tolerant variety Sp75 potentially had stronger stress tolerance depending on ABA signaling pathway than heat- sensitive variety Sp73. Consistently, some AtBSKs were also significantly altered in response to various exogenous hormones (i.e., BL, ABA, JA, GA-3, Auxin, Zeatin, and 1-aminocyclopropane-1-carboxylic acid (ACC, ethylene precursor)) [17], implying that BSKs- mediated pathways had cross-talks with other hormone signaling pathways.

Importantly, our results indicated that the expression patterns of SoBSKs in leaves were altered under certain heat- and/ or cold- stress conditions, and most SoBSKs increased obviously in heat- tolerant variety Sp75 than those in heat- sensitive variety Sp73 (Fig. 8). Taken together with the ABA and drought responses of BSKs from Arabidopsis (i.e. AtBSK3, AtBSK5, and AtBSK8) and maize (ZmBSK1) [17, 24, 35], we supposed that BSKs were involved in the heat response via regulation of ABA- mediated stomatal closure and ROS homeostasis [24, 52]. Thermotolerance assay of WT, bsk134678 mutant, and SoBSK1- overexpressed Arabidopsis seedlings suggested that SoBSKs participated in response to heat treatments redundantly, and SoBSK1 could be a negative regulator under heat conditions (Fig. 10). Besides, their homologous AtBSKs were also significantly altered under heat and cold conditions. Most AtBSKs were reduced under heat stress, but AtBSK1, AtBSK2 and AtBSK9 were induced in Arabidopsis under cold stress after 24 h [17]. Similarly, in Populus tomentosa, PtBSK, PtBRI1, and PtBIN2 were all increased under cold stress [38].

Additionally, the BSKs- mediated cross-talks of BR- signaling pathway and other pathways (e.g., ABA signaling, auxin signaling, ROS signaling, and MAPK cassette) were reported to function in diverse environment responses, such as salts [24, 25, 47, 53, 54], drought [17, 35], low nitrogen [22], exogenous sucrose supply [41], as well as oxidative stress and wounding [17]. In this study, we evaluated the important gene expression in BR signaling and ABA signaling in spinach variety Sp75 and Sp73 under heat stress using RNA-seq analysis. Our results indicated that heat- tolerant variety Sp75 was likely to improve heat resistance by promoting BR- mediated downstream pathways through BR signaling transcription factors (i.e. BZR1, BEH2, and BEH4), and activating ABA signaling through regulating PYL4 and PP2Cs (Fig. 9). However, the key members and functions of BSKs in these fine-tuned modulation mechanisms, as well as the cross talk of BR and ABA signaling still needs to be investigated.

4.1 Identification and cloning of BSKs genes in spinach

To identify the BSK family in spinach, the genome was downloaded from the SpinachBase (http://www.spinachbase.org/). We used 12 AtBSK protein sequences as queries to search against the spinach genome database using BLASTp with an expected value (e-value) cutoff of 1E^-100. Candidate SoBSKs were submitted to the NCBI web CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to verify their conserved kinase domain and TPR domain, and their coding sequences (CDSs) were cloned with the SoBSKs primers (Additional file 5). The CDS and protein sequences of SoBSKs were shown in Table S1.

4.2 Phylogenetic analysis of BSKs from different species

The phylogenetic tree of BSKs in spinach and other six plant species (i.e. sugar beet, Arabidopsis, common tobacco, woodland tobacco, maize, and rice) were constructed with MEGA-X by the NJ method and 1,000 bootstrap replicates. Herein, 12 AtBSKs [11], five OsBSKs [15, 16], two NsBSKs [42], one NtBSK [42], and nine ZmBSKs [35] were reported previously, and all the other protein sequences of BSKs were obtained by BLSATp with AtBSKs as queries to search against the genome databases, including RefBeet-1.2.2 (https://bvseq.boku.ac.at/index.shtml), Ntab-TN90 (https://www.ncbi.nlm.nih.gov/data-hub/taxonomy/4097/), Nsyl (https://www.ncbi.nlm.nih.gov/data-hub/taxonomy/4096/), and Zm-B73-REFERENCE-NAM-5.0 (https://www.ncbi.nlm.nih.gov/data-hub/taxonomy/4577/) for sugar beet, common tobacco, woodland tobacco, and maize, respectively.

4.3 Chromosomal distribution and collinearity analysis of SoBSK genes

The position information (e.g., chromosomal distribution, length, as well as the start and end positions) of SoBSK genes on the chromosome were obtained from SpinachBase, and visualized by MapGene2Chrom web v2 (http://mg2c.iask.in/mg2c_v2.0/). The collinearity analysis of BSKs between the homologs in spinach and Arabidopsis, sugar beet, or maize were verified and visualized using One Stem MCScanX and Dual Systeny Plot for MCScanX in TBtools software, respectively.

4.4 Analyses of gene structure, function domain, conserved motif, and post-modification

The exon-intron structure was analyzed based on the full-length genome sequences and the CDSs of SoBSKs by TBtools. Functional domains of SoBSKs were detected by the NCBI web CD-search tool. The conserved motifs of SoBSKs were determined by MEME (https://meme-suite.org/meme/tools/meme) using the protein sequences with the maximum motif number of 13, and were visualized by TBtools. The conserved phosphorylation, myristoylation, and reported conserved functional sites in SoBSKs were analyzed by sequence alignment with previous researches in Arabidopsis [11, 18, 21, 26, 27, 29, 41] and rice [16]. The S-nitrosylation and ubiquitination sites were predicted by GPS-SNO 1.0 [43] with default parameters and UbiComb (http://nsclbio.jbnu.ac.kr/tools/UbiComb/) with a threshold value of 0.5 [44], respectively.

4.5 Identification of putative cis-regulatory elements in the promoters

The promoter sequences (2,000 bp before the start codon) of all SoBSKs were extracted from the SpinachBase, and were analyzed by using the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and visualized by TBtools.

4.6 Plant materials, RNA extraction, cDNA synthesis, and RT-qPCR

The spinach heat- sensitive variety Sp73 and heat- tolerant variety Sp75 were used for gene expression analysis. The seeds were sown in a tray containing a perlite-matrix (1:1) mixture and grown in a plant growth chamber. The growth conditions were set as 22 ℃ 10 h light/ 18 ℃ 14 h dark and 60% relative humidity [4]. The expression levels of SoBSK genes in various tissues including roots, stems, leaves (i.e. the 1^st, 2^nd, and 3^rd leaves) were detected. Temperature stresses (4 ℃ for cold and 37 ℃ for heat) and exogenous hormone treatments (10 μM BL and 100 μM ABA) were applied for 0, 2, 4, 12, and 24 h with six- leaf stage of seedlings, respectively.

Total RNA was extracted from spinach leaves with TRIzol™ LS Reagent (Invitrogen, USA) [55]. The RNA was reverse transcribed into cDNA using the Prime Script RT reagent kit. The RT-qPCR was performed using the SYBR Premix ExTaq II kit (TRAN, China) with SoARF as an internal control. Specific primer pairs were designed using Primer3 Web tool (http://bioinfo.ut.ee/primer3/) (Additional file 5). RT-qPCR were performed with three independent biological replicates. Each gene was normalized to the SoARF internal control gene, and the relative gene expression was calculated according to the 2^-ΔΔCt method [56].

4.7 Differentially expressed genes (DEGs) analysis by RNA-sequencing

Total RNA was extracted from leaves of the spinach heat- sensitive variety Sp73 and heat- tolerant variety Sp75 after heat treatments at 0, 2, 4, 12, and 24 h using the mirVana miRNA Isolation Kit (Ambion). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The samples with RNA integrity number (RIN) ≥ 7 were subjected to enrich mRNA and construct cDNA libraries using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, USA) according to the manufacturer’s instructions. The libraries were sequenced on the HiSeq 2500 platform (Illumina, USA). The high- quality clean reads were obtained by removing adaptor sequences, empty reads low quality bases (Q < 30) and used for transcriptome de novo assembly by mapping to spinach reference genome using hisat2 [1].

Fragments Per Kilobase of transcript per Million (FPKM) of each gene and read counts value of each transcript (protein_coding) were calculated using bowtie2 and eXpress. Differential expression analysis among the samples was performed using the DESeq (2012) R package. The genes with p value < 0.05 and Fold Change >2 (or < 0.5) across the heat- stressed and control (0 h in heat- sensitive variety Sp73 or heat- tolerant variety Sp75) samples in at least two replicates were designated as DEGs in each variety.

4.8 Subcellular localization of SoBSK1 and SoBSK6

The open reading frames (ORFs) of SoBSK1 and SoBSK6 without stop codon were introduced into the plant expression vector pCAMBIA2300-GFP to produce SoBSK1-GFP and SoBSK6-GFP fusion proteins under the control of the CaMV 35S promoter. The resulting constructs (i.e. pCAMBIA2300-SoBSK1-GFP and pCAMBIA2300-SoBSK6-GFP) and the empty vector pCAMBIA2300-GFP were introduced into Agrobacterium tumefaciens stain EHA105. 35S::SoBSK1-GFP, 35S::SoBSK6-GFP, and 35S::GFP (the vector control) were introduced into six- week- old tobacco leaves by infiltration using needle-less syringes [57]. The transformed tobacco seedlings grew for 24 h in dark, and following under normal conditions (25 ℃ 16 h light/ 20 ℃ 8 h dark) for 24 h. The GFP signals in transformed tobacco epidermal cells were visualized under an Olympus FV3000 confocal laser scanning microscope (Olympus, Japan).

4.9 Thermotolerance analysis of transgenic Arabidopsis

The A. tumefaciens stain EHA105 cells containing pCAMBIA2300-SoBSK1-GFP plasmid were transformed into A. thaliana WT seedlings using the floral dip method [58] to obtain SoBSK1- overexpressed seedlings. The transformed seedlings were screened on 1/2 MS agar medium containing 50 μg/mL kanamycin for three generations. The SoBSK1 expression levels of several independently homozygous T3 lines were evaluated by RT-qPCR with gene-specific primers (Additional file 5), and the highest expressed line were selected for thermotolerance analysis. WT, bsk134678 mutant, and SoBSK1- overexpressed A. thaliana seedlings were grown on 1/2 MS medium in a light incubator (16 h/ 8 h of light/dark, 22 °C/ 20 °C day/ night, and 75% relative humidity) for eight days were control. The heat- treated seedlings were grown under control conditions for three days, then 43 ℃ for 4 h, and following under control conditions for five days. After treatments, the photographs were taken and the survival rates of seedlings from WT, bsk134678 mutant, and SoBSK1-overexpressed A. thaliana was calculated with three biologically independent replicates.

ABA: Abscisic acid; ABA3: ABA deficient3; ABA4: ABA deficient4; ABCC: ABC transporter C family member; ABCG: ABC transporter G family member; ABI5: ABSCISIC ACID-INSENSITIVE 5; ABRE: ABA responsive element; ACC: 1-aminocyclopropane-1-carboxylic acid; AREB3: ABRE binding factor3; BAK1: BRI1-ASSOCIATED RECEPTOR KINASE1; BEH: bri1 EMS SUPPRESSOR1/ BRASSINAZOLE RESISTANT1 homolog protein; BES1: bri1 EMS SUPPRESSOR1; BIN2: BRASSINOSTEROID INSENSITIVE2; BIR1: BAK1-INTERACTING RECEPTOR-LIKE KINASE1; BKI1: BRI1 kinase inhibitor 1; BL: brassinolide; BR: Brassinosteroid; BRI1: BR INSENSITIVE1; BRL2: BRI1-like 2; BSK: BR SIGNALING KINASE; BSU1: BRI1 SUPPRESSOR 1; BZR1: BRASSINAZOLE RESISTANT1; CDS: Coding sequence; DEG: Differentially expressed gene; DRE: Dehydration- responsive element; EFR: ELONGATION FACTOR-TU RECEPTOR; ERE: Ethylene responsive element; ETI: Effector-triggered immunity; e-value: Expected value; FLS2: FLAGELLIN SENSING2; FPKM: Fragments Per Kilobase of transcript per Million; G: Glycine; GA: Gibberellin acid; GE: glucosyl ester; JERE: jasmonate and/or elicitor responsive element; K: Lysine; LYK5: LYSIN-MOTIF RECEPTOR KINASE5; MAPK: Mitogen-activated protein kinase; MAPKKK5: Mitogen-activated protein kinase kinase kinase5; MBS: MYB-binding site; MeJA: Methyl jasmonate; MRS: MYB recognition site; MYB: v-myb avian myeloblastosis viral oncogene homolog; MYC: Myelocytomatosis; NCED: 9-cis-epoxycarotenoid dioxygenase; ORF: Open reading frame; PAMP: Pathogen-associated molecular pattern; PEPR1: PEP1 RECEPTOR1; PIF4: BES1-PHYTOCHROME INTERACTING FACTOR4; PM: Plasma membrane; PP2C: Protein phosphatase 2C; PTI: Pathogen-associated molecular pattern- trigged immunity; PTM: Post-translational modification; PYL: PYRABACTIN RESISTANCE 1-LIKE; PYR: PYRABACTIN RESISTANCE; R: Arginine; RIN: RNA integrity number; RLCK: Receptor-like cytoplasmic kinase; RLK902: RECEPTOR-LIKE KINASE 902; ROS: Reactive oxygen species; RPS2: Ribosomal protein S2; RT-qPCR: Reverse transcription quantitative real-time polymerase chain reaction; S: Serine; SA: Salicylic acid; SnRK: SNF1-related protein kinase; SOBIR1: SUPPRESSOR OF BIR1-1; SRF: STRUBBELIG-RECEPTOR FAMILY; SSP: SHORT SUSPENSOR; STRE: Stress response element; TPR: Tetratricopeptide repeat; WAKL: WALL-ASSOCIATED RECEPTOR KINASE-LIKE; WGD: Whole genome duplication; WT: Wild-type; Y: Tyrosine; ZEP: Zeaxanthin epoxidase.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the National Natural Science Foundation of China (No. 32070300); the Fund of Central Government Guides Local Science and Technology Development, China (No. YDZX20203100003927); and the Fund of Shanghai Engineering Research Center of Plant Germplasm Resources, China (No. 17DZ2252700) to Shaojun Dai and by the National Nature Science Foundation of China (No. 31902012) and the Natural Science Foundation of Shanghai (No. 22ZR1445700) to Meihong Sun.

Author’s contributions

Shaojun Dai and Heng Zhang conceived and designed the experiments. Yang Li and Heng Zhang carried out experiments, data analysis, and wrote the draft. Yongxue Zhang and Haodong Tian participated in RT-qPCR, subcellular localization, and thermotolerance analysis. Yanshuang Liu and Yueyue Li participated in the bioinformatic analysis. Siyi Guo, Meihong Sun, and Zhi Qin helped to draft the manuscript. Heng Zhang and Shaojun Dai finalized the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Wenqiang Tang (Hebei Normal University) for providing the bsk134678 Arabidopsis mutant.

Author details

¹Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China. ²Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin 150040, China. ³State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng 475004, China.

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No competing interests reported.

Additionalfile1.xlsx
Additional file 1: Table S1. List of the 72 BSKs genes from spinach, sugar beet, Arabidopsis, rice, maize, common tobacco, and woodland tobacco.
Additionalfile2.xlsx
Additional file 2: Table S2. List of BSK homologous gene pairs identified in spinach vesca Arabidopsis, sugar beet, and maize.
Additionalfile3.docx
Additional file 3: Figure. S1 Sequence logos of thirteen motifs in SoBSKs. The height of each amino acid represented the relative frequency of the amino acid at that position.
Additionalfile4.xlsx
Additional file 4: Table S3. The promoter cis-acting elements of SoBSK genes.
Additionalfile5.docx
Additional file 5: Table S4. List of primers used in cloning and RT-qPCR analysis for SoBSKs.

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Genome-wide identification and expression analysis reveals spinach brassinosteroid-signaling kinase (BSK) gene family functions in temperature stress response

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