LRR1 involved in the abscisic acid signaling pathway to regulate the early growth and development of Arabidopsis thaliana

Plant materials and growth conditions

The Columbia-0 ecotype of Arabidopsis was used in all assays. The LRR1 (At5g16590) T-DNA insertion line SALK_053366 (lrr1-1), SAIL_412_D10 (lrr1-2), and the KIN7 (At3g02880) T-DNA insertion line SALK_001905 (kin7) were obtained from the European Arabidopsis Inventory Center. The genotyping primers were used as described in Table S1. Seeds were germinated at 22 °C in a standard growth chamber with a 14 h/10 h light/dark cycle. 10-day-old seedlings grown on 1/2 MS plates were transplanted into the soil in a phytochamber with a 16-h/8-h light/dark cycle at 22 °C. we isolated and screened lrr1-1, lrr1-2, kin7 mutant plants, and constructed LRR1ox2, LRR1ox10 overexpression lines, construction of the double mutant lrr1kin7 by single mutants lrr1-1 and kin7 of LRR1 and KIN7.

Determination of germination and greening

Wild-type WT, single mutant lrr1-1, lrr1-2, kin7, double mutant lrr1kin7, overexpression lines LRR1ox2, LRR1ox10 seed materials of Arabidopsis were disinfected and then planted on MS medium plate (no sugar) containing ABA. The control group was MS medium plate (no sugar) without ABA. The seeds were refrigerated at 4 °C for 2–3 days. Then, they were cultured in the light culture room, The time calculation starts from the day of putting them into the light incubator, 24 h later is the first day, and the germination percentage was calculated with the index of two mm of seed radicle exposed, statistics were collected for six days. The cotyledons of the control material turned green in about seven days, and the cotyledon greening assay was counted from the seventh day.

pHBT-LRR-GFP eukaryotic expression vector construction

The gene sequence of CDS was obtained from the Arabidopsis thaliana website (http://www.arabidopsis.org) using a primer with specific primers, primer 5.0 software design based on cDNA template, by PCR amplification of CDS length sequence. Target fragments were acquired through double enzyme digestion (NcoI and SalI). The resulting product was ligated with pHBT-GFP plasmid, which had been cut with the same enzymes, at 16 °C. This ligation product was then transformed into E. coli DH5α recipient cells. Following overnight growth, colonies were subjected to colony PCR analysis to identify positive clones for further confirmation of recombinant plasmids via double enzyme digestion in plasmid PCR assays. The source of plasmids used in this experiment is from the Key Laboratory of Natural Products, Henan Academy of Sciences, China. All of the primers and cloning sites for vector construction are listed in Table S1.

Construction of vectors with LRR1 gene and plant transformation

The CDS sequence of LRR1 was obtained from the Arabidopsis website (https://www.arabidopsis.org/), and targeted primers were designed using Primer 5.0 software. The full-length CDS sequence was amplified by PCR, utilizing cDNA as a template. The CDS sequence of LRR1 was subjected to double digestion using SalI and SpeI enzymes to obtain the target fragment. The target fragment was ligated with the same double-digested pCAMBIA1300-GFP plasmid at 16 °C, and the ligated product was transformed into E. coli DH5α receptor cells. The overnight-grown colonies were subjected to colony PCR, and the positive colonies were selected for plasmid PCR further to confirm the recombinant plasmid through double enzyme digestion. The CDS obtained by double enzyme digestion was recovered, and the sequencing-free plasmid was transformed into Agrobacterium tumefaciens GV3101 receptor cells, which were identified by colony PCR and then infiltrated into Arabidopsis seedlings according to the Agrobacterium flower immersion method. After harvesting the F₁ generation of seeds, the seeds were spotted on screening MS plates containing hygromycin, and the seeds were collected singly when the plants matured, repeating the screening process three times. Screening of pure and transgenic plants is still in progress. To complement lrr1-1, we amplified and cloned the LRR1 coding region into the pCAMBIASP⁺1300 vector to generate 35S:LRR1. The constructs were transformed into lrr1-1 to generate complemented transgenic plants. The 35S:LRR1 plasmids were also transformed into Col-0 to generate LRR1-overexpressing transgenic plants (LRR1ox2, LRR1ox10). For histochemical analysis of LRR1, we amplified approximately 1,000 bp promoter fragment of LRR1 from Col-0 genomic DNA and then cloned it into beta-glucuronidase (GUS) expression vector pCAMBIA1381 (Clontech). The LRR1pro:GUS construct was transformed into Col-0. The source of plasmids used in this experiment is from the Key Laboratory of Natural Products, Henan Academy of Sciences, China. All of the primers and cloning sites for vector construction are listed in Table S1.

Tissue Localization of β-Glucuronidase

T3 transgenic seedlings or tissues were harvested and incubated in freshly prepared buffer containing 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid for 4 h at 37 °C in the dark, then washed with 70% ethanol. Images were observed by a stereo fluorescence microscope.

RNA extraction and RT-PCR assay

Total RNA was extracted from seedlings by RNA extraction kit (Tiangen, Beijing, China). After DNase I treatment, reverse transcription was conducted using PrimeScript II 1st Strand cDNA Synthesis Kit (Tiangen) according to the manufacturer’s protocol.

Through agar-gel electrophoresis, the template amount of each sample was adjusted so that the product amount of ACTIN2 obtained by amplification of different samples with the adjusted template amount was consistent. Then, the LRR1 gene was amplified with the sample’s adjusted template amount to detect the target gene’s expression difference in different samples. The primers utilized in this study are listed in Table S2.

qRT-PCR assay

LRR1 cDNA template in each reaction tube was diluted 3–5 times, and 2 µL of diluted cDNA, SYBR premix EX Taq 10 µL, ROX dye (control dye) 0.4 µL, and upstream and downstream specific primers 0.4 µL (10 µM) were added. Then the reaction was carried out on a qRT-PCR instrument. According to the type of instrument used and the characteristics of the qRT-PCR kit used, the reaction procedure was set as pre-denaturation at 94 °C for 30 s; denaturation at 94 °C for 15 s, annealing at 55 °C for 20 s, 40 cycles of the above two steps; extension at 75 °C for 15 s, and analyzed according to software data after amplification. The amplification efficiency and Ct values were calculated using a program with at least three parallel reactions per sample, repeated three times. Standard computational methods were used to analyze gene expression. The primers utilized in this study are listed in Table S2 (qRT-PCR primer design: through www.ncbi.nlm.nih.gov/tools/primer-blast/).

Yeast two-hybrid assay

Saccharomyces cerevisiae strain Y2HGold (Clontech, Mountain View, CA, USA) was used for co-transformation of the activation domain (AD) and binding domain (BD) constructs. PYL1 to PYL9, SnRK2.1 to SnRK 2.10, ABI1, and ABI2 were cloned into the pGAD-T7 vector to generate the AD-ABI1, AD-ABI2, AD-SnRK(2.1∼2.10), AD-PYL(1∼9) plasmids, and the full-length sequences of LRR1 and KIN7 were cloned into pGBK-T7 to generate the BD-LRR1 and BD-KIN7 plasmids, respectively. A series of diluted co-transformed Y2H Gold cultures were spotted onto synthetic defined plates lacking Trp and Leu or lacking Trp, Leu, His, and Ade, and incubated at 30 °C for 3 days to observe yeast growth. All of the primers for plasmid construction are listed in Table S3.

BiFC assay

The coding regions of ABI1, ABI2, and PYL6 were cloned into the binary BiFC vector pSPYNE173 to produce the ABI1-YFP^N, ABI2-YFP^N, and PYL6-YFP^N plasmids, respectively. The coding regions of LRR1 and KIN7 were cloned into pSPYCE(M) to produce LRR1-YFP^C and KIN7-YFP^C. Constructs including empty vectors were introduced into the Agrobacterium strain GV3101 and then infiltrated into Nicotiana benthamiana leaves. YFP signals were observed using confocal microscopy 2 days after infiltration. All primers used for plasmid construction are listed in Table S3.

GST pull-down assay

The coding region of LRR1 was cloned into pGEX-4T-1 to obtain the LRR1-GST recombinant protein, and the coding region of ABI1 and PYL6 were cloned into pET28a to obtain the ABI1-His and PYL6-His recombinant protein. Glutathione beads containing LRR1-GST were incubated with ABI1-His and PYL6-His proteins in 1 ×PBS buffer at 4 °C for 2 h. The beads were washed five times with wash buffer (1 ×PBS, 0.1% Triton X-100). Proteins retained on the beads were analyzed by immunoblotting with an anti-GST or anti-His antibody. All primers used for plasmid construction are listed in Table S3.

Expression pattern of LRR1

ABA plays an important role in regulating plant growth and development. About 10% of genes in Arabidopsis are expressed by ABA induction, including various protein kinases (Baek, Lim & Lee, 2018). The cascades of LRR-RLK receptor protein kinases are involved in ABA-regulated plant growth and development. The analysis of the phylogenetic tree of LRR-RLKs in Arabidopsis showed that the protein sequences of KIN7 and LRR1 genes exhibited a relatively high degree of similarity (Figs. 1A & 1B). The results analyzed through the TAIR website show that the T-DNA of lrr1-1, lrr1-2, and kin7 were inserted into the promoter region approximately 1,000 bp upstream of the start codon ATG (Fig. 1C). The lrr1-2 mutant appears to produce a lower level of the RT-PCR band than WT, while the lrr1-1 does not appear to have any band. In addition, the kin7 allele also appears to produce some RNA. These results would seem to indicate that the lrr1-2, kin7 mutants are not nulls, but down-regulated expression (Fig. S1).

The expression of the GUS gene driven by the LRR1 promoter in various tissues and organs of Arabidopsis, as shown in Fig. 2A. Among them, the expression of the LRR1 gene was abundant in roots, stems, and petioles. The results indicated that LRR1 is widely expressed in multiple organs. To verify the relationship between LRR1 and ABA, RT-PCR was employed to assess the expression of the LRR1 gene in each material treated with ABA (Fig. S2). Further, the GUS transgenic plants of LRR1 were treated with ABA. GUS activity was lower in seedlings treated for with ABA for longer periods of time. The results showed that LRR1 gene expression was gradually down-regulated with the increase in ABA treatment time (Fig. 2B). qRT-PCR assay: After exogenous ABA treatment, the expression level of LRR1 was down-regulated (Fig. 2C).

Transient expression and GFP fluorescence assay with LRR1

To analyze the subcellular localization of LRR1, the vector pHBT-LRR1-GFP was constructed. After transient expression in Arabidopsis protoplasts, GFP alone is visible in the cytosol, but the LRR1-GFP fusion is detected only in the plasma membrane, suggesting that the endogenous LRR1 protein is protoplast membrane localized. We use confocal laser microscopy to detect the fluorescence of Arabidopsis leaf cell protoplasts (Fig. 3A).

To further analyze the subcellular localization of the LRR1 protein, and determine the expression and distribution of LRR1 in Arabidopsis cells, we constructed the SP1300-GFP transgenic recombinant vector of LRR1. The fluorescence of Arabidopsis roots was detected by confocal (Fig. 3B). LRR1 may be located in the plasma membrane of Arabidopsis plants. Since plants have cell walls, to determine the location of LRR1 in the plasma membrane, the root material of Arabidopsis carrying LRR1-GFP transgenic recombinant vector was treated with 200 mM mannitol overnight (12 h) in this study, and the fluorescence of Arabidopsis roots was detected by confocal (Fig. 3C). LRR1 was located on the plasma membrane in Arabidopsis roots.

Germination assay, growth conditions, ABA stress

LRR1 involved in the regulation of seed germination inhibited by ABA

LRR1 is a leucine-rich repeat receptor protein kinase, LRR-RLK, isolated from Arabidopsis. Its expression is down-regulated by dehydration, hypersaline, and low temperature (Soltabayeva et al., 2022). To determine the role of LRR1 in ABA signaling, we analyzed the effects of ABA treatment on wild-type, single mutants, double mutant, and overexpression lines cultivated on MS medium plates with different concentrations of ABA, either 0.4 µM or 0.8 µM. The control was an MS medium plate without ABA. The seeds were refrigerated at 4 °C for 2–3 days. Then, they were cultured in the light culture room, The time calculation starts from the day of putting them into the light incubator, 24 h later is the first day, and the germination percentage was calculated with the index of two mm of seed radicle exposed, statistics were collected for 6 days. The germination percentage of wild-type WT decreased after treatment with 0.8 µM ABA (Fig. 4A). The germination percentage of mutants lrr1-1, lrr1-2, kin7, lrr1kin7 and overexpression lines LRR1ox2 and LRR1ox10 without ABA treatment were the same. The germination percentage of mutants lrr1-1, lrr1-2, kin7, and lrr1kin7 treated with 0.8 µM ABA showed difference from that treated with 0.4 µM ABA. Under 0.8 µM ABA treatment, the germination of lrr1-1, lrr1-2, kin7, and lrr1kin7 was fast in the first 3 days (Figs. 4B & 4C).

Under 30 µM ABA treatment, the germination percentage of the wild-type was inhibited (Fig. 5A). The germination of double mutant lrr1kin7 was fast in the first 3 days. In addition, the overexpression lines LRR1ox2 and LRR1ox10 were essentially non-germinating (Figs. 5B & 5C). In addition, the germination percentage of the mutant gradually slowed down with the extension of treatment time. These results suggest that LRR1 gene knockout plants are less sensitive to ABA, and the LRR1 gene may be involved in regulating the signal transduction process of ABA inhibition of seed germination.

LRR1 involved in ABA regulation of cotyledon greening

We conducted the cotyledon greening test to validate the seed germination assay. This further elucidates that LRR1 is involved in regulating the ABA signaling pathway. Notably, all cotyledons turned green without ABA in the MS medium. As ABA concentration increased, inhibition of cotyledon greening was observed across all materials tested. Interestingly, mutants lrr1-1 and lrr1-2 exhibited less inhibition while the overexpression lines exhibited greater inhibition when both are compared to wild-type. (Fig. 6A). This study suggests that the sensitivity of LRR1 gene to ABA may implicate its involvement in regulating signal transduction processes associated with ABA-mediated inhibition of cotyledon greening.

This study further designed assays to conduct phenotypic analysis assays. The cotyledon of the materials in the MS medium without ABA had turned green, and the mutant materials lrr1-1, lrr1-2, and kin7 after 0.4 µM ABA treatment had turned green with less inhibition compared with the wild-type WT material. The number of green cotyledons of double mutant lrr1kin7 was more than that of single mutants lrr1-1, lrr1-2, and kin7. In contrast, the overexpression lines LRR1ox2 and LRR1ox10 were more susceptible to ABA inhibition than the wild-type, and the cotyledon did not turn green (Figs. 6B, 6C & 6D). The results of the above phenotyping experiments indicate that LRR1 is involved in the ABA signaling pathway to regulate cotyledon greening.

LRR1 regulates ABA-induced gene expression

We have preliminarily demonstrated that LRR1 is involved in the ABA signaling pathway to regulate seed germination and cotyledon greening during early development in Arabidopsis. To prove the function of LRR1 in the ABA regulation of gene expression, qRT-PCR analysis was performed. The transcription factors ABI3, ABI4, and ABI5, which regulate early growth and development in Arabidopsis downstream in the ABA signaling pathway, were selected as the objects of qRT-PCR amplification. Seed and cotyledon of wild-type WT, single mutants lrr1-1, kin7, and double mutant lrr1kin7 were selected as the target materials. Seed material was treated with 5 µM ABA for three days (the treatment ended on the 3rd day after the seeds began to appear white). cotyledon material was treated with 0.8 µM ABA treatment for nine days (from the beginning of seed whitening to the end of cotyledon green treatment, nine days). As shown in Figs. 7A & 7B, qRT-PCR detection showed that the expression levels of ABI3, ABI4, and ABI5 genes were reduced in single mutants lrr1-1 and kin7 treated by ABA. The expression levels of ABI3, ABI4, and ABI5 genes in double mutant lrr1kin7 after ABA treatment were lower than those in single mutants lrr1-1 and kin7. In addition, ABI3, ABI4, and ABI5 transcription factors play important roles in regulating seed germination and growth and development in the ABA signaling pathway. If the expression level of ABI5 is increased, the seed will be dormant; if the expression level is decreased, the seed will break dormancy and start germination, growth, and development. The ABI5 expression levels of single mutants lrr1-1 and kin7 were lower than those of wild-type WT after ABA treatment, which is by the fact that the germination percentage and cotyledon greening of single mutants lrr1-1 and kin7 were higher than those of wild-type WT.

Proteins binding to LRR1 were identified by in vivo and in vitro protein interaction assays

Previous assays have shown that LRR1 and its homologous gene KIN7 are involved in the ABA signaling pathway to regulate the growth and development of Arabidopsis seeds. However, the mechanism of the function of LRR1 and its homologous protein KIN7 in the ABA signaling pathway as well as the associated proteins, are unknown. To further screen the proteins associated with LRR1 and KIN7 in the ABA signaling pathway, we constructed a yeast two-hybrid vector, using LRR1-BD and KIN7-BD as bait proteins. Protein phosphatases ABI1 and ABI2 (repressors of ABA signaling pathway), protein kinases SnRK2.1 to SnRK2.10 and ABA receptors PYL1 to PYL9 were used as the target proteins (AD-ABI1, AD-ABI2, AD-SnRK(2.1∼2.10), and AD-PYL(1∼9)) to check their potential interaction with BD-LRR1 and BD-KIN7. we found that both BD-LRR1 and BD-KIN7 could interact with AD-PYL6 in yeast cells (Fig. 8A).

The GFP fluorescence of the transformed Arabidopsis protoplast was observed under a 60 × objective lens of a laser confocal scanning microscope. Fluorescence was found in the positive control group (Fig. 8B, top row), and LRR1-YCE and PYL6-YNE were observed. Both KIN7-YCE and PYL6-YNE showed fluorescence, while YNE and YCE were negative controls. YNE and LRR1-YCE, KIN7-YCE; Neither YCE nor PYL6-YNE fluoresces (Fig. 8B). The results show that LRR1 and PYL6 interact in vivo.

In addition, we used Nicotiana benthamiana leaves to verify the protein-protein interaction. The tobacco was injected at room temperature for 2 to 3 h. Two to five days after injection, the leaves of the tobacco were torn off, and the back of the leaves were taken under the 40 × objective lens of the laser confocal scanning microscope to observe the interaction fluorescence after transformation. Fluorescence was found in the positive control group, LRR1-YFP^C, and PYL6- YFP^N were observed. KIN7-YFP^C and PYL6-YFP^N both fluoresce, while LRR1-YFP^C and PYL6-YFP^N fluoresce very strongly, and a specific group is made below. YNE and YCE were the negative controls. YNE and LRR1-YFP^C, KIN7-YFP^C; Neither YCE nor PYL6-YFP^N fluoresces (Figs. 8C & 8D). The assay of bimolecular fluorescence interaction between LRR1-YFP^C and PYL6- YFP^N is also consistent with the above assays. The experiment results show that LRR1 and PYL6 interact with each other.

To further detect the interaction between LRR1 and PYL6, we constructed prokaryotic expression vectors of LRR1-GST, PYL6-His, and ABI1-His. The protein interaction was demonstrated by the GST pull-down technique. PYL6-His was observed in the LRR1-GST pull-down and PYL6-His input control lanes (Fig. 8E). This implies that the interaction between LRR1 and PYL6 facilitates the retention of PYL6, as evidenced by its detection through Western Blot analysis. The assay results demonstrate an in vitro interaction between LRR1 and PYL6.