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. 2022 Apr 25;235(2):563–575. doi: 10.1111/nph.18140

LATERAL BRANCHING OXIDOREDUCTASE, one novel target gene of Squamosa Promoter Binding Protein‐like 2, regulates tillering in switchgrass

Ruijuan Yang 1,2,*, Wenwen Liu 1,*, Ying Sun 1, Zhichao Sun 1, Zhenying Wu 1,2, Yamei Wang 1, Mengqi Wang 1, Honglun Wang 3, Shiqie Bai 4, Chunxiang Fu 1,2,3,✉
PMCID: PMC9321131  PMID: 35383390

Summary

  • Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late‐acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual‐purpose fodder and biofuel crop.

  • We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild‐type and miR156 overexpressing (miR156OE) switchgrass. Co‐expression analysis, quantitative real‐time polymerase chain reaction (qRT‐PCR), transient dual luciferase assay, and chromatin immunoprecipitation‐qPCR were all used to determine the activation of PvLBO by miR156‐targeted Squamosa Promoter Binding Protein‐like 2 (PvSPL2) in regulating tillering of switchgrass.

  • PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2.

  • We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156‐SPL module in switchgrass.

Keywords: LATERAL BRANCHING OXIDOREDUCTASE, miR156, SPL transcription factor, strigolactones, switchgrass, tillering

Introduction

Strigolactones (SLs) are a class of carotenoid‐derived plant hormones that primarily function as germination stimulants for root parasitic plants, symbiotic signals for fungi, and inhibitors of shoot branching/tillering (Cook et al., 1966; Akiyama et al., 2005; Gomez‐Roldan et al., 2008). Strigolactones are synthesized from all‐trans‐β‐carotene by 9‐cis‐β‐carotene isomerase DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7/D17/MAX3, MORE AXILLARY GROWTH 3), and CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8/D10/MAX4). These enzymes are known to convert β‐carotene to carlactone (CL), which is then converted to carlactonoic acid (CLA) by the cytochrome P450 enzyme MORE AXILLARY GROWTH 1 (MAX1) (Booker et al., 2005; Zhang et al., 2014). The conversion of all‐trans‐β‐carotene to CLA is highly conserved and is generally considered to be an important step in SL biosynthesis (Yoneyama et al., 2018). However, the structures of SLs produced by subsequent steps after CLA synthesis vary across plant species (Yoneyama & Brewer, 2021). Compounds with an ABC‐ring system, such as 4‐deoxyorobanchol (4DO), 5‐deoxystrigol (5DS), and orobanchol (ORO), are called canonical SLs, while SLs such as methyl carlactonoate (MeCLA) and hydroxymethyl carlactonoate (1′‐HO‐MeCLA) without typical ABC‐rings belong to noncanonical SLs (Yoneyama et al., 2009; Omoarelojie et al., 2019).

Arabidopsis LATERAL BRANCHING OXIDOREDUCTASE(LBO) encodes a 2‐oxoglutarate‐dependent dioxygenase that oxidizes MeCLA to 1′‐HO‐MeCLA to act during the final steps of noncanonical SLs biosynthesis (Brewer et al., 2016). Although most MeCLA is converted to CLA by LBO in vitro, 1′‐HO‐MeCLA is indeed present as an endogenous compound in Arabidopsis (Yoneyama et al., 2020). Moreover, CL and MeCLA can accumulate in the roots of Arabidopsis lbo mutants, and the morphological characterizations induced by MeCLA accumulation are intermediate between the wild‐type Ws‐4 and the max4 mutant plants. Overexpression of LBO in Ws‐4 plants cannot reduce branching (Brewer et al., 2016). While the 1′‐HO‐MeCLA is highly unstable and difficult to detect in plants, the enzyme activity analysis of LBO recombinant proteins in vitro suggests that the conversion of MeCLA to 1′‐HO‐MeCLA is conserved in Arabidopsis, tomato, maize, and sorghum (Yoneyama et al., 2020). Nevertheless, it remains unclear whether LBO regulates tiller bud outgrowth or shoot architecture in monocotyledons, especially perennial grasses.

Strigolactones are perceived by an α/β hydrolase, DWARF14 (D14) in rice (Chevalier et al., 2014). Upon SL perception, D14 forms a complex with an F‐box component DWARF 3 (D3) of the SCF E3 ubiquitin ligase complex and a repressor DWARF 53 (D53) of SL signaling, triggering the ubiquitination and degradation of D53, resulting in transcriptional changes of downstream genes (Zhou et al., 2013). A similar interaction has been confirmed in Arabidopsis for the orthologs of D14, D3, and D53 (Zhou et al., 2013). During this reaction, D14 is also degraded via proteolysis in response to SLs (Chevalier et al., 2014). Other SL‐responsive genes also have been identified as well, including SL biosynthetic genes, MAX3, MAX4 and SMXL2, SMXL6, SMXL7, SMXL8, and anthocyanin biosynthetic activator genes, PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) and MYB113 in Arabidopsis (Wang et al., 2020); and the bud outgrowth repressor gene, TEOSINTE BRANCHED 1 (TB1) and cytokinin oxidase/dehydrogenase gene, CYTOKININ OXIDASE/DEHYDROGENASE 9 (CKX9) in rice (Lu et al., 2013; Duan et al., 2019).

Strigolactones have been shown to regulate plant branching, leaf shape and senescence, and root development (Braun et al., 2012; Waldie et al., 2014). Strigolactones can also regulate plant tolerance to drought, salt and phosphate starvation stresses as well as the biosynthesis of anthocyanin and flavonols (Mayzlish‐Gati et al., 2012; Waters et al., 2017; Wang et al., 2020). Aside from phytohormones, microRNA (miRNA) is another multi‐function factor involved in plant growth and development, and biotic and abiotic stress responses in plants. Among them, miR156 is one of the conserved master regulators of plant vegetative phase transitions by suppressing its target SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes (Wu & Poethig, 2006). Overexpression of miR156 or the repression of its target SPLs in different plant species can result in severe decrease of apical dominance and produce obvious morphological changes, including increased tiller and leaf numbers, altered inflorescence architecture, accumulated anthocyanin, and reduced lignin content (Chuck et al., 2007; Wang et al., 2008; Miura et al., 2010; Gou et al., 2011; Fu et al., 2012). OsTB1 is an important negative regulator controlling the outgrowth of axillary buds in rice (Takeda et al., 2003). Overexpression of IDEAL PLANT ARCHITECTURE 1 (IPA1, otherwise known as OsSPL14), a key gene repressed by miR156, can decrease rice tiller number through the transcriptional activation of OsTB1 (Lu et al., 2013). A previous study demonstrated that IPA1 and SLs seem to work independently to control rice tillering (Luo et al., 2012). However, recent studies indicate that D53 can interact with IPA1 and inhibit the transcriptional activation of IPA1. Furthermore, IPA1 binds to the promoter of D53, activates its expression, and plays a critical role in the feedback regulation of SL‐induced D53 degradation in bread wheat and rice (Liu et al., 2017; Song et al., 2017). Taken together, the earlier reports suggest that the miR156‐SPL module has a complex crosstalk with the SL signaling pathway during tillering.

Switchgrass (Panicum virgatum L.) is a C4 perennial tall grass in the Gramineae family and is native to North America. It is a dual‐purpose forage and bioenergy crop (Lovell et al., 2021). Tillering is one of the most important agronomic traits that determine the biomass yield of switchgrass. The CRISPR/Cas9‐induced mutation of PvTB1, one SL signaling pathway gene, can increase the tiller number and biomass yield of switchgrass (Liu et al., 2020). However, few SL biosynthetic or signaling genes in switchgrass have been identified and characterized. In addition, previous studies have demonstrated that overexpression of miR156 in switchgrass can significantly increase aerial axillary bud outgrowth, tiller number, and biomass yield (Chuck et al., 2011; Fu et al., 2012). Downregulation of miR156‐targeted PvSPL4 can elevate the expression of LAX PANICLE1 (LAX1) and MONOCULM1 (MOC1) and therefore increase the tiller number in switchgrass (Gou et al., 2017). Moreover, our previous study found that suppressing of miR156‐targeted PvSPL2 activity in switchgrass via the chimeric repressor gene silencing technology increases tiller number and reduces lignin accumulation as well (Wu et al., 2016). However, the direct downstream genes of PvSPL2 that are involved in switchgrass tillering remain largely unknown.

In this study, we found that PvLBO was significantly downregulated in the miR156 overexpressing (miR156OE) transgenic switchgrass plants, and that overexpression of PvLBO in the miR156OE transgenic line can reduce tiller number to even less than that of control switchgrass plants. Based on these results, we propose that PvLBO functions in miR156‐SPL mediated tillering regulation. Overexpression of PvLBO in switchgrass significantly reduced the tiller density, while downregulation of PvLBO led to a substantial increase in tiller number. Moreover, we demonstrate for the first time that PvSPL2 was able to bind to the PvLBO promoter directly, activate its transcription, and therefore repress switchgrass tillering. Therefore, our results indicate that PvLBO acts as a crucial downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156‐SPL2 module during the regulation of switchgrass tillering.

Materials and Methods

Plant growth conditions

A lowland‐type switchgrass cultivar, Alamo (2n = 4x = 36), was employed for genetic transformation and molecular and biochemical analysis. Plants were grown in a glasshouse under a 16‐h light photoperiod with supplementary lighting supplied by parabolic aluminized reflector lamps (390 µmol m−2 s−1). The temperature in the glasshouse ranged from 25 to 29°C. Plants were watered two times a week and fertilized with Peters N/P2O5/K2O‐20‐10‐20 fertilizer (J.R. Peters Inc., Allentown, PA, USA). Primary transgenic plants were propagated by transferring the same numbers of tillers into each pot. Three copies of each line were grown in three‐gallon pots. The development of switchgrass in the glasshouse was divided into three vegetative stages (V1, V2 and V3), five elongation stages (E1, E2, E3, E4 and E5) and three reproductive stages (R1, R2 and R3) according to previously outlined method (Hardin et al., 2013). The control and modified plants at E3 stage were trimmed back and then treated with 10 ml of 2 μM rac‐GR24 and 4 μM TIS108 (Coolaber, Beijing, China) or an equal volume of water every other 2 d. After 35 d, we took photographs and analyzed tiller numbers.

Gene expression quantification

Total RNA was extracted from different tissues and organs of switchgrass and were reverse transcribed into complementary DNA (cDNA) after treatment with genomic DNA (gDNA) Eraser. Total RNAs SYBR Premix ExTaq™ (Takara, Dalian, China) was employed for quantitative real‐time polymerase chain reaction (qRT‐PCR). Mature miR156 levels were detected and quantified using a highly sensitive qRT‐PCR method (Varkonyi‐Gasic et al., 2007). The primers used for qRT‐PCR are listed in Supporting Information Table S1.

Identification and cloning of PvLBO

PvLBO was identified through searching the switchgrass genome database v.4.1 (http://www.phytozome.org/) using the AtLBO sequence (Table S2). Alignment of multiple sequences and phylogenetic tree analysis of PvLBO and its orthologs in Arabidopsis thaliana, Medicago  truncatula, Populus  trichocarpa, Oryza  sativa and Zea mays were conducted using megax software suite (http://www.megasoftware.net). The full‐length sequence cDNA of PvLBO was isolated from switchgrass stem by PCR and was subjected to sequencing.

Generation of transgenic switchgrass plants

The overexpressing vectors of pANIC6A‐PvLBO and pANIC6D‐PvLBO and the RNA interference (RNAi) vector of pANIC8B‐PvLBO were constructed as described (Mann et al., 2012). These vectors were individually transformed into Agrobacterium  tumefaciens strain EHA105. A high‐quality embryogenic callus line with a single genotype was established by the large‐scale screening of switchgrass Alamo seed‐induced calli and was used for Agrobacterium‐mediated transformation following the procedure described by Xi et al. (2009). To produce double transformed switchgrass lines, the calli induced from inflorescences of the selected TmiR156OE‐33 transgenic line (Fu et al., 2012) was used to retransform the pANIC6D‐PvLBO construct into the miR156 overexpression background.

Transcriptomic analysis

RNA‐sequencing (RNA‐Seq) was performed using an Illumina Novaseq™ 6000 (LC Sciences, Houston, TX, USA). Transcripts were assembled using hisat2 software and quantified using StringTie (https://ccb.jhu.edu/software/stringtie/gffcompare.shtml). gffcompare software was used to construct a comprehensive transcriptome. The differentially expressed genes (DEGs) were selected for gene ontology (GO) enrichment analysis. DEGs from the RNA‐Seq data were used for GO category enrichment analysis. Twenty enriched pathways of DEGs were performed.

Determination of carotenoids and chlorophyll contents

The leaf blade and leaf sheath of internode 4 were harvested from transgenic and control switchgrass plants at the E5 stage, and ground in liquid nitrogen. The total contents of carotenoid, chlorophyll a, and chlorophyll b were examined as described (Sumanta et al., 2014).

Total lignin content analysis

Stems of the control and PvLBOOE transgenic switchgrass plants at the E5 stage were harvested. The resulting samples were ground in liquid nitrogen and lyophilized. Lyophilized extractive‐free cell wall residue (CWR) was used for lignin analysis. The AcBr method was employed to quantify total lignin content (Hatfield et al., 1999).

Luciferase (LUC) reporter assay

The vectors of pCAMBIA1300‐rPvSPL2 and pCAMBIA1300‐rPvSPL4 were transformed into EHA105 and used as effectors. These promoter sequences are listed in Table S3. PvLBOpro::LUC, mPvLBOpro::LUC, S1::LUC, and S2::LUC were transformed into the EHA105 cells using the helper plasmid pSoup19. In addition, the PvD53 promoter, with five GTAC motifs, was amplified by PCR and cloned into the pGreenII 0800‐LUC and transformed with pSoup19 as a reporter. The transformed Agrobacterium cultures were individually infiltrated into different regions of the same Nicotiana benthamiana leaves (Thompson et al., 1991; Hellens et al., 2005). The treated leaves were analyzed for luciferase (LUC) fluorescence activity assay.

Chromatin immunoprecipitation‐quantitative polymerase chain reaction (ChIP‐qPCR) assay

The ORF sequence of rPvSPL2 was amplified, fused to an eGFP sequence, and cloned into the pANIC6B vector. Transgenic switchgrass lines overexpressing pANIC6B‐rPvSPL2‐eGFP were selected for chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR) assays using the previously described methods (Gendrel et al., 2005). Briefly, 1 g leaf tissue was harvested from each 4‐wk‐old seedling and ground in liquid nitrogen. The samples were individually crosslinked using 1% (v/v) formaldehyde under the vacuum for 5 min. Chromatin complexes were isolated from individual samples, sonicated for 30 s, 10 times (left on ice for 30 s between each sonication), and then incubated in an anti‐green fluorescent protein (anti‐GFP) polyclonal antibody solution (632569; Takara). The recovered DNA from ChIP and 10% input DNA control was then used for qPCR analyses.

Yeast one‐hybrid (Y1H) and yeast two‐hybrid (Y2H) assays

The full‐length cDNAs of rPvSPL2 and rPvSPL4 were fused to the pGADT7 (AD) vector respectively. The sequence fragments of S1 and S2 described earlier were individually fused to the pHIS2.1 vector. The construct groups were transformed into the yeast strain Y187. The yeast one‐hybrid (Y1H) assay was performed according to the manufacturer’s instructions.

The full‐length PvSPL2 coding sequence was cloned into the pGBKT7 vector, and the full‐length PvD53 coding sequence was cloned into the pGADT7 vector. Plasmid pGBKT7‐PvSPL2 and pGADT7‐PvD53 were co‐transformed into AH109 yeast cells and screened on the SD/‐Leu‐Trp medium. The yeast two‐hybrid (Y2H) assay was performed according to the manufacturer’s instructions.

Electrophoretic mobility shift assay

The full‐length cDNAs of rPvSPL2 and PvrSPL4 were fused to the pGEX4T‐1 expression vector, respectively. The pGEX4T‐1‐rPvSPL2 construct was transformed into Escherichia coli Transsetta, and protein expression was induced at 16°C for 12 h with 0.2 mM isopropyl β‐d‐thiogalactoside (IPTG; Sigma‐Aldrich, St Louis, MO, USA). Reactions were performed using a chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.

In situhybridization

The probe sequence of PvLBO was amplified by PCR and cloned into the pSPT18 vector. Digoxigenin‐labeled sense and antisense probes were synthesized with SP6 and T7 RNA polymerase (Roche, Shanghai, China). The samples of control and transgenic plants were prepared following pre‐treatment and hybridization methods as described (Zhang et al., 2007).

Results

PvLBOwas downregulated in miR156OE transgenic switchgrass plants

The miR156 and SLs both regulate branching/tillering in various plant species. Our previous results have shown that overexpression of miR156 in switchgrass can significantly increase the number of tillers (Fu et al., 2012). To study the relationship between miR156 and SLs, we treated the wild‐type and miR156OE transgenic switchgrass plants with rac‐GR24, one synthetic SL analog, and TIS108, one SL‐biosynthesis inhibitor. As the results exhibited, rac‐GR24 repressed tillering in both wild‐type and miR156OE transgenic plants, implying that the biosynthesis of SLs might be impaired in miR156OE transgenic switchgrass plants (Fig. 1a,b). In contrast, TIS108 only promoted tillering in the wild‐type (Fig. 1a,b). It further suggested that overexpression of miR156 in switchgrass might disrupt the function of SL biosynthetic genes acting downstream of MAX1 since TIS108 has been considered as an efficient inhibitor of MAX1 (Ito et al., 2013). Interestingly, further analysis revealed that the transcripts of one downstream SL biosynthetic gene, PvLBO (gene number: Pavir.5NG619800) were negatively correlated with the abundances of mature miR156 in the miR156OE transgenic plants (Figs 1c, S1). These results imply that PvLBO could play an important role in the miR156‐PvSPL module when regulating switchgrass tillering.

Fig. 1.

Fig. 1

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Overexpression of miR156 compromises strigolactone (SL) synthesis. (a) Gross morphologies of the control and miR156OE transgenic switchgrass plants at the V3 stage with treatment by 2 μM rac‐GR24 and 4 μM TIS108. (b) Statistical analysis of tiller number of (a). Values are means ± SE (n = 3). Student’s t‐test, *, P < 0.05; **, P < 0.01; ns, no significance. (c) Correlation between miR156 and PvLBO expression levels in miR156OE transgenic switchgrass lines. The control and group A transgenic plants had comparable levels of miR156 and exhibited normal growth and tiller number. The levels of miR156 and tiller number were significantly increased in groups B, C and D transgenic plants. Among these miR156OE transgenic lines, relatively low levels of miR156 were observed in group B plants, moderate levels of mature miR156 were found in group C lines, high levels of miR156 were observed in group D.

To further clarify the functions of PvLBO, the coding sequence (CDS) was cloned from lowland‐ecotype switchgrass cv Alamo. Other LBOs were then retrieved from maize, rice, Arabidopsis, M. truncatula and poplar. Phylogenetic analysis showed that PvLBO is clustered together with maize and rice LBO sequences (Fig. 2a). Moreover, alignment of amino acid sequences revealed that PvLBO shared high sequence identity with AtLBO and ZmLBO, and contained the hallmark amino acid residues (i.e. His‐250, Asp‐252, and His‐307) required for iron‐binding, and Arg‐317 for the interaction with the substrate (red asterisks) in the 2‐oxoglutarate and iron(II)‐dependent dioxygenases superfamily domain (Fig. 2b). The results of qRT‐PCR showed that PvLBO was expressed ubiquitously in different switchgrass tissues, and with the highest transcript level in the E2L2 tissue (leaf 2 of switchgrass plants at the E2 stage) followed by the E3L2 tissue (leaf 2 of switchgrass plants at the E3 stage), and then crown buds and roots (Fig. 2c). These findings are consistent with the research into AtLBO, and suggest that PvLBO can also have the ability to oxidize MeCLA to 1′‐HO‐MeCLA in switchgrass.

Fig. 2.

Fig. 2

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Phylogenetic analysis, conserved domain alignment and expression pattern of PvLBO. (a) A phylogenetic tree showing the relationship between PvLBO and the LBOs from different plant species. The phylogenetic tree was constructed based on the alignment using the megax software. (b) Protein sequences alignment shows the differences among the AtLBO (AT3G21420), PvLBO and ZmLBO. (c) Expression levels of PvLBO in crown buds, root, internodes, and leaves of the wild‐type switchgrass. The quantitative real‐time polymerase chain reaction (qRT‐PCR) data were normalized against the expression level of switchgrass PvUbq2. The values are the means ± SE (n = 3). E2I2, internode 2 of the stem elongation 2 stage; E3I2, internode 2 of the stem elongation 3 stage; E4I2, internode 2 of the stem elongation 4 stage; E3L2, leaf 2 of the stem elongation 3 stage; E4L2, leaf 2 of the stem elongation 4 stage. Crown buds and roots were collected from the wild‐type switchgrass at the E4 stage.

To demonstrate the roles of PvLBO in the miR156‐PvSPL module, we overexpressed PvLBO in a miR156OE transgenic switchgrass line (miR156OE‐33) that exhibited characteristic morphological changes resulting from miR156 overexpression (Fig. 3a). Among these double transformed switchgrass plants, 27, 4 and 7 lines showed a significant stepped‐up increase in PvLBO transcripts (Fig. 3b). The tiller numbers were decreased dramatically in the double transformed switchgrass plants compared with miR156OE‐33 transgenic plants, and even with the control switchgrass plants (Fig. 3a,c). These results indicated PvLBO is likely a critical downstream effector contributing to the miR156‐PvSPL module during the regulation of switchgrass tillering.

Fig. 3.

Fig. 3

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Overexpression of PvLBO reduced the increased tillering in miR156 overexpressing switchgrass. (a) Overexpression of PvLBO in the miR156OE‐33 transgenic plants reduced the tiller number. Bar, 20 cm. (b) The expression levels of PvLBO were determined through quantitative real‐time polymerase chain reaction (qRT‐PCR). The expression level of PvUbq2 was used as an internal control. Values are means ± SE (n = 3). Student’s t‐test, *, P < 0.05; **, P < 0.01. (c) Statistical analysis of tiller numbers of the control, miR156OE‐33 and LBOOE‐miR156OE transgenic switchgrass plants at the reproductive two stage of the control plants. Values are means ± SE (n = 3–7). The letters above the error bars indicate significant differences determined by one‐way ANOVA (P < 0.05, Duncan's multiple‐range test). Control plants were produced with pANIC6B empty vector from the same experimental batch.

PvLBO regulates switchgrass tillering

Then the coding sequence of PvLBO driven by ZmUbi1 promoter was introduced into the wild‐type switchgrass by Agrobacterium‐mediated transformation. Six positive transgenic lines were divided into two groups (i.e. LBOOEI and LBOOEII) based on their number of tillers (Figs 4a, S2). The qRT‐PCR analysis showed that the LBOOEII plants had much higher expression levels of PvLBO and displayed much stronger reductions in tiller number compared with the control plants (Fig. 4b). Additionally, the stems of the LBOOEII plants were thicker, the node numbers of LBOOEII plants were increased, and the leaves were wider and longer compared with the control plants (Fig. S3). Moreover, we found that the tiller number of each plant was negatively correlated with the expression level of PvLBO (Fig. 4b). Alternatively, the expression levels of the SL‐responsive gene, PvTB1, were significantly upregulated in the LBOOEI and LBOOEII plants (Fig. S4a). This finding indicates that PvLBO is likely a key enzyme of the SL biosynthetic pathway and plays a vital role in regulating the tillering of switchgrass.

Fig. 4.

Fig. 4

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PvLBO regulated tillering of switchgrass. (a) Phenotypic characterizations of the control, LBOOEII and LBORNAi transgenic switchgrass plants at the reproductive two stage of control plants. Bar, 20 cm. (b) The expression levels of PvLBO and tiller numbers of control and transgenic plants at the E4 stage. The expression levels of PvLBO were determined through quantitative real‐time polymerase chain reaction (qRT‐PCR). The expression level of PvUbq2 was used as an internal control. Values are means ± SE (n = 3). The columns represent the expression levels of PvLBO. The red triangular markers represent tiller numbers. (c) Overview of upregulated and downregulated genes in the control and LBOOE transgenic plants, determined through RNA‐sequencing. The numbers represent the altered genes in various samples. The numbers in the overlapped areas represent the genes observed in both libraries. (d) A diagram showing both strigolactone (SL) biosynthesis and signaling pathways. Blue letters mean that genes were downregulated in the transgenic plants compared to the control plants, while the red letters mean that genes were upregulated. The blunt ended arrow means tillering inhibition. Control plants were produced with the empty vector from the same experimental batch.

To further investigate the function of PvLBO, we generated PvLBO downregulation transgenic switchgrass lines using RNAi technology. As expected, the LBORNAi transgenic lines exhibited higher tiller numbers but semidwarf phenotypes with shorter internodes (Fig. 4a,b). It is noteworthy that downregulation of PvLBO promoted the elongation of axillary buds and produced axillary shoots at some nodes (Fig. S5). In addition, we treated LBORNAi and LBOOE transgenic switchgrass plants with rac‐GR24 and TIS108, respectively. As anticipated, the tiller number was significantly reduced in the LBORNAi plants after treatment with 2 μM rac‐GR24, whereas more tillers formed from the LBOOE transgenic plants after treatment with 4 μM TIS108 (Fig. S6). The treatment results specifically support a role for PvLBO in SL biosynthesis, downstream of TIS108 inhibition.

Numerous genes were changed in PvLBOOE transgenic switchgrass plants

To assess the global effects of PvLBO overexpression on SL biosynthesis, signaling pathways, and responses, we extracted RNA from the stems of the representative LBOOEI and LBOOEII transgenic lines and performed transcriptome analysis by high‐throughput RNA‐Seq. RNA‐Seq data revealed a total of 1943 genes were significantly upregulated, and 2018 genes were significantly downregulated in the LBOOEI plants compared to the control plants. In the LBOOEII plants, 3294 genes were significantly upregulated and 2166 genes were significantly downregulated compared to the control plants (Fig. 4c; Tables S4, S5). Previous studies in Arabidopsis and poplar have shown that the SL biosynthetic genes are involved in the negative feedback on the SL biosynthesis (Mashiguchi et al., 2009), whereas the SL perception and signaling genes participate in the positive feedback (Katyayini et al., 2019). We also found that the SL biosynthetic genes, such as PvD27, PvCCD7 and PvMAX1 were downregulated in the LBOOEI and LBOOEII transgenic plants (Fig. 4d; Table S6). As expected, the SL perception and signaling genes, PvD14 and PvMAX2, were upregulated in the LBOOEI and LBOOEII transgenic plants (Fig. 4d; Table S6), suggesting that more bioactive SL compounds were synthesized in the LBOOE transgenic switchgrass. This result was confirmed by qRT‐PCR (Fig. S4b). Furthermore, GO category enrichment analysis of DEGs showed that plant hormone signal transduction was the most enriched item in the biological process. Other processes such as response to karrikin, lignin catabolism, salicylic acid catabolism, and regulation of phenylpropanoid metabolism were also enriched (Fig. S7). These results demonstrate that PvLBO is an effective gene involved in SL biosynthesis in switchgrass.

PvLBO affected carotenoid, chlorophyll, and lignin biosynthesis in switchgrass

Strigolactones are a group of isoprenoid‐based phytohormones derived from carotenoids. In this study, the total carotenoid content decreased by 25–40% in the LBOOEI and LBOOEII plants compared with the control plants (Fig. S8). Therefore, the rate of SL biosynthesis could be promoted as the concentration of substrate increases in the overexpression transgenic switchgrass plants. Additionally, the contents of Chla and Chlb, which share the geranylgeranyl pyrophosphate precursor with carotenoids, were significantly decreased in the LBOOEI and LBOOEII plants, while Chla was accumulated in LBORNAi plants (Fig. S8). Most surprisingly, the transcriptome analysis showed that numerous genes involved in the lignin biosynthetic pathway, as well as the transcriptional activator PvMYB103, PvMYB42, PvMYB61, and PvMYB63, were downregulated in LBOOE transgenic switchgrass plants (Fig. S9). In contrast, the expression levels of the transcriptional suppressor PvMYB4 were upregulated in the LBOOE plants (Fig. S9b). Based on the results, we hypothesized that overexpression of PvLBO in switchgrass could affect lignin biosynthesis. The content of acid‐insoluble lignin in the LBOOE plants decreased by approximately 33% compared with the control plants (Fig. S10). In contrast, the contents of acid‐insoluble lignin were increased in the LBORNAi transgenic switchgrass plants (Fig. S10). Taken together, these findings suggest that SLs could regulate lignin biosynthesis of stems in switchgrass.

PvSPL2 directly bound to the PvLBO promoter directly and activated its transcription in switchgrass

Our previous study has shown that miR156‐targeted PvSPL2 plays an essential role in switchgrass tillering (Wu et al., 2016). Expression profiles of PvSPL2 and PvLBO in various tissues and phases of switchgrass revealed an appreciable co‐expression between PvSPL2 and PvLBO in the tissues of the roots, crown and nodes at the E4 stage (Fig. 5a). It suggests that PvSPL2 has the potential to regulate PvLBO expression temporally and spatially. Furthermore, we found there are two SBP binding motifs (GTAC) in a 1397 bp upstream sequence of the PvLBO promoter (Fig. 5b). Dual‐LUC reporter assays showed that 35S::rPvSPL2 and PvLBOpro::LUC co‐infiltrating significantly increased the luminescence intensity, while 35S::rPvSPL2 and mutated mPvLBOpro::LUC co‐infiltrating had even lower luminescence intensity than the negative control (Fig. 5c,d), suggesting that PvSPL2 can bind to the motif of the PvLBO promoter and activate its transcription. Furthermore, ChIP‐qPCR analysis of PvSPL2‐GFP revealed an apparent enrichment of site 2 in the PvLBO promoter (Fig. 5e). About 200 bp regions of PvLBO promoter containing sites 1 and 2 (referred to as S1 and S2), respectively, were used for further identification of the core motif recognized by PvSPL2. The LUC reporter assays and Y1H showed that site 2 was necessary and sufficient for PvSPL2‐responsive transcription (Fig. 5f,g). Moreover, EMSA showed that PvSPL2 bound to probe 1 and probe 2 (44 bp length sequence around site 1 and site 2) in vitro (Fig. 5h). These results revealed that PvSPL2 was an efficient transcriptional activator of PvLBO, suggesting the presence of putative crosstalk between the SL biosynthesis and miR156‐SPL module during switchgrass tillering regulation.

Fig. 5.

Fig. 5

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PvLBOexpression was activated by PvSPL2 in switchgrass. (a) Correlations between expression levels of PvSPL2 and PvLBO in different tissues and organs of switchgrass. The representative probe sets of PvSPL2 (AP13CTG29191_at) and PvLBO (AP13ITG40875_at) were retrieved from the switchgrass gene expression atlas. (b) Schematic presentation of putative SPL binding sites in the PvLBO promoter. (c) Dual‐luciferase (LUC) reporter assay showing the activation of PvLBO expression by the PvSPL2 effector compared to the control effector. mLBOpro::LUC shows that two GTAC motifs in PvLBO promoters were mutated to ACGT. The empty vector pCAMBIA 1300 (EV) was employed as the control effector. Values are means ± SE (n = 3). Student's t‐test, *, P < 0.05; **, P < 0.01. (d) Image of the LUC reporter assay presenting the fluorescence intensity, 1, pCAMBIA1300 and LBOpro::LUC; 2, SPL2 and LBOpro::LUC; 3, SPL2 and mLBOpro::LUC. (e) Chromatin immunoprecipitation‐quantitative polymerase chain reaction (ChIP‐qPCR) analysis of rPvSPL2‐GFP overexpressing transgenic switchgrass. Crude chromatin extracts were immunoprecipitated with anti‐green fluorescent protein (anti‐GFP) antibodies. Purified ChIP and input DNAs were used for qPCR analyses. rPvSPL2, miR156‐resistant PvSPL2. Values are means ± SE (n = 3). Student's t‐test, *, P < 0.05; **, P < 0.01. (f) The normalization of the LUC : REN ratio in the LUC assay indicates relative luciferase activity of different fragmenst in the PvLBO promoter. The empty vector pCAMBIA 1300 (EV) was used as the control effector. Values are means ± SE (n = 3). Student’s t‐test, *, P < 0.05; **, P < 0.01. (g) Growth of yeast cells on SD/‐Trp‐Leu‐His supplemented with 3‐AT. pHIS2.1‐ProS1 plus pGADT7 (50 mM 3‐AT) and pHIS2.1‐ProS2 plus pGADT7 (150 mM 3‐AT) served as the negative controls. (h) Electrophoretic mobility shift assay (EMSA) showing PvSPL2 binding to its target sequence in PvLBO promoter. Panel 1 shows that the GST tag does not bind to the biotin‐labeled probe 1; panel 2 shows that PvSPL2 binds to its target probe 1, and panel 3 shows competition of binding by the unlabeled probe 1. Panel 4 shows that the GST tag does not bind to the biotin‐labeled probe 2; panel 5 shows that PvSPL2 binds to its target probe 2, and panel 6 shows competition of binding by the unlabeled probe 2.

PvLBO and SL signaling genes were upregulated in PvSPL2 overexpressing transgenic switchgrass plants

To confirm the regulation of PvLBO by PvSPL2 in vivo and the spatial expression pattern of PvLBO in the control, SPL2OE and miR156OE transgenic switchgrass plants were performed by in situ hybridization (Fig. 6a,b). The PvLBO gene was preferentially expressed on the apex of the shoot apical and floral meristems of control switchgrass plants. Furthermore, in situ hybridization analysis revealed that more PvLBO transcripts accumulated in SPL2OE plants. In contrast, the transcript abundance of PvLBO was obviously reduced in miR156OE plants (Fig. 6a,b). Moreover, the results of the qRT‐PCR showed that the expression level of PvLBO as well as three SL signaling genes, PvMAX2, PvD14, and PvTB1 were consistently upregulated in all SPL2OE transgenic lines analyzed by this study (Figs 6c, S11).

Fig. 6.

Fig. 6

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Expression levels of strigolactone (SL) biosynthetic and signaling genes among in SPL2OE transgenic switchgrass compared to control plants. RNA in situ hybridization of PvLBO with antisense probes in shoot apical (a) and floral (b) meristems of the control, SPL2OE, and miR156OE transgenic switchgrass plants. Bar, 200 μm. (c) The expression level of PvUbq2 was used as an internal control. Values are means ± SE (n = 3). Significant differences were determined using Student's t‐test, P < 0.05. Oblique line square shows there were no significant difference between the control and SPL2OE transgenic switchgrass plants, 1# to 7# means seven individual SPL2OE transgenic lines. The blue square means that genes were downregulated, while the red square means that genes were upregulated in the SPL2OE transgenic switchgrass plants. Control plants were produced with pANIC6B empty vector from the same experimental batch.

Additionally, our Y1H and LUC reporter assay also suggested that PvSPL4 could bind to the PvLBO promoter and then activate its transcription as well. However, there are irregular changes in the expression levels of PvLBO in SPL4OE transgenic switchgrass plants, indicating that there is a complicated relationship between PvSPL4 and PvLBO (Fig. S12). In addition, Y2H analysis and LUC reporter assay revealed that PvSPL2 can interact with PvD53 (Fig. S13). Altogether, our results suggest that PvLBO is a novel target gene of PvSPL2 and functions in the crosstalk between the SL biosynthetic pathway and the miR156‐SPL2 module in switchgrass (Fig. 7).

Fig. 7.

Fig. 7

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A proposed tillering regulation model mediated by strigolactones (SLs) and miR156‐SPL module in switchgrass. In the presence of miR156, SPL2 transcripts are cleaved by miR156, while D53 interacts with SPL2 which further inhibits the transcriptional activation of SPL2 to downstream genes, such as PvTB1 and PvLBO. When miR156 levels decreased, the increased PvSPL2 can directly bind to the promoter of LBO to activate its expression, and then produce more bioactive SLs. The increased SLs trigger the ubiquitin‐mediated degradation of D53. Therefore, more PvSPL2 is released to activate the expressions of downstream genes involved in tillering.

Discussion

The noncanonical SL biosynthetic gene LBO is present in many seed plant species and acts in the later steps after CLA synthesis (Yoneyama et al., 2020). It is a novel branching‐associated gene originally identified in Arabidopsis (Brewer et al., 2016). The LBOs from Arabidopsis, tomato, maize, and sorghum can convert MeCLA to 1′‐HO‐MeCLA in vitro (Yoneyama et al., 2020). Metabolite analysis has suggested that noncanonical SLs are likely predominant in Arabidopsis (Brewer et al., 2016). The biosynthesis of SLs has been widely studied in rice as well (Mashiguchi et al., 2021). However, in contrast to Arabidopsis, the canonical SL 4DO is a major endogenous inhibitor of tillering in rice (Mashiguchi et al., 2021). Thus, the function of LBO in monocots is still elusive. Switchgrass is an important perennial monocotyledonous grass, which is used to produce fodder and biofuels (Lovell et al., 2021). Previous studies have demonstrated that the miR156‐SPL module can control switchgrass tillering (Chuck et al., 2011; Fu et al., 2012; Wu et al., 2016; Gou et al., 2017). Similar observations have been reported in maize and rice (Xie et al., 2006; Chuck et al., 2007). In this study, we found that PvLBO plays an important role in the control of switchgrass tillering. The upregulation of PvLBO in switchgrass dramatically inhibited tiller bud outgrowth, while the downregulation of PvLBO led to substantial increase in the tiller number. The 1′‐HO‐MeCLA and its derivatives converted by LBO are highly unstable and hard to detect in plants (Yoneyama et al., 2020). However, the expression levels of SL‐responsive genes, MAX2, TCP14a, and TB1, were significantly induced when PvLBO were overexpressed, whereas the SL biosynthetic genes, CCD7 and MAX1, were repressed due to the feedback loop of SL biosynthesis. These findings indicate that PvLBO could participate in the biosynthesis of active SLs and regulate switchgrass tillering.

Identification and characterization of the transcription factors regulating LBO expression are crucial for deciphering the SL biosynthesis in plants. Our previous studies have shown that overexpression of miR156 or PvSPL2SRDX in switchgrass can result in drastic increase of tiller number (Fu et al., 2012; Wu et al., 2016). Moreover, the miR156‐targeted SPL4 can repress the initiation of aerial axillary buds and tillering as well (Gou et al., 2017). However, the molecular mechanism of switchgrass tillering remains largely unknown, especially in the miR156‐SPL module. In this study, we found that the miR156‐targeted PvSPL2 directly bound to the promoter of PvLBO and activated its transcription in switchgrass. Therefore, it is reasonable to propose that the miR156‐PvSPL2 module is involved in controlling the noncanonical SL synthesis through the transcriptional regulation of PvLBO in switchgrass. In addition, the miR156‐targeted SPL is a downstream component of D53 in the SL signaling pathway (Liu et al., 2017; Song et al., 2017). Previous study suggests that D53 acts with IPA1 and represses its transcriptional activity by recruiting the TPL–TPR complex in rice. Meanwhile, IPA1 can activate D53 expression by directly binding to the promoter of D53. The feedback regulation between IPA1 and D53 could guarantee the precise regulation of SLs on tiller bud outgrowth in rice (Lovell et al., 2021). However, the miR156 targeted SPLs play a vital role in many crucial biological processes, including tillering, vegetative‐to‐reproductive phase transition, and flowering. Therefore, an extra mechanism is required to remove or alleviate D53 inhibition in plants. Based on our model, PvSPL2 could lead to the D53 ubiquitin degradation via the transcriptional regulation of SL biosynthesis and therefore remove and/or alleviate D53 inhibition in switchgrass. The released PvSPL2 will promote the transcriptional activation of its downstream genes, including PvTB1, which efficiently inhibits tiller bud outgrowth (Fig. 7). Previous study has demonstrated the switchgrass miR156‐targeted SPL4, one ortholog to rice IPA1, is also involved in tillering (Gou et al., 2017). Our Y1H and LUC reporter assay indicate that PvSPL4 also has potential to activate the expression of PvLBO. Unfortunately, no consistent changes were detected in the expression levels of PvLBO and PvSPL4 in the SPL4OE transgenic switchgrass lines, implying a complex network of regulatory interactions between PvSPL4 and PvLBO. Additionally, PvSPL2 and PvSPL4 belong to different subfamilies of which the SPL members exhibit functional redundancy and diversity (Wu et al., 2016; Gou et al., 2017). Moreover, PvSPL4 regulates the expression of LAX and MOC1 rather than TB1 in switchgrass (Gou et al., 2017). Previous studies found that MOC1 is a critical component required for tillering (Li et al., 2003). Therefore, we suspect that PvSPL2 and PvSPL4 play different roles in switchgrass tillering regulation.

GR24 is a synthetic analog of SLs and its activity triggers SL signaling in plants. The tiller number of miR156OE transgenic switchgrass plants significantly decreased after treatment with a moderate level (2 μM) of rac‐GR24. Our results are consistent with the observation in the miR156 overexpressing transgenic rice plants treated with high concentrations (10 μM) of rac‐GR24 (Luo et al., 2012). In contrast, Song et al. (2017) found that low levels (1 μM) of rac‐GR24 slightly inhibited tiller bud outgrowth in the miR156 overexpressing transgenic rice plants. These results suggest that the inhibition of SLs on tiller bud outgrowth in plants is a dosage dependent. In rice, the miR156‐SPL engages in crosstalk with the SL signaling pathway through the interaction between D53 and IPA1. Exogenous application of rac‐GR24 degrades D53 and releases more upregulated IPA1 (Song et al., 2017). D53 and PvSPL2 also interacted in switchgrass, implying a similar mechanism responsible for tillering regulation in switchgrass. However, our results further indicate that the dramatically increased tiller number of miR156 overexpressing transgenic switchgrass plants was due to both a decreased expression of miR156‐targeted PvSPL2 and impaired biosynthesis of SLs. Thus, we believe that PvSPL2 is not only the common target of miR156 and the SL signaling pathway, but also functions as a crucial regulator that fine‐tunes the biosynthesis of SLs in switchgrass. However, due to the structural diversity of SLs, the further studies are required to confirm whether the SPL‐LBO module exists in other plant species.

Tillering is an important agronomic trait affecting the biomass yield of forage and bioenergy grasses. Both SLs and the miR156‐SPL module are involved in the regulation of tiller number in monocot grass species. Our results demonstrate that PvLBO is a crucial enzyme in the noncanonical SL biosynthetic pathway and strongly affected switchgrass tillering. Moreover, PvLBO was identified as a novel downstream gene of PvSPL2 in miR156‐SPL mediated tillering in switchgrass. Further studies such as SL measurements, SL precursor feeding assays, and heterologous enzyme assays are required to confirm the function of PvLBO in future. However, our study facilitates a better understanding of the regulatory mechanism of grass tillering mediated by SLs and the miR156‐SPL module. It will provide novel targets for improving plant architecture and biomass yield of forage and bioenergy crops through molecular breeding.

Author contributions

CF, WL and RY designed the research; RY, WL, YS, ZS, ZW, YW and MW performed the experiments; CF, WL, RY, HW and SB analyzed the data; CF, WL and RY wrote the manuscript. RY and WL contributed equally to this work.

Supporting information

Fig. S1 Morphological characterization of the control and miR156OE transgenic switchgrass plants.

Fig. S2 Phenotypic characterizations of the control and LBOOEI transgenic switchgrass plants.

Fig. S3 Comparison of the leaves and internodes harvested from control and LBOOEII plants.

Fig. S4 Analysis of the expression levels of SL‐related genes in the control and LBOOE transgenic switchgrass plants.

Fig. S5 Downregulation of PvLBO produced axillary shoots at some nodes.

Fig. S6 Gross morphologies of the LBORNAi and LBOOE transgenic switchgrass plants after treatment with 2 μM rac‐GR24 and 4 μM TIS108, respectively.

Fig. S7 Gene ontology enrichment of differentially expressed genes.

Fig. S8 The contents of carotenoid and chlorophyll in the control, LBOOEI, LBOOEII, and LBORNAi transgenic switchgrass plants.

Fig. S9 Differentially expressed lignin‐related genes in LBOOE transgenic switchgrass plants.

Fig. S10 The contents of AcBr lignin in the control, LBOOEI, LBOOEII, and LBORNAi transgenic switchgrass plants.

Fig. S11 Expression levels of strigolactone biosynthetic and signaling genes in SPL2OE transgenic switchgrass.

Fig. S12 PvLBO expression was activated by PvSPL4.

Fig. S13 Interaction between PvSPL2 and PvD53.

Table S1 List of primer pairs used in this study.

Table S2 Amino acid sequences of LBO in different plant species.

Table S3 Probe sequences for luciferase reporter and yeast one‐hybrid assays.

Table S4 Upregulated genes in PvLBOOE RNA‐Seq libraries compared to the control.

Table S5 Downregulated genes in PvLBOOE RNA‐Seq libraries compared to the control.

Table S6 The differentially expressed SL‐related genes in the LBOOE RNA‐Seq libraries.

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Click here for additional data file. (922.1KB, pdf)

Acknowledgements

The authors thank Prof. C. Neal Stewart Jr for providing the pANIC vectors, Dr Xinshun Ding, Prof. Chuanxiao Xie and Prof. Wen‐Xue Li for critical reading and discussion of the manuscript. This work was supported by the Agricultural Variety Improvement Project of Shandong Province (no. 2019LZGC010), the National Natural Science Foundation of China (nos. 31672479, 31872879, and 31801429), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA26030301), and QIBEBT and Dalian National Laboratory for Clean Energy (DNL), CAS (Grant: QIBEBT I201930).

Data availability

Sequence data from this article can be downloaded from the Phytozome database (https://phytozome‐next.jgi.doe.gov/). The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Morphological characterization of the control and miR156OE transgenic switchgrass plants.

Fig. S2 Phenotypic characterizations of the control and LBOOEI transgenic switchgrass plants.

Fig. S3 Comparison of the leaves and internodes harvested from control and LBOOEII plants.

Fig. S4 Analysis of the expression levels of SL‐related genes in the control and LBOOE transgenic switchgrass plants.

Fig. S5 Downregulation of PvLBO produced axillary shoots at some nodes.

Fig. S6 Gross morphologies of the LBORNAi and LBOOE transgenic switchgrass plants after treatment with 2 μM rac‐GR24 and 4 μM TIS108, respectively.

Fig. S7 Gene ontology enrichment of differentially expressed genes.

Fig. S8 The contents of carotenoid and chlorophyll in the control, LBOOEI, LBOOEII, and LBORNAi transgenic switchgrass plants.

Fig. S9 Differentially expressed lignin‐related genes in LBOOE transgenic switchgrass plants.

Fig. S10 The contents of AcBr lignin in the control, LBOOEI, LBOOEII, and LBORNAi transgenic switchgrass plants.

Fig. S11 Expression levels of strigolactone biosynthetic and signaling genes in SPL2OE transgenic switchgrass.

Fig. S12 PvLBO expression was activated by PvSPL4.

Fig. S13 Interaction between PvSPL2 and PvD53.

Table S1 List of primer pairs used in this study.

Table S2 Amino acid sequences of LBO in different plant species.

Table S3 Probe sequences for luciferase reporter and yeast one‐hybrid assays.

Table S4 Upregulated genes in PvLBOOE RNA‐Seq libraries compared to the control.

Table S5 Downregulated genes in PvLBOOE RNA‐Seq libraries compared to the control.

Table S6 The differentially expressed SL‐related genes in the LBOOE RNA‐Seq libraries.

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Data Availability Statement

Sequence data from this article can be downloaded from the Phytozome database (https://phytozome‐next.jgi.doe.gov/). The data that support the findings of this study are available from the corresponding author upon reasonable request.

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