Impact of Bikinin on Brassinosteroid Signaling Pathway and its Influence on Barley Development

doi:10.21203/rs.3.rs-4157821/v1

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Impact of Bikinin on Brassinosteroid Signaling Pathway and its Influence on Barley Development

https://doi.org/10.21203/rs.3.rs-4157821/v1

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Impact of bikinin, a Glycogen Synthase Kinase 3 (GSK3) inhibitor, on long-term response of barley after spraying. Using three different concentrations of 10 µM, 50 µM, and 100 µM bikinin led to distinct phenotypic changes in shoot and root growth, leaf development, and photosystem II efficiency. Transcriptomic analysis revealed genotype-dependent responses, with Golden Promise exhibiting more significant changes than Haruna Nijo. Expression pattern of genes controlling the Brassinosteroid signaling pathway, as well as Western blot analysis, showed constitutive expression of HvGSK genes and HvGSK2.1 kinase activity in barley after bikinin treatment. On the other hand, analysis showed varied phosphorylation levels of HvBZR1 in response to high concentrations of bikinin, particularly in Golden Promise. The study contributes to understanding the intricate role of GSK3 in barley growth and the genotype-dependent effects of bikinin. Additionally, the research highlights the potential of bikinin as a tool for studying the Brassinosteroid-dependent pathways. This study provides valuable insights into the molecular mechanisms underlying barley responses to bikinin, shedding light on the complex interplay between GSK, BZR1, and Brassinosteroids in monocotyledonous plants.

GSK3

Golden Promise

Haruna Nijo

BZR1

RNA-seq

The world's growing population is putting pressure on agriculture, breeding companies, and scientists who are expected to produce high-productivity (high-yielding), disease-resistant crops that will provide essential nutrients (Hemathilake and Gunathilake 2022). On the other hand, the European Commission puts pressure on the implementation of organic crops, which increases production costs, which in turn limits the availability of food for less affluent communities. These goals included in many breeding programs require the use of precise tools, including in the aspect of implementing methods of new genomic techniques to improve grain quality. Breeding barley for improved agronomic traits is primarily focused on yield, disease tolerance, and resistance, but also on making the best use of plant energy resources. Improvements in several important traits were observed after the application of Brassinolide (BL), the most active phytohormone in the Brassinosteroid (BR) group.

The Glycogen Synthase Kinase 3 (GSK3) family is highly conserved in many species, including mammals. About 70% similarity of GSK3β human protein was detected in Arabidopsis (Arabidopsis thaliana L.) Brassinosteroid Insensitive 2 (BIN2) kinase (Saidi et al. 2012; Li et al. 2001; Li and Nam 2002), the best-characterized kinase from the second group of plant GSK3s. In plants, the GSK3s family is represented by four groups (Saidi et al. 2012). Ten genes are known in Arabidopsis (Charrier et al. 2002), nine genes in rice (Oryza sativa L.) (Yoo et al. 2006), eleven in maize (Zea mays L.) (Li et al. 2022), and seven in barley (Hordeum vulgare L.) (Groszyk et al. 2018). Similar to mammals, GSK3s in plants control various developmental processes, mainly by regulating transcription factors (TFs). These TFs influence the development of the root, stomata, floral organs, seeds, hypocotyl elongation, fruit ripening, sugar content in fruits, as well as responses to environmental stress such as salt, drought, cold, and biotic stress (Mao et al. 2021). Among the important regulatory functions of GSK3s are cell division and elongation, as well as phytohormonal regulation. At low concentrations of BRs, GSK3s phosphorylates Brassinazole Resistant 1 (BZR1) TF, leading to its proteasomal degradation (He et al. 2002). BZR1 controls several steps of BR biosynthesis by inhibiting consecutive steps of the biosynthesis pathway (Rozhon et al. 2014). During the low activity of BZR1, BRs are highly produced. However, the end-product of BR biosynthesis, BL, regulates consecutive steps of the BR biosynthesis pathway by the regulation of the BR signaling pathway. BL molecules are recognized and bound by Brassinosteroid Insensitive 1 (BRI1), leading to conformational changes in this receptor and the disconnection of BRI1 Kinase Inhibitor 1 (BKI1) (Wang and Chory 2006), and association with BRI1 Associated Receptor Kinase 1 (BAK1). The BRI1-BAK1 transmembrane receptor initiates a cascade of phosphorylation and dephosphorylation of cytoplasmic relay proteins, leading to the dephosphorylation and inactivation of GSK3s. Maintaining the constitutive activity of this and other proteins is essential for proper plant development; however, due to its growth in natural conditions, the plant is exposed to many stress factors, and this effectively disrupts this arrangement.

Given the numerous positive changes resulting from modifying the expression of genes from the GSK3 family, it is crucial to understand the range of kinase activity in which their participation is essential. Limiting it to the range were reducing protein levels or their activity positively affects plant biomass and yield is necessary. Plants with inhibited expression of OsGSK1 gene exhibited an enhanced response to drought in rice (Oryza sativa L.) (Koh et al. 2007) and increased grain size (Che et al. 2015). In barley (Hordeum vulgare L.), reduced expression of HvGSK1.1 gene was associated with a higher thousand-grain weight and improved salt stress response (Kloc et al. 2020). A lower concentrations of 24-EBL (0.1 nM–2.0 nM) have been observed to enhance root growth in potato (Solanum tuberosum L.) (Hu et al. 2016) and apple seedlings (Malus hupehensis Rehd.) (Mao et al. 2017). In the case of carrot (Daucus carota L.) plants treated with 0.001 µM 24-EBL had an increase number of petioles and improved aboveground weight (Que et al. 2017). Additionally, the application of 0.0001 µM 24-EBL under salinity stress conditions stimulated stem elongation in potato, accompanied by enhanced activity of antioxidant enzyme (POD, SOD, and CAT) and altered K⁺/Na⁺ content in the stem (Hu et al. 2016).

Bikinin was identified as one of 10,000 compounds that induced constitutive responses in Arabidopsis by the inhibiting the BIN2 (De Rybel et al. 2009). When using bikinin, a significant increase in hypocotyl length, long and bending petioles, and blade-shaped, pale-green leaves was observed. These effects were comparable to those obtained with treatment with BL at micromolar concentrations, resulting in reduced lateral root density (De Rybel et al. 2009). Subsequent analyses with bikinin and its inactive variant revealed treatment-induced petiole and hypocotyl elongation under both light and darkness-grown conditions. Similar changes were observed in barley during the lamina joint inclination (LJI) test and germination with dilutions of 24-EBL and bikinin (Groszyk and Szechyńska-Hebda 2021b). These observations were consistent in both the leaf LJI test and the phenotype characterization of five-day-old barley seedlings. In summary, GSK3s play an essential role in shaping plant architecture. Although the role of the GSK3 genes has been well studied in Arabidopsis, the function of the GSK3 genes in monocots, including barley, remains to be elucidated.

Transcript profiling via RNA sequencing is a quantitative method used to compare transcriptional differences over the developmental time course and under various treatments within and between crop plants (Croucher et al. 2009; Xiao et al. 2013). Under biotic and abiotic stresses, plants exhibit significant alterations in their gene expression profiles and metabolism to resist the damage caused by negative stress factors (Smita et al. 2020). Firstly, plants receive environmental cues and then respond by swiftly altering gene expression patterns, inducing or suppressing the activity of various kinases, and initiating the production and signaling of hormones and hormone-like molecules to timely respond to stress and minimize damage (Smita et al. 2020). Therefore, transcriptional changes represent the first step in a plant’s response to environmental stress, followed by physiological and phenotypic changes. Analysis of gene expression networks and pathway enrichment after bikinin application can help identify genes and regulatory pathways related to GSK3s inhibition during barley development.

Harmonizing the above requires a thorough understanding of gene functions. In this study, we present the effects of applying bikinin in the form of a spray, a method hitherto unexplored in monocot species. We hypothesized that the application of a specific inhibitor for GSK3 would allow us to determine the role of this kinase family in barley development and the shaping of traits desirable in breeding. We present here the effects at the phenotype and genotype levels after applying three concentrations of bikinin as a spray. These results are discussed in the context of earlier studies using these genotypes, demonstrating that strong inhibition of the BR signaling pathway can lead to undesirable traits. Both barley varieties have been utilized in previous research (Groszyk and Przyborowski 2022; Groszyk and Szechyńska-Hebda 2021b, a).

Plant materials and growth conditions. The barley (Hordeum vulgare L.) cultivars Golden Promise (GP) and Haruna Nijo (HN) were obtained from gene banks, namely the United States Department of Agriculture, GRAIN-Global, USA (accession number 343079), and Gene Bank Dept., CRI Prague-Ruzyně (accession number 03C0602168), respectively. Both cultivars served as parental stains to obtain single seed descent lines, which were utilized in this study. The grains were placed in a single Petri dish with three layers of filter paper, and 10 ml of spring water was added for each cultivar. They were imbibed at 4°C in dark conditions for 72 hours and then transferred to 23°C for an additional 72 hours. Eight seedlings with similar lengths of coleoptile and roots were planted in 1 dm³ pots (height = 13 cm), filled with a soil substrate and sand mixture (4:1). The plants were cultivated in a growth chamber under a 16-hour photoperiod at 20°C/18°C (day/night) with a daylight intensity of about 200 µmol photons m² s^− 1 and 70% humidity.

Bikinin treatment. Bikinin (Sigma) was dissolved in DMSO (Sigma). For bikinin treatments, eight/nine-day-old barley seedlings at the BBCH12 stage (stage of two developed leaves) were subjected to 10 µM, 50 µM, and 100 µM bikinin treatment, each with 0.3% Tween 20 (Sigma). An identical concentration of the DMSO, serving as a solvent solution with 0.3% Tween 20, was utilized as a control treatment (CK).

Barleys’ analyses. GP and HN at BBCH12 were treated with CK, BK10, BK50, and BK100 as previously (Groszyk and Przyborowski 2022). Plants were analyzed ten days after treatment. The analysis at this stage included measurements of shoot and root fresh and dry weight, length, area and length of consecutive leaves, chlorophyll a fluorescence of the 4th leaf, transcriptome analysis (CK, BK10, and BK100) in the 3rd and 4th leaves combined, expression profile of the BR signaling genes, HvGSK2.1 amount, and HvBZR1 activity.

RNA extraction. Total RNA was extracted from 3rd and 4th leaves ground into powder in liquid nitrogen using TRI Reagent Solutions (Invitrogen). Verification of RNA quality and quantity was conducted using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies) and electrophoresis in 1.5% agarose gels. Total RNA extraction process was performed in triplicate for each biological sample.

RNA sequencing. A total of 3 µg of total RNA was subjected to genomic DNA removal using DNase I, RNase-free (Thermo Fisher Scientific) for CK, BK10, and BK100 samples. Verification of RNA quality and quantity was carried out using a 2100 Bioanalyzer (Agilent). Library preparation and sequencing were conducted by BGI Co. Ltd using the BGI 500 platform. The barley genome and gene information for reference cultivar GPv1 (GCA_902500625.1) (Schreiber et al. 2020) were obtained from the Ensemble Plants database during the alignment setup (Yates et al. 2022). HISAT2 was employed for mapping quality and saturation analysis (Kim et al. 2019). DESeq2 analysis was subsequently performed to determine differential expression (Love et al. 2014). The analyses were executed using the Interdisciplinary Centre for Mathematical and Computational Modelling UW and Galaxy software (Afgan et al. 2022), respectively. Results were imported into Integrated Differential Expression and Pathway analysis (iDEP 1.13) software (Ge et al. 2018) for filtering with log2 fold change ≥ 2 or ≤ -2, FDR ≤ 0.01. Gene Ontology analysis after bikinin treatment was conducted using this software.

cDNA synthesis and Real-Time PCR were conducted as follows: 3 µg of total RNA underwent genomic DNA removal using DNase I, RNase-free (Thermo Fisher Scientific). Subsequently, 1 µg of total RNA was reverse-transcribed to cDNA using the Revert Aid cDNA Synthesis Kit (Thermo Fisher Scientific) following the to manufacturer’s protocol. Real-Time PCR analysis was carried out using the 5x HOT FIREPol EvaGreen qPCR Mix Plus (noROX) (Solis BioDyne) and a Rotor Gene 6000 Series thermal cycler (Corbett) according to the manufacturer's protocol. The cDNA synthesis was performed in triplicate for each biological sample, and Real-Time PCR had three technical repeats for each sample. Barley's ADP-ribosylation factor and GAPDH genes served as internal controls to calculate the relative expression level of the investigated genes. The gene specific primers used for Real-Time PCR were published by (Groszyk et al. 2018) and (Groszyk and Szechyńska-Hebda 2021a).

Western blot detection of HvGSK2 and HvBZR1 was conducted as follows: The 3rd and 4th leaves of GP and HN were collected with three biological replicates. Subsequently, the samples were ground into powder in liquid nitrogen and used for protein extraction with 1x SDS sample buffer (5 µl per 1 mg) as previously (Groszyk and Szechyńska-Hebda 2021a). Total protein was separated under SDS-PAGE and utilized for immunoblot analysis, as described in the above study. Commercial anti-OsGSK2 (Beijing Protein Innovation Co) and anti-OsBZR1 (Agrisera) polyclonal antibodies were employed for the detection of HvGSK2.1 and HvBZR1, respectively. Detection was performed using a PVDF membrane (Bio-Rad), AgriseraECL Super Bright (Agrisera), and Ilford Delta 400 photographic film (Herman Technologies). The film was scanned using an Epson Perfection V850 Pro scanner.

Physiological traits were measured as follows: Plants from both experimental setups were subjected to phenotypical analysis. Initially, the plants were photographed for documentation. Chlorophyl a fluorescence was measured in the 4th leaf using a fluorometer (FluorPen FP 100, ICT International) and leaf-clips with a window diameter of approximately 3 mm, according manufacturer’s protocol with an adaptation to darkness of about 30 min. Subsequently, the fresh weight of both shoot and root was measured. Following the fresh weight measurement, the plants were subjected to drying, initially at 100°C for 24 hours, followed by an additional 24 hours at 30°C. Finally, the dry weight of the respective shoot and root was determined. For the leaves from the first experimental setup, a scanning process was conducted, and the area and length of the leaves were analyzed using ImageJ software. The data collected were organized in Microsoft Excel and utilized for subsequent statistical analysis.

Relative water content analysis was conducted using 5 cm fragments of the 3rd as previously (Groszyk and Przyborowski 2022). Fragments were consistently cut from the same region of the leaf across all tested plants. The procedure involved several steps: Fresh Weight (FW): Initially, the fresh weight of the leaf fragments was measured; Incubation: The leaves were then incubated under specific conditions (4°C for 3 hours); Turgid Weight (TW): After the incubation period, the turgid weight of the leaves was measured; Drying: Subsequently, the leaves were dried under specific conditions (80°C for 24 hours); Dry Weight (DW): The dry weight of the leaves was measured after the drying process. The RWC was calculated using the formula: ((FW - DW) / (TW - DW)) * 100 (Barrs and Weatherley 1962). This analysis provides insights into the water status of the barley leaves under different experimental conditions.

Phenotypic analysis of different barley cultivars after bikinin application

To assess the function of the Glycogen Synthase Kinase 3 (GSK3) family in barley development, we applied three different concentrations of the bikinin, to two unrelated barley cultivars with distinct sensitivities to exogenous Brassinosteroids (BRs): Golden Promise (GP) and more BR-sensitive Haruna Nijo (HN) (Groszyk and Szechyńska-Hebda 2021b). Phenotypic differences between both barley genotypes were examined during the stage before tillering, up to the beginning of the 5th leaf development stage (in the development phase BBCH20), as previously described (Groszyk and Przyborowski 2022). In this study, we focus on the long-term response of two barley cultivar seedlings treated with 10 µM bikinin (BK10), 50 µM bikinin (BK50), and 100 µM bikinin (BK100). The eight-day-old GP seedlings and nine-day-old HN seedlings (BBCH12) were sprayed with bikinin and 0.11% DMSO (CK). After ten days science spraying, the plants were phenotypically characterized, including measurement of the fresh and dry weight of shoot and root; length of shoot and root; length and area of the 1st, 2nd, 3rd, and 4th leaf; relative water content; chlorophyll fluorescence; HvBZR1 and HvGSK2.1 protein activity. Additionally, for selected samples, a transcriptome analysis was performed.

Similarly, to the previous study using these two cultivars, after applications of bikinin concentrations, we observed genotype- and treatment-dependent reactions determined by barley phenotypic traits (Table 1). The application of various concentrations of bikinin led to genotype- (p < 0.000) and treatment-dependent (p < 0.002) reactions, resulting in phenotypic changes in both barley cultivars (Fig. 1a, b). For GP, the fresh weight of the shoot increased in BK10 to 116.6% and decreased in BK100 to 93.9%, with no significant changes in BK50 plants (Fig. 1c). All bikinin concentrations inhibited the accumulation of fresh weight in the HN shoot, resulting in 97.8%, 99.6%, and 80.5% weight for BK10, BK50, and BK100 barley, respectively (Fig. 1c). The reaction of the barley genotypes to bikinin application was significantly reflected in the dry weight of the GP (BK10, 121.6%; BK50, 108.6%; BK100, 101.7%; r = 0.967, p ≤ 0.000) and HN (BK10, 94.1%; BK50, 91.5%; BK100, 75.7%%; r = 0.849, p ≤ 0.000) shoot (Fig. 1d). Compared to the weight parameters, changes after consecutive doses of bikinin were observed during the shoot length analysis (Fig. 1e). The lower concentration of bikinin led to the development of shorter shoots than the highest concentrations of bikinin in GP (BK10, 106.0%; BK50, 107.5%; BK100, 120.0%). In HN, a longer shoot developed in BK10 and BK50 plants (105.3% and 113.1%, respectively), while in BK100 led to results similar to CK plants (99.7%). The response of barley genotypes to bikinin treatment significantly differed for each genotype and bikinin concentration when analyzing the length and area of consecutive leaves (Fig. 2). The use of 50 µM and 100 µM bikinin significantly reduced the development of the first two leaves of HN. The length of the first and second leaf reduced to 80.7%-89.2%, while the area decreased to 82.7%-89.0% compared to control plants (Fig. 2a, b, e, f). Analysis of the first two leaves of GP after all concentrations of bikinin and BK10 HN resulted in parameters comparable to CK. Subsequently, newly unfolded leaves in HN showed negligible changes in the 3rd leaf length and area (Fig. c, g); slightly larger differences were observed in the 4th leaf, which was longer (108.3%) and had a larger area (108.8%) in BK10 (Fig. 2d, h). Significant changes were observed in BK50 HN, where application resulted in an increase in 4th leaf length (115.6%) and area (124.6%), as well as after the application of 100 µM bikinin that inhibited both parameters to 95.8% and 82.3%, respectively (Fig. 2d, h). In this cultivar, the length of the 4th leaf determined shoot length (r = 0.716, p ≤ 0.000). A greater variation in response to different concentrations of bikinin was observed in sequentially developing GP leaves. While the following has been applied, the length and area of the 3rd and 4th leaf were treatment-dependent (Table 1). 10 µM bikinin application resulted in negligible changes 3rd leaf (Fig. 2c) and longer 4th leaf with a greater area (135.4% and 158.0%, respectively) (Fig. 2d, e). 50 µM bikinin resulted in a significantly longer 3rd leaf (110.4%) about a larger area (106.3%) (Fig. 2c, g), with slightly reduced parameters of the 4th leaf, i.e. length to 95.3% and area to 89.5% (Fig. d, h). The greatest changes in the 3rd and 4th leaves were detected in BK100 plants (Fig. 2c, d, g, h). The 3rd and 4th leaves were significantly longer (132.4% and 122.3%, respectively), with the area of 3rd leaf increased to 108.2%, but the 4th leaf reduced to 91.7%. The data indicate that the length of the 3rd GP leaf significantly influences the length of the GP shoot (r = 0.812, p ≤ 0.000). Relative water content indicates that all plants present good conditions (Supplemental Fig. S1).

Table 1. Results of two-way ANOVA for two barley genotypes Golden Promise and Haruna Nijo treated with bikinin in three different concentrations, i.e., 10 µM, 50 µM, and 100 µM compared to plants treated with 0.11% DMSO calculated for aboveground and below-ground organ traits.

Parameters	Genotype			Treatment			*Genotype Treatment**
Parameters	MS	F	p	MS	F	p	MS	F	p
Shoot FW	4.5993	55.53	0.0000	0.3926	4.74	0.0024	0.1225	1.54	0.2054
Shoot DW	0.0996	132.22	0.0000	0.0048	6.31	0.0003	0.0037	4.91	0.0020
Shoot length	192.6042	29.04	0.0000	26.8413	4.05	0.0061	41.1229	10.62	0.0000
1^st leaf length	517.9635	365.71	0.0000	3.8981	2.75	0.0372	6.4365	6.34	0.0003
2^nd leaf length	294.1653	166.37	0.0000	2.8450	1.61	0.1866	7.8889	4.46	0.0037
3^rd leaf length	4.7540	1.67	0.2020	28.2101	9.92	0.0000	24.3518	8.56	0.0000
4^th leaf length	722.6969	97.78	0.0000	28.2535	3.82	0.0083	43.9528	9.84	0.0000
1^st leaf area	524.7965	554.07	0.0000	2.6711	2.82	0.0346	2.8786	3.04	0.0255
2^nd leaf area	546.1494	273.25	0.0000	4.8476	2.77	0.0374	5.0683	2.89	0.0314
3^rd leaf area	30.6664	16.23	0.0002	0.4590	0.24	0.9127	5.0400	3.08	0.0242
4^th leaf area	520.7232	105.84	0.0000	68.6702	13.96	0.0000	52.0185	10.57	0.0000
Root FW	0.0005	0.03	0.8537	0.1423	10.77	0.0000	0.0168	1.30	0.2820
Root DW	0.0064	50.31	0.0000	0.0015	11.52	0.0000	0.0001	0.60	0.6664
Root length	8.8935	0.38	0.5407	20.9898	0.90	0.4738	37.4568	1.60	0.1895
RWC	9.0561	8.30	0.0057	4.2324	3.88	0.0077	1.4891	1.41	0.2454
DWF4	0.0000	2.15	0.1577	0.0000	1.72	0.1856	0.0000	3.49	0.0255
BRI1	0.0003	4.43	0.0460	0.0001	0.69	0.6042	0.0001	1.16	0.3576
BAK1	0.0006	330.42	0.0000	0.0000	0.71	0.5919	0.0000	1.51	0.2385
BSU1	0.0000	0.00	0.9447	0.0001	0.44	0.7781	0.0001	0.42	0.7916
GSK1.1	0.0028	0.83	0.3702	0.0009	0.28	0.8908	0.0025	0.70	0.5994
GSK1.2	0.0021	0.91	0.3486	0.0010	0.42	0.7932	0.0034	1.62	0.2072
GSK1.3	0.0020	4.55	0.0433	0.0001	0.19	0.9394	0.0004	0.87	0.4991
GSK2.1	0.0273	8.06	0.0091	0.0007	0.21	0.9302	0.0029	0.84	0.5135
GSK2.2	0.0017	10.86	0.0030	0.0001	0.86	0.5026	0.0002	1.54	0.2292
GSK3.1	0.0010	2.80	0.1072	0.0001	0.41	0.8030	0.0005	1.57	0.2222
GSK4.1	0.0074	3.07	0.0952	0.0021	0.89	0.4883	0.0044	1.81	0.1670
BZR1	0.0000	1.02	0.3243	0.0000	0.47	0.7537	0.0000	0.59	0.6713
F_V/F_M	0.0008	34.26	0.0000	0.0001	4.78	0.0030	0.0000	0.86	0.4937
φP₀	0.0008	34.26	0.0000	0.0001	4.78	0.0030	0.0000	0.86	0.4937
Ψ₀	0.0175	94.85	0.0000	0.0007	3.85	0.0098	0.0007	3.55	0.0143
φE₀	0.0149	106.25	0.0000	0.0007	4.70	0.0033	0.0005	3.48	0.0158
φD₀	0.0008	34.26	0.0000	0.0001	4.78	0.0030	0.0000	0.86	0.4937
PI_ABS	4.3672	78.27	0.0000	0.2853	5.11	0.0020	0.0760	1.36	0.2640
ABS/RC	0.0274	11.25	0.0018	0.0175	7.18	0.0002	0.0079	3.24	0.0215
TR₀/RC	0.0063	4.80	0.0343	0.0081	6.14	0.0006	0.0061	4.64	0.0036
ET₀/RC	0.0235	53.66	0.0000	0.0023	5.31	0.0016	0.0051	11.67	0.0000
DI₀/RC	0.0073	27.96	0.0000	0.0020	7.79	0.0001	0.0001	0.50	0.7334

Two-way ANOVA indicates that depending on bikinin concentrations, shoot weight influenced root weight (p ≤ 0.001) (Table 1). The application of 10 µM bikinin in long-term response resulted in nonsignificant changes; however, 50 µM and 100 µM bikinin led to a reduction in fresh weight (BK50: 85.7% and 68.6%; BK100: 67.4% and 50.3%) and dry weight (BK50: 89.0% and 79.7%; BK100: 60.2% and 66.8%) in GP and HN, respectively (Fig. 1f, g). The length of the roots in GP and HN with consecutive concentrations of bikinin was not significant, with one exception; HN had longer roots in BK10 (108.3%, p ≤ 0.05) (Fig. 1h).

Barley transcriptome analysis after bikinin treatment

To elucidate the molecular mechanisms of barley after BK treatment, 3rd and 4th leaves were collected from GP and HN plants following CK, BK10, and BK100 variants. Each biological samples underwent three replicate treatments, resulting in a total of 18 libraires constructed for transcriptome analysis. Utilizing DNB sequencing technology (BGI Genomics Co., Ltd.), each sample generated over 60 million raw pair-end reads with a size 100 bp (Table 2). Following quality control, each sample yielded a minimum of 60,187,800 (GP BK100_1) and a maximum 73,790,912 (GP BK100_3) clean reads, with an average Q20 of approximately 96% (Table 2), indicative of high-quality sequencing data in the RNA-seq experiments. After the removal of adaptor sequences and low-quality reads, all clean reads were mapped to the barley reference genome GPv1 (Schreiber et al. 2020) using HISAT2 software (Kim et al. 2019). The results demonstrated that, on average, 32.89 million reads were mapped to the reference genome, with approximately 30.94 million reads being uniquely mapped. We identified 58,815 coding type genes, 3,126 nontranslating CDS, and 127 identified as a pseudogene (Supplemental Fig. S2). Principal component analysis (PCA) was performed on the RNA-seq dataset of 18 samples. The control samples of barley cultivars (HN and GP) and two doses of bikinin, 10 µM (BK10) and 100 µM bikinin (BK100) were distinctly differentiated by the first principal component (PC1), explaining 50.35% of the total variation, and PC2, which accounted for 11.89% of the total variation (Fig. 3a).

Table 2

Summary of RNA sequencing results.
Genotype	Treatment	Clean reads	Clean beases	Clean bases (GB)	Q20 (%)	GC (%)
GP	CK	68538918	6853891800	5.36	95.91%	54.68%
GP	CK	70336000	7033600000	5.48	95.94%	54.34%
GP	CK	72421912	7242191200	5.61	96.11%	53.92%
GP	BK10	64398886	6439888600	5.02	95.81%	54.43%
GP	BK10	65148758	6514875800	5.07	96.12%	54.58%
GP	BK10	73043922	7304392200	5.66	96.22%	53.94%
GP	BK100	60187800	6018780000	4.67	95.90%	54.62%
GP	BK100	65715288	6571528800	5.07	96.24%	54.47%
GP	BK100	73790912	7379091200	5.72	96.15%	54.11%
HN	CK	64174626	6417462600	4.92	96.28%	53.78%
HN	CK	64348198	6434819800	4.95	96.19%	54.10%
HN	CK	71423906	7142390600	5.53	96.12%	54.83%
HN	BK10	67858502	6785850200	5.22	96.24%	53.40%
HN	BK10	68091566	6809156600	5.28	96.13%	54.08%
HN	BK10	67886934	6788693400	5.26	96.09%	54.44%
HN	BK100	73010038	7301003800	5.60	96.25%	53.73%
HN	BK100	71828166	7182816600	5.55	96.32%	54.52%
HN	BK100	71812666	7181266600	5.54	96.23%	53.98%

Identification of DEGs regulated by bikinin

Differentially expressed genes (DEGs) between the combinations GP_BK10-GP_CK, GP_BK100-GP_CK, GP_BK100-GP_BK10, HN_BK10-HN_CK, HN_BK100-HN_CK, HN_BK100-HN_BK10 were identified for FDR < 0.01 and log2FC| > 2) (Fig. 3b). The DEGs were identified by comparing the expression levels of genes in GP and HN barley in CK, BK10 and BK100 variants, thereby elucidating genes associated with the long response of barley to bikinin treatment. Among the total 272 significant DEGs, 160 genes were upregulated (Fig. 3c), and 112 genes were downregulated (Fig. 3d) in GP, while in HN, 37 genes were upregulated (Fig. 3e), and 49 genes were downregulated (Fig. 3f) after bikinin treatment. When DEGs were compared between all the above bikinin variants, 2 upregulated and 2 downregulated genes in GP were common for BK10 and BK100 (Fig. 3c, d). In contrast to the common DEGs, 13 upregulated and 2 downregulated genes were specific for BK10 GP; 145 upregulated and 108 downregulated genes were specific for BK100 GP; 26 upregulated and 16 downregulated genes were specific for BK10 HN; and 11 upregulated and 33 downregulated were specific for BK100 HN (Fig. 3e, f). Moreover, by comparing the transcriptome profiles of barley cultivars in BK10 and BK100, a total of 78 upregulated and 162 downregulated genes were identified in GP, and 68 upregulated and 234 downregulated genes were identified in HN, suggested treatment-dependent reaction of barley to bikinin concentration. Furthermore, a total of 54 induced and 59 repressed genes in GP, and 5 induced and 23 repressed genes in HN were identified as common DEGs between barley treated with 100 µM bikinin, suggesting that both concentrations of bikinin lead to opposite reactions.

Functional annotation of bikinin-induced DEGs

To elucidate the function of bikinin-responsive genes in barley cultivars, an enrichment analysis of Gene Ontology (GO) function was conducted on DEGs. The results revealed that 5 DEGs specifically detected in BK10 GP, annotated to 10 GO terms in the molecular function category (Fig. 4a). Among the upregulated genes, annotations fell under 3 GO terms: intramolecular lyase activity, isomerase activity, and hydroxymethylglutaryl-CoA reductase (NADPH) activity. 1 of the 2 downregulated genes was annotated under 4 GO terms, including alpha-glucosidase activity, glucosidase activity, sucrose alpha-glucosidase activity, and beta-fructofuranosidase activity. A total of 9 DEGs in BK10 HN were annotated to 5 GO terms in the molecular function category (Fig. 4b). The upregulated genes were annotated to DNA-binding transcription factor activity, transcription regulator activity, and glutathione transferase activity. Moreover, 10 µM bikinin regulated the activities of carbon-oxygen lyase activity pathways in both barley genotypes but in specific parts. In GP, 1 gene regulated 3 GO terms: tryptophan synthase activity, hydro-lyase activity, and carbon-oxygen lyase activity. In HN, the gene regulated terpene synthase activity, and carbon-oxygen lyase activity acting on phosphates. After 100 µM bikinin treatment, a greater number of DEGs were identified in GP compared to HN. In GP after the application of 100 µM bikinin, 67 DEGs were detected and annotated to 41 GO terms across 3 categories: biological processes, molecular function, and cellular components (Fig. 4c). Under biological processes, 37 genes were related to the organic substance biosynthetic process, with 35 genes of them annotated to the biosynthetic process and cellular biosynthetic process. The most abundant terms were amine and peptide biosynthetic processes, translation, and cellular amide and peptide metabolic processes. In the molecular function category, 20 genes belonged to oxidoreductase activity, and 15 genes were associated with structural constituent of ribosome. In the cellular component category, the 24 genes mainly fell under intracellular non-membrane-bounded organelle, ribosome, and extracellular region. For BK100 HN, 3 genes were uniquely differentially expressed and functionally annotated. These DEGs were enriched in 8 GO terms across the biological processes and molecular functions categories (Fig. 4d). 1 gene was annotated to the biological process of terpenoid metabolic process, and the other 2 genes were associated with molecular functions, including inositol heptakisphosphate kinase activity and fatty-acyl-CoA reductase (alcohol-forming) activity. The overall distribution between GP and HN with bikinin exhibited differences, with the number of genes involved in each GO term and the number of GO terms being genotype- and treatment-dependent.

Photosystem II efficiency

A total of 2 genes uniquely differentially expressed in the GP treated with 100 µM bikinin were functionally annotated to biological processes under GO terms, specifically related to photosynthetic electron transport in photosystem II (PSII) (Fig. 4c). Analysis of normalized data counts showed that the transcript levels of these genes were upregulated after 100 µM bikinin application, reaching 141.61% and 123.43% in GP and 117.81% and 112.67% in HN, respectively (Fig. 6a). Evaluation of consecutive parameters of chlorophyl fluorescence (Fig. 6b) revealed that GP treated with BK100 exhibited higher light absorption (ABS/RC) compared to HN. Simultaneously, increased dissipation of light energy as a heat (DI₀/RC, φD₀) resulted in a reduced performed index (PI_ABS) in GP. Lower trapped energy flux (ET₀/RC, TR₀/RC) in HN contributed to a decreased photosynthetic electron transport (Fig. 5b, c). The quantum yield of the primary PSII photochemistry and electron transport (φP₀, φE₀, Ψ₀) was negligible in the leaves of both barley cultivars. Conversely, changes in PSII efficiency under bikinin treatment in the fourth leaf were impaired, suggesting that plants were readjusting to homeostasis. Statistical analysis indicated that the parameters of the efficiency of PSII, such as quantum yield of the primary PSII photochemistry (F_V/F₀), efficiency and flow of energy (F_V/F_M, ψ₀, φP₀, φE₀, φD₀), and performance index (PI_ABS), depended on genotype, bikinin concentrations, and barley responses to bikinin treatment (Table 1).

Gene expression analysis and protein activity

Consecutive Rael-Time PCR analysis of few genes controlling the BR biosynthesis pathway, HvDWF4 (Fig. 6a), BR signaling pathway, HvBRI1, HvBAK1, HvBSU1, HvGSK1.1, HvGSK1.2, HvGSK1.3, HvGSK2.1, HvGSK2.2, HvGSK3.1, HvGSK4.1 (Fig. 6b-k), and transcription factor, HvBZR1 (Fig. 6L) indicates, that only five of them – HvBRI1, HvBAK1, HvGSK1.3, HvGSK2.1, and HvGSK2.2 – exhibit a genotype-dependent response (Table 1); however, none of them shows a reaction dependent on bikinin concentrations (Table 1).

To elucidate the role of bikinin in the regulation of HvGSK2.1 and HvBZR1 activity, Western blot analysis was conducted using the 3rd and 4th leaves from both GP and HN treated with 10 µM, 50 µM, and 100 µM bikinin. Similarly to results obtained in five-day-old barley shoots (Groszyk and Szechyńska-Hebda 2021a), in leaves, we detected HvGSK2.1 and phosphorylated form of HvBZR1 at two negligible different levels (Fig. 7). As in five-days-old barley seedlings, HvGSK2.1 was detected at a consistently level in all samples. Contrary to previous study, in HN leaves, the dephosphorylated form of HvBZR1 was undetected, even after bikinin treatment. However, in GP leaves, both 50 µM and 100 µM bikinin treatments resulted in a reduction of pBZR1 accumulation. The HvGSK2.1 levels remained comparable across all samples.

To date, numerous positive effects induced by the application of 24-epibrassinolide (24-EBL) have been demonstrated, among which notable improvements include stress resistance and increased biomass or plant yield. Similar results have been achieved in transgenic plants with reduced or induced expression of GSK3 genes, encoding Glycogen Synthase Kinase 3 (GSK3) family. Rozhon et al. (Rozhon et al. 2014) showed that bikinin is an effective and specific inhibitor of the GSK3 family, eliciting a phenotypic response in Arabidopsis (Arabidopsis thaliana L.) plants treated with bikinin similar to that observed after treatment with 24-EBL. While there are a few reports on the lamina joint inclination (LJI) and the influence of Brassinosteroids (BRs) (Liu et al. 2018; Sun et al. 2015), there is a lack of work examining the long-term influence of bikinin in monocotyledonous plants. Thus far, it has been demonstrated that 50 µM bikinin and 0.5 µM 24-EBL lead to comparable phenotypic changes in barley during the LJI test; however, lower or higher dosages than these resulted in different genotype-dependent LJI (Groszyk and Szechyńska-Hebda 2021b).

Our research supports the theory that BRs play a crucial role in various developmental processes, and bikinin can be employed to analyze the role of GSK3 in barley development. In this study, we present the results of treatment with three doses of bikinin and the barley plants' response ten days after spraying. At this stage, the plants developed new leaves, specifically the 3rd and 4th leaf, which exhibited a delayed response to bikinin treatment. The objective of our experimental setup was to enhance our understanding of GSK3 involvement barley growth and to identify key elements in the transcriptome that are either downregulated or upregulated by bikinin. Contrary to our initial hypothesis, this study confirmed genotype- and treatment-dependent reactions in response to our interference in the BRs signaling pathway using bikinin (Table 3), as demonstrated in our previous research (Groszyk and Szechyńska-Hebda 2021b, a). Additionally, we observed limited common elements between GP and HN (Fig. 4). Consistent with the LJI results determined for GP and HN after incubation in bikinin solutions (Groszyk and Szechyńska-Hebda 2021b), these genotypes exhibited distinct phenotypical changes for tested dosages (Fig. 1, 2). As observed, spraying 100 µM bikinin led to a more significant reduction in shoot and root weight parameters, elongation of the 3rd leaf (Fig. 1a, Fig. 2c, g), changes in the activity of more than 257 DEGs (Fig. 4c), and HvBZR1 activity in GP compared to HN. For the same bikinin dosage, transcriptome analysis of HN revealed altered expression of 302 DEGs (Fig. 3b); however, phenotypical changes were less conspicuous. Analysis of the bikinin-treated barley transcriptome indicated the activation of other genotype-dependent pathways and displayed a milder response at the phenotype level in HN, a genotype considered more sensitive to 24-EBL and bikinin than GP (Groszyk and Szechyńska-Hebda 2021b). The impact of a high concentration of 24-EBL on the long-term response of plants has not been tested in monocots to date, making it challenging to relate the results of the 100 µM bikinin dose to the work of other researchers. However, elongation or inhibition of different plant parts after the application of BR signaling pathway inhibitors has been observed in the last decade. While 1 µM BL induced epicotyl elongation, 10 µM reduced them in soybean (Glycine max) (Yin et al. 2019). Stimulation of root development was observed in barrel medic (Medicago truncatula Gaertn.) after application of 0.005 µM to 0.05 µM EBL; however, higher dosages inhibited it (Cheng et al. 2017). The 1 µM 24-EBL led to elongation of the coleoptile in HN, while the same concentration resulted in the reduction of shoot, coleoptile, and root length in GP (Groszyk and Szechyńska-Hebda 2021b). In the same study, 1 µM and 10 µM bikinin stimulated root elongation in both cultivars, even during salinity stress induced 150 mM NaCl (Groszyk and Szechyńska-Hebda 2021b). On the other hand, 50 µM bikinin reduced all measured parameters, similar to 10 µM and 50 µM Brassinazole, an inhibitor of BR biosynthesis (Groszyk and Szechyńska-Hebda 2021b). These changes, primarily phenotypic, are akin to those observed in soybean (Glycine max L.) after treatment with 1 µM and 5 µM Propiconazole, another inhibitor of BRs biosynthesis (Song et al. 2019). The results clearly indicate impaired growth, suggesting that GSK3s play an extremely important role in plant development.

In accordance with the hypothesis, the results of Western blot analysis confirmed that GSK3 is a constitutively active kinase (Xu et al. 2009; Li and Nam 2002). BZR1 in the 3rd and 4th leaves was detected in a phosphorylated form at two levels (Fig. 7), while the active form of BZR1 in barley leaves was undetectable (Groszyk and Szechyńska-Hebda 2021a). The results might suggest that the application of bikinin in the highest concentration (100 µM) resulted in the deactivation of GSK3 by binding with LEYV and MEYV motifs, a bikinin-binding domains characteristic for second (MEYV), and first and third (LEYV) groups of GSK3 (De Rybel et al. 2009), respectively, leading to lower amounts of pBZR1 in BK50 and BK100 GP plants (Fig. 7). Based on the findings of similar studies, a more plausible explanation is that active BZR1 in leaves may exist at low and undetectable concentration (Groszyk and Szechyńska-Hebda 2021a), and the phosphorylation of the BZR1 by GSK3 is one of the mechanisms that stabilize BZR1 in the cytoplasm. To date, several security proteins for BZR1 have been identified (Gampala et al. 2007; Zhang et al. 2016; Srivastava et al. 2020; Oh et al. 2014). Moreover, similar to previous research with GP and HN, HvBZR1 was detected in greater amounts than HvGSK2.1 (BIN2 ortologs’), and contrary to that, the expression level of GSK3 genes were from 4- fold to 11-fold higher than HvBZR1. All genes controlling BR biosynthesis, HvDWF4; BR signaling pathway, HvBRI1, HvBAK1, HvBSU1, HvGSK1.1, HvGSK1.2, HvGSK1.3, HvGSK2.1, HvGSK2.2, HvGSK3.1, HvGSK4.1; and transcription factor, HvBZR1 were unchanged after bikinin treatment (Fig. 6). The experiment provides new insight into the relationship between activity of HvGSK2.1 and HvBZR1 (Fig. 7), gene expression, and barley phenotype (Fig. 1, 2). An important confirmation of this thesis can also be found in the fact that the GSK3 gene orthologs identified so far in barley, despite phenotypic changes in plants, are expressed at a constant genotype-dependent level.

While our previous research from the same experimental setup has focused on bikinin alleviation of the salinity effects (Groszyk and Przyborowski 2022), these results demonstrate that bikinin in high concentration can lead to inhibition of barley growth. Moreover, these results demonstrate that direct inhibitor of GSK3, bikinin, can improve our knowledge about role of these kinase in monocots growth. Due to the lack of data on the time between barley spraying and the day of the performed analysis, the results cannot confirm changes in plant phenotype in the consecutive developmental stage between BBCH12 and BBCH20. However this parts of results present important role of GSK3 during barley development. Further research is needed to establish how inhibition of GSK3 can influence barley development after the tillering stage, spike development, and ultimately, barley yield.

Acknowledgments: The Galaxy server that was used for some calculations is in part funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry of Education and Research (BMBF grants 031 A538A/A538C RBC, 031L0101B/031L0101C de.NBI-epi, 031L0106 de.STAIR (de.NBI)).

Funding: This research was funded by the Plant Breeding and Acclimatization Institute–National Research Institute, Statutory Grant for Young Scientist 2020 (Radzików, Poland), grant number 1-1-04-3-01 (J.G.); National Science Centre (Kraków, Poland), grant number DEC-2020/04/X/NZ9/00037 (J.G.); and Interdisciplinary Centre for Mathematical and Computational Modelling Warsaw University (Poland), grant number G84-42 (J.G.).

Data Availability Statement

The data presented in this study will be openly available in NCBI SRA at (will be downloaded) (day-month-year); reference data, xxx; temporary submission ID: xxx; release date, xxx.

Conflicts of Interest

The authors declare no conflict of interest.

AUTHOR CONTRIBUTION STATEMENTS

Conceptualization: J.G.; Methodology: J.G., M.P.; Validation: J.G.; Formal analysis: J.G.; Investigation: J.G.; Resources: J.G.; Data Curation: M.P., J.G.; Writing - Original Draft: J.G.; Writing - Review & Editing: M.P., J.G.; Visualization: J.G.; Supervision: J.G.; Project administration: J.G.; Funding acquisition: J.G.

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Impact of Bikinin on Brassinosteroid Signaling Pathway and its Influence on Barley Development

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