# Overexpression of bHLH95, a basic helix-loop-helix transcription factor family member, impacts trichome formation via regulating gibberellin biosynthesis in tomato

Yao Chen, Dan Su, Jie Li, Shiyu Ying, Heng Deng, Xiaoqing He, Yunqi Zhu, Ying Li, Ya Chen, Julien Pirrello, et al.

► To cite this version:

Yao Chen, Dan Su, Jie Li, Shiyu Ying, Heng Deng, et al. Overexpression of bHLH95, a basic helix-loop-helix transcription factor family member, impacts trichome formation via regulating gibberellin biosynthesis in tomato. Journal of Experimental Botany, 2020, 71 (12), pp.3450-3462. 10.1093/jxb/eraa114. hal-02916187

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RESEARCH PAPER

# Overexpression of bHLH95, a basic helix-loop-helix transcription factor family member, impacts trichome formation via regulating gibberellin biosynthesis in tomato

Yao Chen<fnref n="1" />, Dan Su<fnref n="1" />, Jie Li<fnref n="2" />, Shiyu Ying<fnref n="1" />, Heng Deng<fnref n="1" />, Xiaoqing He<fnref n="1" />, Yunqi Zhu<fnref n="1" />, Ying Li<fnref n="1" />, Ya Chen<fnref n="1" />, Julien Pirrello<fnref n="3" />, Mondher Bouzayen<fnref n="3" />, Yongsheng Liu<fnref n="1" /> and Mingchun Liu<fnref n="1" />,*, ID

Received 30 October 2019; Editorial decision 7 February 2020; Accepted 28 February 2020

Editor: Angus Murphy, University of Maryland, USA

## Abstract

Trichomes are epidermal protuberances on aerial parts of plants known to play an important role in biotic and abiotic stresses. To date, our knowledge of the regulation of trichome formation in crop species is very limited. Through phenotyping of the *Solanum pennellii*×S. lycopersicum (cv. M82) introgression population, we identified the SlbHLH95 transcription factor as a negative regulator of trichome formation in tomato. In line with this negative role, SlbHLH95 displayed a very low expression in stems where trichomes are present at high density. Overexpression of SlbHLH95 resulted in a dramatically reduced trichome density in stems and a significant down-regulation of a set of trichome-related genes. In addition to the lower trichome density, overexpressing lines also showed pleiotropic alterations affecting both vegetative and reproductive development. While most of these phenotypes were reminiscent of gibberellin (GA)-deficient phenotypes, expression studies showed that two GA biosynthesis genes, S/GA20ox2 and S/KS5, are significantly down-regulated in SlbHLH95-OE plants. Moreover, in line with a decrease in active GA content, the glabrous and dwarf phenotypes were rescued by exogenous GA treatment. In addition, yeast one-hybrid and transactivation assays revealed that SlbHLH95 represses the expression of S/GA20ox2 and S/KS5 via direct binding to their promoters. Taken together, our study established a link between SlbHLH95, GA, and trichome formation, and uncovered the role of this gene in modulating GA biosynthesis in tomato.

**Keywords:** Gibberellin, gibberellin biosynthesis, tomato, transcription factor, transcriptional regulation, trichome.

## Introduction

Plant trichomes are specialized epidermal cell structures present on aerial parts of plants including leaves, stems, and peduncles. Trichomes play important roles in plant physiology and development by providing physical or chemical barriers that protect plants against insect predation, UV radiation, and excessive transpiration

(Ishida et al., 2008). Depending on the species, trichomes are classified as unicellular or multicellular, glandular or non-glandular, and branched or unbranched (Werker, 2000; Pattanaik et al., 2014).

Arabidopsis trichomes are typically unicellular, branched, and non-glandular. The initiation and regulation mechanisms

<footnote n="1">¹ Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, Sichuan, PR China</footnote>

<footnote n="2">² Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK</footnote>

<footnote n="3">³ GBF Laboratory, Université de Toulouse, INRA, Castanet-Tolosan, 31320, France</footnote>

<footnote>* Correspondence: mcliu@scu.edu.cn</footnote>
underlying trichome formation have been thoroughly studied in Arabidopsis, and genetic and molecular analyses demonstrated that a complex network of transcription factors (TFs) is involved in this process. The R2R3 MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WDR) proteins, form a trimeric MYB-bHLH-WDR complex that promotes the formation and development of trichomes by activating the expression of downstream target genes (Rerie *et al.*, 1994; Johnson *et al.*, 2002; Ishida *et al.*, 2007). Other TFs including TRANSPARENT TESTA 8 (TT8) and AtMYC1, NAC, and NTM1-LIKE 8 (NTL8) proteins also play pivotal roles in trichome development (Maes *et al.*, 2008; Symonds *et al.*, 2011; Zhao *et al.*, 2012; Tian *et al.*, 2017). In addition, phytohormones, especially gibberellins (GAs), jasmonates (JAs), and cytokinins (CKs), regulate trichome formation by modulating the expression of key regulatory genes in Arabidopsis (Schellmann and Hulskamp, 2005; Yang and Ye, 2013; Qi *et al.*, 2014). Although the regulatory network of unicellular trichome formation in Arabidopsis has been extensively studied, our knowledge of the initiation and development of trichomes in crop species remains scarce.

Considering the wide diversity of trichome types in tomato (Solanum lycopersicum), this species can potentially serve as a model system for studying multicellular and glandular trichomes, due to the extensive genetic resources available, the high-quality genome sequence, and other genomics resources along with a handful of reported trichome mutants among which are *chi1*, *coi1*, *hairless*, *odorless-2*, and *inquieta* (Li *et al.*, 2004; Kang *et al.*, 2010a, b, 2014; Jeong *et al.*, 2017). Tomato produces eight types of trichomes which can be divided into glandular (types I, IV, VI, and VII) and non-glandular (types II, III, V, and VIII) whose characterization is giving rise to an increasing number of studies (Luckwill, 1943; Simmons and Gurr, 2005; Glas *et al.*, 2012; Xu *et al.*, 2018). The HD-Zip protein Woolly was reported to physically interact with a B-type cyclin protein SlCycB2 to regulate type I trichome formation (Yang *et al.*, 2011). Moreover, the C2H2 zinc finger protein Hair (H), interacting with Woolly, was recently identified as a key regulator of type I trichome formation in tomato (Chang *et al.*, 2018). SIMX1, a MIXTA-like MYB TF, was shown to be involved in the formation of both glandular and non-glandular trichomes in tomato (Ewas *et al.*, 2016, 2017). In addition, down-regulation of *SlIAA15* or *SlARF3*, two auxin-related TF genes, led to reduced trichome density in tomato (Deng *et al.*, 2012; Zhang *et al.*, 2015). More recently, it was shown that down-regulation of *SIMYC1*, a bHLH TF gene, resulted in the production of smaller type VI glandular trichomes at lower densities, and the complete knockout of this gene led to total absence of type VI trichomes (Xu *et al.*, 2018), indicating that SIMYC1 is instrumental in type VI glandular trichome development in tomato. In addition, it was reported that JA is required for the formation and development of type VI glandular trichomes in tomato (Li, 2004; Yan *et al.*, 2013; Bosch *et al.*, 2014). However, the role of other hormones such as GAs in tomato trichome formation remains unclear. In spite of the progress made in uncovering some of the factors controlling trichome development in tomato, our understanding of the

regulatory mechanisms underlying the development of different types of tomato trichomes remains largely incomplete.

Here, we report on a new bHLH TF, SlbHLH95, acting as a putative regulator of trichome formation identified by screening the *S. pennellii*×*S. lycopersicum* introgression population for altered trichome phenotypes. Overexpression of *SlbHLH95* in tomato resulted in reduced trichome density in stem, leaf, and young fruit. Moreover, *SlbHLH95*-overexpressing (OE) lines exhibited GA-deficient phenotypes that were rescued by exogenous GA application. The data provide new insight into the role of bHLH TFs in controlling trichome formation in tomato through the regulation of GA biosynthesis.

## Materials and methods

### Plant materials and growth conditions

Tomato (*S. lycopersicum*, Micro-Tom) plants were grown under standard greenhouse conditions set as follows: 14 h day/10 h night cycle, 25 °C/20 °C day/night temperature, 80% relative humidity, and 250 µmol m⁻² s⁻¹ light intensity.

### Plant transformation

*Agrobacterium tumefaciens*-mediated tomato plant transformation was performed as described in Wang *et al.* (2005). The transformed lines were then selected on a kanamycin-containing medium. Homozygous lines from the F₃ or later generations were used in all experiments.

### Subcellular localization of SlbHLH95 protein

To investigate the subcellular localization of the SlbHLH95 protein, the coding sequence of *SlbHLH95* without the stop codon was amplified by PCR and then cloned into pART2a to generate the 35S::SlbHLH95-GFP (green fluorescent protein) fusion gene. The vector bearing the fusion construct SlbHLH95-GFP and the control GFP vector were individually transformed into leaves of *Nicotiana benthamiana* using a 2 ml needleless syringe. The transformed samples were incubated in the dark at 22 °C for 48 h, then the subcellular localization of each expressed protein was visualized using a confocal microscope (Leica, TCS SP5 II). Images of transformed tobacco cells were captured using a ×40 objective lens, using bright field, GFP (excitation/emission: 488/498–548 nm), and DAPI (excitation/emission: 405/421–523 nm) filters.

### RNA extraction and RT-qPCR

Total RNAs from different tissues were isolated using a Plant RNA Purification Reagent (Invitrogen, cat. no. 12322-012) according to the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio, Kusatsu, Japan, AK4201). cDNA products were diluted to 2.5 ng µl⁻¹ and used as templates for the qPCR. Quantitative reverse tanscription–PCR (RT–qPCR) was performed using the Bio-Rad CFX384 device. Each reaction (10 µl) consisted of 5 µl of iTaq™ Universal SYBR Green Supermix (Bio-Rad, Hercules, USA, #172-5124), 1 µl each of forward and reverse primers, and 3 µl of cDNA. Thirty-nine amplification cycles were performed (pre-incubation at 95 °C for 2 min followed by each cycle consisting of 5 s at 95 °C, 10 s at 60 °C, and added melting curve analysis during 65–95 °C). The results were calculated using Bio-Rad CFX Manager software. The relative expression of each gene was calculated by the ΔΔCt method (Schefe *et al.*, 2006) using *SlActin* as internal control. The primer pairs for RT-qPCR were designed using Primer3Plus (http://www.primer3plus.com) and blasted at the NCBI database to check their specificity (the primers used for qRT-PCR are listed in Supplementary Table S3 at JXB online).
## Fruit shape analysis

Full size maturing (7 d post-breaker stage) freshly harvested fruits were cut with a new razor blade longitudinally, scanned at 300 dpi, and analyzed using Tomato Analyzer v3.0 (Rodríguez et al., 2010). Then fruit shape index and fruit shape triangle, two parameters characterizing the shape of the fruit, were obtained after analysis. Fruit shape index is the ratio of the maximum height length to the maximum width of the fruit, and fruit shape triangle is the ratio of the proximal end width to the distal end width (Brewer et al., 2007).

## SEM analysis

Fresh tomato leaves or stems (three biological replicates) collected from wild-type (WT) and transgenic plants were fixed for 24 h in a solution of 2.5% paraformaldehyde, 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4 (Electron Microscopy Sciences, Hatfield, PA, USA). Samples were dehydrated in the following ethanol concentrations (30, 50, 70, 80, 90, 100, and 100%), each treatment lasting 10 min. Samples were then critical point dried with liquid CO₂ (Quarum K850 CPD), mounted, and sputter coated with a 5 nm thin layer of platinum (LEICA EM MED 020). Images were acquired with a scanning electron microscope (SEM Quanta 250 FEG FEI) at 5 kV, spot size 3 or 2 with a working distance of 1 cm.

## RNA-Seq analysis and data processing

Global expression of tomato genes was determined by replicated strand-specific Illumina RNA sequencing (RNA-Seq). Paired-end RNA-Seq was carried out using Hiseq 2500 (150 bp paired ends) by Novogene (China). WT and *SlbHLH95-OE83* RNAs were extracted from 4-week-old leaves with three biological replicates. Prior to sequencing, the quality of purified RNA was checked with the Agilent2100 Bioanalyzer (RIN ≥6.3).

Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adaptor, reads containing poly-N (>10%

unknown base per read), and low-quality reads (>50% of Q<sub>phred</sub> ≤20 bases per read) from raw data. At the same time, Q20 (probability of incorrect base call, 1 in 100), Q30 (probability of incorrect base call, 1 in 1000), and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Trimmed reads were then mapped to the *S. lycopersicum* reference genome and gene annotation ITAG2.4 (Tomato Genome Consortium, 2012) using TopHat-2.0.12 (Trapnell et al., 2009) calling Bowtie 2.2.3 (Langmead and Salzberg, 2012).

Differential expression analysis was performed using the DESeq R package (1.18.0) (Anders and Huber, 2010). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting *P*-values were adjusted using the Benjamini-Hochberg's approach (Benjamini and Hochberg, 1995) for controlling the false discovery rate. Genes with an adjusted *P*-value < 0.05 found by DESeq were assigned as differentially expressed genes (DEGs).

Gene Ontology (GO) enrichment analysis of DEGs was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with a corrected *P*-value <0.05 were considered as significantly enriched within DEGs (Young et al., 2010). Then, we carried out the statistical enrichment of the differential expression of DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using KOBAS software (Mao et al., 2005; Kanehisa et al., 2008).

All raw-sequence reads data were uploaded in the NCBI Sequence Read Archive (SRA; http://www.ncbi.nlm.nih.gov/Traces/sra) with accession number SRP144705.

## GA content analysis

GA measurements were conducted by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). Leaf material was collected from WT and *SlbHLH95-OE* plants at the 4-week-old stage. Three replicates were performed for each genotype. Frozen plant materials were ground using a mixer mill (MM 400, Retsch, Germany) for 1 min at 30 Hz. Then, 200 mg

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Different trichome phenotypes displayed by domesticated tomato (*S. lycopersicum*), a wild relative (*S. pennellii*), and introgression lines. (a) Introgression line IL10-2 showed altered trichome density in stem compared with the parental line M82. The white bars represent 1 cm. IL10-1, IL10-2, IL10-2-2, and IL10-3 are different introgression lines. (b) RT-qPCR analysis of mRNA levels of *SlbHLH95* and *SlbHLH65* in M82 and the introgression line IL10-2. Values are means ±SD. Statistical significance was determined by Student's *t*-test: **0.001<P<0.01. (This figure is available in colour at JXB online.)</figcaption></figure>
of the powder sample was extracted overnight at 4 °C with 1500 µl of 70% (v/v) acetonitrile, and ultrasound-assisted extraction was carried out for 30 min at room temperature with vortexing (15 s) and centrifugation (14 000 rpm for 10 min). From each sample, 1000 µl of supernatant was collected, then evaporated to dryness under a nitrogen gas stream at room temperature, reconstituted in 100 µl of 80% (v/v) methanol, and diluted to 800 µl with water. The extracts were passed through the SPE cartridge (200 mg, 3 ml; CNW) and evaporated to dryness under a nitrogen gas stream at room temperature (Ma et al., 2008; Hao et al., 2015). Samples were reconstituted in 200 µl of 80% (v/v) methanol and filtrated (PTFE, 0.22 µm; Anpel), then analyzed using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, http://www.shimadzu.com.cn/; MS, Applied Biosystems 6500 Triple Quadrupole, http://www.appliedbiosystems.com.cn/). A Waters Acquity UPLC BEH C₁₈ column (2.1 mm×100 mm×1.8 µm) was used. The instrument was operated, and multiple reaction monitoring (MRM) transitions were assessed (Li et al., 2017). For all samples, peak areas for endogenous and labeled GAs were compared and combined with 200 mg of powdered sample to calculate ng g⁻¹.

## GA treatment

For application of GA to young plants growing on soil, gibberellic acid (GA₄, Sangon Biotech) were first dissolved in absolute ethanol and then diluted with the nutrient solution (Miracle-Gro® Pour & Feed Plant Food, Item #1006002). A solution of 10⁻⁵ M GA₄ was sprayed on the plants twice a week starting 14 d post-germination. After 2 weeks of

treatment, the treated plants were compared with the control plants (treated with the same solution without GA₄).

## Dual-luciferase transient expression assay

The *SIKS5* and *SIGA20ox2* promoters were cloned into the pGreenII 0800-LUC double-reporter vector, while *SlbHLH95* was cloned into the pGreenII 62-SK vector effector, as described by Hellens et al. (2005). The constructed reporter and effector plasmids were transiently expressed in tobacco (*N. benthamiana*) leaves as described by Hellens et al. (2005). A dual-luciferase assay kit (Promega) was used to analyze the transient expression in tobacco leaves on the third day after infiltration. Absolute firefly luciferase (LUC) reporter and renilla luciferase (REN) values were measured in a Luminoskan Ascent Microplate Luminometer (Thermo Scientific) according to the manufacturer's instructions, with a 5 s delay and 15 s integrated measurements. The transcriptional activation activity of SlbHLH95 on the promoters of GA biosynthesis-related genes was indicated by the ratio of LUC to REN. At least six biological repeats were conducted.

## Yeast one-hybrid assays

The full-length cDNA sequence of *SlbHLH95* was amplified and inserted into the pDEST22 vector containing the GAL4 activation domain (AD) to form the construct pDEST22-SlbHLH95. Three tandem copies of the E-box from *SIKS5* and *SIGA20ox2* promoters and the promoter fragment were each cloned into the pHis-Leu2-GW vector. Various combinations of pDEST22-SlbHLH95 and different promoters

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Expression pattern of *SlbHLH95* and subcellular localization of SlbHLH95 protein. (a) Expression levels of the *SlbHLH95* gene in different tissues. TPM, transcripts per kilobase of exon model per million mapped reads. (b) Relative mRNA levels of *SlbHLH95* in different tissues. Accumulation of *SlbHLH95* transcripts was assessed by RT-qPCR in root, stem, leaf, flower, mature green fruit (MG), breaker fruit (Br), 3 d post-breaker fruit (Br+3), and 7 d post-breaker fruit (Br+7). The relative mRNA levels of *SlbHLH95* in the root were standardized to 1.0, referring to the *SIActin* gene as the internal control. Values are means ±SD of three biological replicates. (c) GFP-tagged fusion proteins were transiently expressed under the control of the 35S promoter in tobacco cells after transfection. (This figure is available in colour at JXB online.)</figcaption></figure>
were co-transformed into yeast strain Y187. Empty pDEST22 with the two promoters was used as a negative control. The transformants were cultivated on SD/-Trp/-Leu medium for 3 d. DNA-protein interactions were determined by the growth of clones on SD/-Trp/-Leu/-His with 10 mM 3-AT (3-amino-1,2,4-triazole).

## Results

### Identification of SlbHLH95 as a putative regulator of tomato trichome formation

Phenotyping of the *S. pennellii*×*S. lycopersicum* (cv. M82) introgression population identified one specific introgression line (IL) on chromosome 10 (IL10-2) showing altered trichome density in the stem compared with the M82 control parental line (Fig. 1a). This suggested the presence of a putative trichome regulatory gene(s) in the replacement region specific to the IL10-2 line. The search for putative genes present in the *S. pennellii* genome region introgressed in IL10-2 identified 56 potential gene units resident in this region,

and among these two were predicted to putatively encode bHLH TFs (Solyc10g079050, SlbHLH95 and Solyc10g079070, SlbHLH65; Supplementary Table S1). Considering that bHLH family genes have been shown to regulate trichome formation in Arabidopsis and given that SlbHLH95 displayed 15 times higher expression at the transcript level in stems of IL10-2 than in M82 (Fig. 1b), whereas SlbHLH65 exhibited similar expression in the two tomato accessions (Fig. 1b), we postulated that SlbHLH95 represents a realistic candidate gene underpinning the observed trichome phenotype. We therefore sought to address the functional significance of SlbHLH95 in the regulation of trichome formation in tomato.

RNA-Seq and RT-qPCR studies revealed that SlbHLH95 was constitutively expressed in all tissues tested, with the highest transcript levels observed in ripe fruit and the lowest in stems where trichomes are present at high density (Fig. 2a, b), indicating that SlbHLH95 expression may negatively correlate with the presence of trichomes in the tissue. Confocal laser scanning microscopy analysis showed that SlbHLH95 strictly

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** SlbHLH95 overexpression lines show reduced trichome number on stems and leaves. (a) RT-qPCR analysis of SlbHLH95 transcripts in SlbHLH95-OE lines. (b) Light microscopy and SEM observation of trichomes on stems. Arrows show type I trichomes. (c) Light microscopy and SEM observation of leaf trichomes. Observations were made on the adaxial surface of leaves at the same stage. Arrows show type V and type VI trichomes. (d) Density of trichomes on adaxial leaves from WT and SlbHLH95-OE plants. (e) The densities of the non-glandular type V trichome and the glandular type VI trichome are shown for the adaxial leaves. (f-j) RT-qPCR relative expression of trichome-associated genes in WT and SlbHLH95-OE lines in leaves at 4 weeks old. Values are means ±SD. Statistical significance was determined by Student's t-test: \*0.01\*P\*0.05; \*\*0.001\*P\*0.01; \*\*\*P\*0.001. OE83, OE5, and OE18 are three SlbHLH95-OE independent lines. (This figure is available in colour at JXB online.)</figcaption></figure>
localized in the nucleus (Fig. 2c), consistent with its putative
TF activity.

Overexpression of SlbHLH95 results in reduced trichome formation

Since the expression level of *SlbHLH95* is very low in stems
showing a high density of trichomes, we generated to-
mato overexpressing lines to uncover the role of this TF in
trichome formation. Twelve 35S::*SlbHLH95* independent
homozygous lines were obtained that showed similar pheno-
types.Three lines exhibiting 100- to 120-fold increases in the
*SlbHLH95* transcript level were selected for further studies
(Fig. 3a). *SlbHLH95*-OE plants exhibited a dramatic reduc-
tion in trichome number in stems compared with WT plants.
Both light microscopy and SEM analyses showed that the de-
crease in trichome number mainly affected type I trichomes
(Fig. 3b). Moreover, these analyses also revealed that the total
trichome number was significantly decreased in leaves due to
the decrease of type V trichomes (Fig. 3c-e) and in 7-day-old
fruit of *SlbHLH95*-OE lines (Supplementary Fig. S1a, b). To
gain insight into the molecular features underlying the reduced

trichome phenotype, we assessed by RT-qPCR the transcript
accumulation of genes known to be involved in trichome for-
mation. *SlCycB2*, a B-type cyclin gene, shown to regulate to-
mato trichome formation, was significantly down-regulated
in the *SlbHLH95*-OE lines (Fig. 3f). Other trichome-related
genes, such as *SlGAMYB2*, *SlGASA4*, and *SlANT1*, also dis-
played a significant down-regulation in *SlbHLH95*-OE plants
(Fig. 3g-i). In contrast, the transcript levels of *SlWoolly*, another
gene reported to control trichome formation, showed no sig-
nificant alteration (Fig. 3j).

*SlbHLH95* overexpression lines display multiple
vegetative and reproductive phenotypes

In addition to the reduced trichome formation, *SlbHLH95*-OE
lines exhibited a stunted phenotype from early developmental
stages with a severe reduction in plant size (Fig. 4a). The
average height of *SlbHLH95*-OE adult plants was less than half
of that of WT plants due to shorter internodes (Fig. 4b, c). Leaf
morphology was dramatically altered in the *SlbHLH95*-OE
lines, with twisted leaf margins and wrinkled lamina, and leaf
size was significantly reduced due to smaller epidermal cells

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Vegetative phenotypes of *SlbHLH95* overexpression lines. (a) Dwarf phenotype of *SlbHLH95*-OE plants. Photographs were taken at 80 d after germination. (b) Reduced plant size of 80-day-old *SlbHLH95*-OE plants. Values are means ±SD (n≥15) of three replicates. (c) Shorter internodes of 80-day-old *SlbHLH95*-OE plants. Values are means ±SD (n≥15) of three replicates. (d) Leaf morphology of *SlbHLH95*-OE plants. (e) Light microscopy observation of a leaf epidermal cell in WT and *SlbHLH95*-OE plants. (f) Epidermal cell size in WT and *SlbHLH95*-OE leaves. \*0.01\*P\*0.05 (Student's *t*-test). *OE83*, *OE5*, and *OE18* are three independent *SlbHLH95*-OE lines. (g) Axillary bud phenotype of *SlbHLH95*-OE plants. Photographs were taken at the 8-week-old stage. (h) *SlbHLH95*-OE plants have more axillary buds than the WT. Values are the mean ±SD (n≥15) of three replicates. \*\*P\*0.001, (Student's *t*-test). (This figure is available in colour at JXB online.)</figcaption></figure>
(Fig. 4d–f). In addition, *SlbHLH95-OE* transgenic plants exhibited more axillary buds than WT plants (Fig. 4g). Unlike the WT that produced 1–2 axillary buds, *SlbHLH95-OE* lines had 3–5 axillary buds per plant at the 8-week-old stage (Fig. 4h).

*SlbHLH95-OE* plants also showed large alterations in re-productive development. The time from germination to an-thesis was delayed by ~20 d compared with the reference WT plants (Fig. 5a, b). Remarkably, although *SlbHLH95-OE* lines produced normal flower buds, the majority of these never reach the anthesis stage (Supplementary Fig. S2a), which is reminiscent of phenotypes due to a defect in GA (Nester and Zeevaart, 1988). Furthermore, the *SlbHLH95-OE* lines exhibited a dramatic reduction in fruit set, leading to mark-edly lower fruit number per plant (Fig. 5c). The fruit set rate was 15–20% in the *SlbHLH95-OE* lines, while 95% of the flowers successfully set fruit in WT plants grown under the same cultivation conditions (Fig. 5d). Cross-fertilization as-says showed that pollen viability and female organ fertility were not affected in overexpressing lines (Supplementary Fig. S2b).

Although fruits of *SlbHLH95-OE* lines showed no signifi-cant difference compared with WT lines with respect to fruit development and ripening (Supplementary Fig. S2c, d), the fruit were more elongated with increased fruit shape index and decreased fruit shape triangle compared with the WT (Fig. 5e–g). The average fruit weight was also reduced, with a higher proportion of small fruits in *SlbHLH95-OE* lines (Fig. 5h). Moreover, the number of seeds per fruit was signifi-cantly reduced in *SlbHLH95-OE* lines (Fig. 5i).

Genome-wide transcriptomic profiling of
*SlbHLH95-OE* lines

To further unveil the molecular basis of the phenotypes ob-
served in *SlbHLH95-OE* lines, we performed a global gene
expression profiling of 4-week-old leaves with three bio-
logical replicates. The number of raw reads ranged from 43 to
55 million with an error rate of ~0.02%, producing 6.11–7.84
G clean bases (Supplementary Data S1). On average, 96% of
these reads were mapped to the ITAG-2.4 tomato reference

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Reproductive development was dramatically altered in *SlbHLH95-OE* lines. (a, b) Delayed flowering of *SlbHLH95-OE* plants compared with the WT. DAG, days after germination. The white bars represent 5 cm. (c, d) Reduced fruit set in *SlbHLH95-OE* plants. (e) Altered fruit shape in *SlbHLH95-OE* plants. The white bars represent 1 cm. (f) Fruit shape index as the ratio of length to width in three representative independent lines. (g) Fruit shape triangle as the ratio of proximal end width to distal end width in three representative independent lines. (h) Distribution of fruit weight in WT and *SlbHLH95-OE* lines. *n*=60 fruits in WT and transgenic lines. (i) Seed number per fruit in WT and *SlbHLH95-OE* lines. *n*=60 fruits in WT and transgenic lines. Values are means ±SD. Statistical significance determined by Student's *t*-test: \*0.01\*P\*0.05; \*\*0.001\*P\*0.01; \*\*\*P\*0.001. (This figure is available in colour at JXB online.)</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** RNA-Seq profiling of *SlbHLH95-OE* lines. (a) Venn diagram of the numbers of expressed genes in WT and *SlbHLH95-OE* lines. (b) Volcano plot showing the DEGs between two different libraries. *q*<0.05 was used as the threshold to determine the significance of DEGs. Dots in section A show down-regulated genes, dots in section B represent up-regulated genes, and dots below the dash-dotted line (section C) indicate transcripts that did not change significantly in the *SlbHLH95-OE* library compared with the WT. (c) GO enrichment analysis of DEGs between *SlbHLH95-OE* and the WT. The most enriched GO terms are shown. Asterisks indicate significantly enriched GO terms (*q*<0.05). (d) KEGG pathway enrichment analysis of DEGs between *SlbHLH95-OE* and the WT. The left y-axis shows the KEGG pathway. The x-axis shows the Rich factor. A high *q*-value is represented by light gray, and a low *q*-value is represented by black (*q*<0.05). (This figure is available in colour at JXB online.)</figcaption></figure>

genome, yielding 38–49 million unique mapping reads based on the sample examined (Supplementary Data S2). The expression values, indicated by fragments per kilobase of transcript per million mapped fragments (FPKM) (Supplementary Data S3), showed high correlations (Pearson correlation coefficient >0.97) among biological replicates (Supplementary Data S4) (Trapnell et al., 2010). Distribution plots showed that the distribution of the normalized expression levels was comparable between WT and OE lines (Supplementary Fig. S3).

A total of 20 854 and 20 699 genes were expressed (averaged FPKM ≥1) in WT and OE lines, respectively. Among all these genes, 484 and 329 genes were uniquely expressed in WT and OE lines, respectively (Fig. 6a). A total of 1368 DEGs were identified between the WT and OE lines, of which 812 were up-regulated and 556 down-regulated in *SlbHLH95-OE* lines (Fig. 6b). The putative functions of the DEGs were predicted using GO analysis. The DEGs were categorized into three main categories: biological

process; molecular function; and cellular component (Fig. 6c; Supplementary Data S5). Using the KEGG annotation pathway for the DEGs of *SlbHLH95-OE* versus the WT, those related to ‘Biosynthesis of secondary metabolites’, ‘Plant hormone signal transduction’, and ‘Phenylpropanoid biosynthesis’ were over-represented (Fig. 6d; Supplementary Data S6). Consistent with the reduced trichome number, most of the genes previously reported to regulate trichome formation in tomato were down-regulated in the *SlbHLH95-OE* lines (Supplementary Fig. S4).

Considering that the phytohormone GA was reported to regulate trichome formation in Arabidopsis and that the *SlbHLH95-OE* lines displayed GA-deficient phenotypes such as reduced plant size, reduced trichome number, increased axillary buds, delayed flowering time, and reduced fruit set, we analyzed the expression of GA-related genes. Interestingly, among the top 20 DEGs that show down-regulation, we found two GA biosynthesis genes. Indeed, (–)-ent-kaurene
<table><caption>**Table 1.** Top 20 down-regulated genes in *SlbHLH95-OE* lines</caption><thead><tr><th>Solyc number</th><th>Log<sub>2</sub>fold</th><th>Description</th></tr></thead><tbody><tr><td>Solyc12g056690</td><td>-7.9933</td><td>Serine/threonine-protein phosphatase 7 long form homolog</td></tr><tr><td><strong>Solyc03g006550</strong></td><td><strong>-4.6366</strong></td><td><strong>(-)-ent-kaurene synthase (SIKS5)</strong></td></tr><tr><td>Solyc10g085240</td><td>-3.9082</td><td>UDP-glucosyltransferase</td></tr><tr><td>Solyc06g009240</td><td>-3.8717</td><td>Cation/H(+)-antiporter 15</td></tr><tr><td>Solyc07g006560</td><td>-3.6676</td><td>Hypersensitive response assisting protein</td></tr><tr><td>Solyc08g080710</td><td>-3.3537</td><td>Carboxyl-terminal peptidase</td></tr><tr><td>Solyc06g083470</td><td>-3.0144</td><td>Tropinone reductase-like protein 16</td></tr><tr><td>Solyc01g094870</td><td>-2.9991</td><td>MRNA clone RAFL21-92-I07</td></tr><tr><td>Solyc04g005600</td><td>-2.8636</td><td>MYB transcription factor</td></tr><tr><td>Solyc11g021060</td><td>-2.7435</td><td>Proteinase inhibitor</td></tr><tr><td>Solyc03g121680</td><td>-2.5768</td><td>β-Fructofuranosidase</td></tr><tr><td>Solyc06g075460</td><td>-2.5287</td><td>TGF-β receptor type I/II extracellular region</td></tr><tr><td>Solyc02g077300</td><td>-2.2942</td><td>Peroxidase 73</td></tr><tr><td>Solyc01g108610</td><td>-2.2776</td><td>Cyclin-dependent kinase inhibitor 12</td></tr><tr><td><strong>Solyc06g035530</strong></td><td><strong>-2.1946</strong></td><td><strong>Gibberellin 20-oxidase-2 (SIGA20ox2)</strong></td></tr><tr><td>Solyc09g075790</td><td>-2.1653</td><td>Long-chain fatty acid CoA ligase</td></tr><tr><td>Solyc03g114830</td><td>-2.1535</td><td>MADS box transcription factor</td></tr><tr><td>Solyc09g072700</td><td>-2.1518</td><td>Peroxidase 57</td></tr><tr><td>Solyc04g014410</td><td>-2.1116</td><td>Serine/threonine protein kinase-like</td></tr><tr><td>Solyc04g056450</td><td>-2.0013</td><td>Cyclopropane-fatty-acyl-phospholipid synthase</td></tr></tbody></table>

All the genes were allocated a *q*-value <0.05.

synthase (SIKS5, Solyc03g006550) and gibberellin 20-oxidase-2 (SIGA20ox2, Solyc06g035530) showed significantly lower expression at the transcript level in *SlbHLH95-OE* lines (Table 1), suggesting that GA content might be decreased in *SlbHLH95-OE* plants.

## SlbHLH95-OE lines exhibit decreased gibberellin contents

Transcript accumulation corresponding to SIKS5 and SIGA20ox2 genes was further assessed by RT-qPCR, showing a significant decrease in *SlbHLH95-OE* lines compared with the WT (Fig. 7a, b). Assessing the GA content indicated that although GA₃, the active GA form, displayed no change, GA₄, the final product of the GA bioactive form, and its precursor GA₉ were significantly decreased in *SlbHLH95-OE* plants (Fig. 7c). These results suggested that the decreased GA levels may account for the inhibition of trichome formation and for some of the vegetative and reproductive developmental alterations in *SlbHLH95-OE* lines.

To further investigate the contribution of the reduced GA levels to the phenotypes of low trichome density and altered vegetative and reproductive development in *SlbHLH95-OE* plants, we applied exogenous GA₄ to 2-week-old plants since GA₄ levels were found to be significantly decreased in the transgenic lines. The plant size of *SlbHLH95-OE* plants was fully rescued by GA₄ treatment (Fig. 7d, e), with the internode length becoming comparable with those of untreated WT plants (Fig. 7f). Moreover, trichome density was enhanced in *SlbHLH95-OE* lines treated with exogenous GA₄ (Fig. 7g). Interestingly, the fruit shape of GA-treated plants

became more round compared with untreated *SlbHLH95-OE* plants (Fig. 7h) and, accordingly, both fruit shape index and fruit shape triangle were significantly decreased (Fig. 7i, j). These results indicated that the reduced trichome density, dwarf plant phenotype, and elongated fruit shape displayed by *SlbHLH95-OE* lines are likely to be due to the decreased GA levels.

## *SlbHLH95* directly regulates SIGA20ox2 and SIKS5 via binding to their promoters

The decreased GA content together with the down-regulated expression of SIKS5 and SIGA20ox2 in *SlbHLH95-OE* lines motivated the investigation of a possible regulation of SIKS5 and SIGA20ox2 transcription by SlbHLH95. An *in silico* search for typical regulatory motifs in the SIKS5 and SIGA20ox2 promoter sequences revealed the presence of conserved E-box (CANTTG) and E-box like cis-elements known as putative targets of bHLH type TFs (Supplementary Table S2). To examine whether SlbHLH95 could repress the transcription of a reporter gene driven by the SIKS5 and SIGA20ox2 promoters, transactivation assays using the dual-luciferase reporter system were performed. The dual luciferase reporter plasmids harboring the SIKS5 and SIGA20ox2 promoters that contain the E-box-like motif were fused to the LUC reporter, and the REN reporter driven by the 35S promoter was used as an internal control. The expression of SlbHLH95 resulted in reduced activity of both SIKS5 and SIGA20ox2 promoters, indicating that SlbHLH95 exerts a negative regulation on the transcription of the two GA biosynthesis genes (Fig. 8a, b).

To assess whether SlbHLH95 directly binds to the SIKS5 and SIGA20ox2 promoters, we then expressed SlbHLH95 fused to the GAL4 AD in a yeast one-hybrid system to test its ability to bind to the two promoters fused to the HIS2 reporter. The results showed that SlbHLH95 significantly reduced yeast growth compared with the control (Fig. 8c, d), whereas when challenged with mutated E-box sequences SlbHLH95 failed to significantly affect yeast growth (Fig. 8c, d), indicating that SlbHLH95 represses the activities of SIKS5 and SIGA20ox2 promoters via binding to the E-box cis-elements. Collectively, these data indicate that SlbHLH95 acts as a negative regulator of GA biosynthesis through direct binding to the E-box motif within the SIKS5 and SIGA20ox2 promoters. This is probably the causal factor leading to reduced trichome formation and other vegetative and reproductive developmental phenotypes.

## Discussion

Development of unicellular non-glandular trichomes is known to be regulated by a network of TFs in Arabidopsis (Schellmann and Hulskamp, 2005; Yang and Ye, 2013; Qi et al., 2014), and phytohormones such as GA, JA, and CK were reported to play an essential role in this process acting in a TF complex-dependent manner (Pattanaik et al., 2014). In contrast, less is known about regulatory networks that control the development of trichomes in crop species. Our present study shows that the TF SlbHLH95 is involved in the regulation of trichome development in tomato through the negative regulation of GA
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Application of exogenous GA rescued the dwarf phenotype in *SlbHLH95-OE* plants. (a, b) Relative expression of GA biosynthesis-related genes in the WT and *SlbHLH95-OE* lines in leaves at 4 weeks old. Statistical significance was determined by Student's *t*-test: **0.001<*P*<0.01; ***$P<0.001$. (c) GA content was decreased in *SlbHLH95-OE* plants. **0.001<*P*<0.01 (Student's *t*-test). (d) GA application rescued *SlbHLH95-OE* plant size. (e) Plant size of *SlbHLH95-OE* compared with WT plants after GA application. Values are means ±SD (n≥15) of three replicates. The error bars and letters in the graph indicate the SEs among samples and significant differences in plant size evaluated by Tukey's test (α<0.05). (f) Internode length in *SlbHLH95-OE* plants treated with exogenous GA compared with that of WT plants. Values are the mean ±SD (n≥15) of three replicates. (g) Trichome density was partially rescued by exogenous GA application in *SlbHLH95-OE* lines. The white bars represent 2 mm. (h) Fruit shape rescued by exogenous GA application in *SlbHLH95-OE* lines. The white bars represent 1 cm. (i) The fruit shape index of GA₄-treated and untreated plants. Values are means ±SD (n≥10) of three replicates. The error bars and letters in the graph indicate the SEs among samples and significant differences in plant size evaluated by Tukey's test (α<0.05). (j) The fruit shape triangle of GA₄-treated and untreated plants. Values are means ±SD (n≥10) of three replicates. The error bars and letters in the graph indicate the SEs among samples and significant differences in plant size evaluated by Dunnett's T3 test (α<0.05). (This figure is available in colour at JXB online.)</figcaption></figure>

biosynthesis. Overexpression of *SlbHLH95* resulted in reduced trichome density, reduced plant size, increased axillary buds, delayed flowering time, poor fruit set, and elongated fruit shape that resemble GA-deficient phenotypes. Supporting this notion, GA content was significantly reduced in *SlbHLH95-OE* lines due to lower expression of two key GA biosynthesis genes, *SIGA20ox2* and *SIKS5*. The role of GA is further sustained by the rescue of the phenotypes by exogenous treatment of *SlbHLH95-OE* lines with the hormone. Yeast one-hybrid and transient expression assays showed that SlbHLH95 regulates the expression of *SIGA20ox2* and *KS5* via binding to their promoters. Altogether, these data validate the link between SlbHLH95, GA, and trichome formation in tomato.

It was reported that the bHLH TFs GL3 and EGL3 regulate trichome development in a partially redundant manner (Payne et al., 2000; Zhang, 2003), and other members of the bHLH gene family, such as AtMYC1 and TRANSPARENT TESTA 8 (TT8), have also been shown to positively regulate trichome

development in Arabidopsis (Maes et al., 2008; Zhao et al., 2012). The present study indicates that bHLH-type TFs are not only essential for trichome formation in Arabidopsis, but they are also involved in this process in tomato. This finding is consistent with recent reports on the role of SlMYC1, another bHLH family gene, that positively regulates type VI glandular trichome formation in tomato (Xu et al., 2018). Interestingly, unlike other reported bHLH TFs, such as SlMYC1 which play a positive role in type VI trichome development (Payne et al., 2000; Zhang, 2003; Maes et al., 2008; Zhao et al., 2012; Shangguan et al., 2016; Xu et al., 2018), overexpression of SlbHLH95 in tomato resulted in dramatically reduced type I trichomes on stems and significantly fewer type V trichomes on leaves.

Mutation in the C2H2 ZFP gene (*Solyc10g078970*) has been suggested to account for the glabrous phenotype of IL10-2 and the *hair absent* (*h*) mutant LA3172 (*Chang et al.*, 2018). Our results demonstrate that the TF gene *SlbHLH95*
biosynthesis via reducing expression of *AtGA20ox2* (Andrés *et al.*, 2014). In contrast, very little is known about the direct regulation of GA biosynthesis genes in tomato. Our data reveal that SlbHLH95 regulates the expression of two key GA biosynthesis genes, *SlGA20ox2* and *SlKS5*, via direct binding to their promoters, thus extending our understanding of the regulation of GA biosynthesis in tomato. While the present data reveal that GA is involved in trichome development, additional studies are needed to uncover the molecular mechanisms underlying GA regulation of trichome formation in tomato. In particular, it remains to be investigated whether trichome formation involving SlbHLH95 is also mediated by a complex similar to the MYB-bHLH-WDR complex described in Arabidopsis.

## Supplementary data

Supplementary data are available at JXB online.

Fig. S1. The glabrous phenotype exhibited by *SlbHLH95-OE* young fruit.

Fig. S2. Reproductive development of *SlbHLH95-overexpressing* lines.

Fig. S3. The gene expression distribution (FPKM) in WT and *SlbHLH95-OE* lines.

Fig. S4. Expression levels of trichome-related genes in WT and *SlbHLH95-OE* lines.

Table S1. Genes contained in the *S. pennellii* genome fragment introgressed in M82 to give rise to the IL10-2 line.

Table S2. Putative bHLH-binding cis-elements present in the promoter regions of *SlGA20ox2*, *SlKS5*.

Table S3. List of the primers used in the study.

Dataset S1. Quality assessment of sequencing data.

Dataset S2. Reads mapping to the reference genome.

Dataset S3. The expression levels of genes in *SlbHLH95-OE* and wild-type (WT) lines.

Dataset S4. Pearson correlation coefficient analysis.

Dataset S5. GO enrichment analysis of DEGs between *SlbHLH95-OE* (*b95_OE*) and WT lines.

Dataset S6. KEGG enrichment analysis of DEGs between *SlbHLH95-OE* (*b95_OE*) and WT lines.

Dataset S7. The expression levels of MYB, bHLH and WD40 family genes in *SlbHLH95-OE* and WT lines.

## Acknowledgements

This research was supported by the National Key R&D Program of China (2016YFD0400100) and the National Natural Science Foundation of China (31772372) to ML, and this project is also supported by the Fundamental Research Funds for the Central Universities (SCU2019D013). Yao Chen was sponsored by the China Scholarship Council.

## Author contributions

ML planned and designed the research; Yao Chen, DS, JL, SY, and HD performed the experiments; XH, YZ, YL, Ya Chen, JP, and YL analyzed the data; and YC, ML, and MB wrote the manuscript.

## Data availability

All relevant data are within the paper and its supplementary files.

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