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Article

Identification of Target Gene and Interacting Protein of Two LaSCL6 Alternative Splicing Variants Provides Novel Insights into Larch Somatic Embryogenesis

by
Qiao-Lu Zang
1,2,
Zha-Long Ye
1,
Li-Wang Qi
1 and
Wan-Feng Li
1,*
1
State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of the National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
2
College of Horticulture, Shanxi Agricultural University, Jinzhong, 030801, China
*
Author to whom correspondence should be addressed.
Plants 2024, 13(21), 3072; https://doi.org/10.3390/plants13213072
Submission received: 28 September 2024 / Revised: 20 October 2024 / Accepted: 26 October 2024 / Published: 31 October 2024
(This article belongs to the Special Issue Molecular Biology and Bioinformatics of Forest Trees)
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Abstract

:
Somatic embryogenesis is valuable for clonal propagation and genetic improvement, and it also serves as an ideal system for studying plant development mechanisms. In Larix kaempferi, microRNA171 and its target gene L. kaempferi SCARECROW-LIKE6 (LaSCL6), which has two alternative splicing variants, can regulate somatic embryogenesis; however, the underlying molecular mechanism is still unknown. In this study, we overexpressed these two LaSCL6 variants in Oryza sativa and Arabidopsis thaliana and then used the RNA-Seq method to screen genes from O. sativa and A. thaliana, whose expression patterns are related to those of LaSCL6 variants. The screened genes were then used to search L. kaempferi proteins to identify the candidate target genes of LaSCL6. After yeast one-hybrid and dual- luciferase transcriptional activity assays, cytochrome P450, family 89, subfamily A, polypeptide 5 (CYP89A5), and wall-associated receptor kinase-like 20 (WAKL20) were confirmed to be the target genes of LaSCL6-var1; in addition, WAKL20 and UDP-glycosyltransferase 85A3 (UGT85A3) were confirmed to be the target genes of LaSCL6-var2. Moreover, APETALA2-like protein 2, a transcription factor from the AP2/ERF family, was shown to interact with LaSCL6-var1 and LaSCL6-var2. Taken together, our results suggest a regulatory network of miR171-LaSCL6. The findings presented here not only provide novel insights into the regulation of the miR171-LaSCL6 module but also explain the mechanism underlying larch somatic embryogenesis and other biological processes.

1. Introduction

Somatic embryogenesis is a valuable technique in the clonal propagation and genetic improvement of plants, and it plays a critical role in larch breeding programs [1,2,3,4]. The process of conifer somatic embryogenesis is divided into two main stages: pro-embryogenic masses and somatic embryo [5]. In the pro-embryogenic masses stage, the cell passes from a primitive single-cell aggregate to aggregates of several cells and then to cellular clusters; these pro-embryogenic masses then differentiate to form a somatic embryo, which is a bipolar structure that has no vascular connection with the maternal tissue [6,7]. Somatic embryogenesis is regulated by a number of external and internal factors; these include genotype, explant status, plant growth regulators, and sources of carbon and nitrogen [8,9]. The induction of embryogenic cultures and the maintenance of embryogenic potential are both important for somatic embryogenesis; however, the occurrence of non-embryogenic cultures limits its efficient utilization [5,10,11]. Further studies of the molecular basis of these processes are therefore required to better understand and improve this technique.
MicroRNA affects somatic embryogenesis by regulating its target gene at the post-transcription level [12,13,14,15]. The miR171 family is highly conserved and functions via regulation of its target gene SCARECROW-LIKE6 (SCL6, also known as HAIRY MERISTEM or LOST MERISTEMS), which is a transcription factor from the GRAS (GAI-RGA-SCR) family [16,17,18]. In citrus, lily, and larch, miR171-SCL6 takes part in the development of embryogenic cultures and in the maintenance of embryogenic potential [10,19,20,21]. However, the molecular mechanism by which miR171-LaSCL6 regulates larch somatic embryogenesis has not yet been elucidated.
The molecular mechanisms of SCL6 have been demonstrated in the regulation of other biological processes. For example, in Arabidopsis thaliana, SCL6 interacts with WUSCHEL (WUS) to regulate meristem through the CLAVATA3-WUS pathway [22,23] and interacts with SQUAMOSA promoter-binding-like protein (SPL) to control flowering and trichome initiation [24]. LbrSCL6 also interacts with WUSCHEL-related homeobox4 (LbrWOX4) to regulate maturation in Lilium [25]. In addition, SCL6 interacts with the DELLA protein, and their interaction reduces the binding activity of SCL6 to the promoter of protochlorophyllide oxidoreductase C (PORC), which regulates the biosynthesis of chlorophyll [26]. Moreover, in tea plants, CsSCL6-4 directly promotes the expression of four drought-resistance genes, peroxiredoxin (CsPrx), short-chain dehydrogenase/reductase (CsSDR), omega-3 fatty acid desaturase (CsFAD7), and eceriferum (CsCER1), by binding motifs in their promoter regions in tea plants [27]. With regard to the existence of these molecular mechanisms in L. kaempferi, however, nothing is yet known.
Notably, two LaSCL6 alternative splicing variants exist in L. kaempferi and have different expression patterns during L. kaempferi somatic embryogenesis [28], adding more complexity to any study of the functional mechanism of SCL6. In addition, no information is yet available regarding the target gene and interacting protein of LaSCL6. In the present study, we aimed to identify the target genes and interacting proteins of two LaSCL6 alternative splicing variants to provide more information about the functional mechanism of SCL6. The results presented here further enhance our understanding of somatic embryogenesis.

2. Results

2.1. Thirty Larix Genes Were Screened as the Candidate Target Genes of LaSCL6 after Analyzing the Transcriptomic Responses of O. sativa and A. thaliana to LaSCL6 Overexpression

Given the challenging nature of obtaining transgenic larch plants and the conservative regulation of genes between species, the over-expression vectors of LaSCL6-var1 and LaSCL6-var2 were separately constructed and transformed into O. sativa and A. thaliana. Transcriptome sequencing of wild-type (WT) and transgenic plants was then performed (Parts 1 and 3 in Supplementary Table S1).
After different comparisons, the differentially expressed genes (DEGs) were obtained (Parts 2 and 4 in Supplementary Table S1). Totals of 1024 and 1129 DEGs were identified in the comparison of LaSCL6-var1 vs. WT in O. sativa and A. thaliana, respectively (Figure 1A); totals of 2517 and 186 DEGs were identified in the comparison of LaSCL6-var2 vs. WT in O. sativa and A. thaliana, respectively (Figure 1B); and totals of 660 and 658 DEGs were identified in the comparison of LaSCL6-var2 vs. LaSCL6-var1 in O. sativa and A. thaliana, respectively (Figure 1C). In addition, the DEGs in the comparisons of LaSCL6-var1 vs. WT and LaSCL6-var2 vs. WT in both O. sativa and A. thaliana were also identified as potential target genes (Figure 1D).
Values for the Pearson correlation coefficient (PCC) between the expression patterns of the DEGs and LaSCL6-var1 or LaSCL6-var2 were calculated; those DEGs with a PCC value of ≥0.9 or ≤−0.9 were considered to have the same or opposite expression pattern as LaSCL6-var1 or LaSCL6-var2 and were used for blast analysis.
Totals of 94 and 182 DEGs showed patterns that were the same as or opposite to LaSCL6-var1 overexpressing O. sativa and A. thaliana, respectively (Figure 1A); these were then used to blast with Larix protein sequences. Ultimately, 75 and 163 Larix genes were identified, in which four genes were shared, and the corresponding homologous genes in O. sativa and A. thaliana had almost the same expression patterns (Figure 1A, Supplemental Table S2).
Totals of 831 and 19 DEGs showed patterns that were the same as or opposite to LaSCL6-var2 overexpressing O. sativa and A. thaliana, respectively (Figure 1B); these were then used to blast with Larix protein sequences. Ultimately, 570 and 17 Larix genes were identified, in which one gene was shared and the corresponding homologous genes in O. sativa and A. thaliana had almost the same expression patterns (Figure 1B, Supplemental Table S2).
In the comparisons of LaSCL6-var1 overexpressing O. sativa vs. LaSCL6-var2 overexpressing O. sativa and of LaSCL6-var1 overexpressing A. thaliana vs. LaSCL6-var2 overexpressing A. thaliana, 660 and 658 DEGs were obtained, respectively; these were then used to blast with Larix protein sequences. Ultimately, 393 and 483 Larix genes were identified, in which 13 genes were shared, and the corresponding homologous genes in O. sativa and A. thaliana had almost the same expression patterns (Figure 1C, Supplemental Table S2).
To identify the potential target genes regulated by both LaSCL6-var1 and LaSCL6-var2, Venn analyses were performed with the DEGs obtained from O. sativa and A. thaliana after correlation analysis. A total of 19 DEGs were obtained from O. sativa, while no DEGs were obtained from A. thaliana (Figure 1D). These 19 DEGs were then used to blast with Larix protein sequences, and 15 Larix genes were ultimately identified (Figure 1D, Supplemental Table S2).
Three genes appear in two comparisons: Larix43329 (identified in the comparison of LaSCL6-var2 vs. LaSCL6-var1 and LaSCL6-var1 vs. WT), Larix12010 (identified in the comparison of LaSCL6-var1/2 vs. WT and LaSCL6-var2 vs. LaSCL6-var1), and Larix22537 (identified in the comparison of LaSCL6-var1 vs. WT and LaSCL6 var1/2 vs. WT). Finally, a total of 30 Larix genes (Part 1 in Supplementary Table S3) were obtained and considered as the candidate target genes of LaSCL6-var1 or LaSCL6-var2; these corresponded to 39 O. sativa genes and 20 A. thaliana genes (Parts 2 and 3 in Supplemental Table S3).

2.2. Three Candidate Target Genes Were Confirmed to Be Regulated by LaSCL6

We assumed that if a gene was controlled by LaSCL6, the GRAS binding motif would exist in its promoter sequence. We therefore analyzed the numbers of GRAS binding motifs in the promoter sequences of 30 Larix genes, 39 O. sativa genes, and 20 A. thaliana genes (Supplemental Table S3). The results showed that three Larix genes and two O. sativa genes had no GRAS binding motif in their promoter sequences, while all A. thaliana genes had a GRAS binding motif in their promoter sequences. Therefore, 27 Larix genes were used for further study. After cloning, two promoter sequences were not obtained. Finally, the promoter sequences of 25 Larix genes were obtained (Supplemental Table S4) and used to test their relationships with LaSCL6-var1 and LaSCL6-var2.
A total of 50 yeast one-hybrid (Y1H) assays were performed between LaSCL6-var1 or LaSCL6-var2 and 25 promoters. The relationships between LaSCL6-var1 and LaCYP89A5, LaWAKL20, LaENODL1, LaPIC30, and LaCYP84A1 were confirmed (Figure 2A,B). The relationships between LaSCL6-var2 and LaCYP89A5, LaWAKL20, LaCCA1, and LaUGT85A3 were also confirmed (Figure 3A,B). Dual-LUC assays further confirmed that LaSCL6-var1 increased the promoter activity of LaCYP89A5 and LaWAKL20 (Figure 2C–H) (p ≤ 0.05) and LaSCL6-var2 increased the promoter activity of LaWAKL20 and LaUGT85A3 (Figure 3C–G) (p ≤ 0.05).

2.3. LaSCL6-var1 and LaSCL6-var2 Could Interact with LaAP2L2 in the Nucleus

LaSCL6-var1 and LaSCL6-var2 both contained the putative nuclear localization signal (NLS) that was also found in AtSCL6 [17] (Figure 4A). Confocal microscopy analysis revealed that LaSCL6-var1-GFP/LaSCL6-var2-GFP and GFP-LaSCL6-var1/GFP-LaSCL6-var2 were localized in the nucleus (Figure 4B).
Yeast two-hybrid (Y2H) screen assays in the larch cDNA library were performed using five fragments of LaSCL6 as baits (Figure 5A). The numbers of binding proteins screened by these five fragments were 15, 4, 43, 7, and 2, respectively (Figure 5B). After blast analysis of these 71 sequences, one was annotated as transcription factor encoding an AP2 protein (LaAP2L2, GenBank accession No. KU355275.1) (Figure 5B). Furthermore, this interaction between LaSCL6 and LaAP2L2 was verified via Y2H and BiFC assays (Figure 5C,D).

3. Discussion

At present, ChIP-seq is the most effective method for finding the target gene of a transcription factor at the whole-genome level; however, high-quality genomic information and a stable transformation system are essential for the successful application of this method [29]. It is almost impossible to find the target gene of a larch transcription factor using ChIP-seq because the larch genome is large and complex [30,31] and a rapid, efficient and stable larch transformation system is still lacking.
In the present study, a larch transcription factor, LaSCL6, was overexpressed in model plants; their transcriptomes were then analyzed to determine the DEGs. Next, following the discovery of homologous genes of these DEGs from larch, and the subsequent determination of their relationships with LaSCL6, three target genes (LaCYP89A5, LaUGT85A3, and LaWAKL20) of LaSCL6 were identified. This method provides a new way to find the target genes of a transcription factor at the whole -genome level in species that are difficult to transform and for which high-quality genomic information is unavailable. In addition, a transcription factor, LaAP2L2, was found to interact with LaSCL6. Taken together, these results help to explain the function and mechanism of LaSCL6 in larch somatic embryogenesis.
The process of somatic embryogenesis in conifers mainly includes the induction of pro-embryogenic masses, proliferation of embryonic cells, maturation and germination of somatic embryos, and the growth of somatic embryo seedlings [5,11,32]. Consequently, somatic embryogenesis in larch also involves these processes, in which LaSCL6 and its regulators play important roles.
The induction and proliferation of embryonic cells depend on cell cycle, division, and proliferation. The post-transcriptional regulation of LaSCL6 by miR171 participates in the regulation of the cell-division mode and the maintenance of embryogenic potential [33]. The heterologous expression of LaAP2L2 in Arabidopsis affects cell proliferation and meristem activity [34]; in the present study, its protein was found to interact with LaSCL6. Likewise, LaSCL6 target genes might also be involved in the early processes of somatic embryogenesis because their homologous genes have been found to function in the above-mentioned cellular processes. For example, UGT85U1, a homologous gene of LaUGT85A3, modifies the expression of cell cycle-related genes [35]; in addition, WAKL functions in cell wall formation [36], which is a very important part of cell reconstruction. Therefore, if embryogenic cells were induced and proliferated, LaSCL6 might work in concert with these genes.
The proliferation of embryonic cells and the maturation of somatic embryos are known to occur under conditions of darkness; however, the underlying molecular basis has rarely been studied. In the present study, on the deduction that darkness influences the biosynthesis of chlorophyll, we offered some molecular cues. It is known that Arabidopsis scl6 mutant plants show increased chlorophyll accumulation [18] and that SCL6 functions via regulating its target genes, namely CYP89A9 [37,38], UGT85A1, and UGT85A5 [39,40]. In this study, we also found the involvement of LaSCL6 regulation of its target genes (LaCYP89A5 and LaUGT85A3) in larch somatic embryogenesis, indicating that SCL6-mediated chlorophyll biosynthesis constitutes the molecular basis of darkness treatment of embryonic cultures.
Together, the identified target genes and interacting protein of LaSCL6 help explain the mechanism of the miR171-LaSCL6 module in larch somatic embryogenesis. In addition, the regulatory network of LaSCL6 was improved (Figure 6). At the genomic level, simple sequence repeats and single nucleotide polymorphisms influence LaSCL6 expression [41]. At the post-transcriptional level, LaSCL6 is regulated by miR171 and the alternative splicing [28,33,42,43]. At the translation level, the interacting protein (LaAP2L2) and target genes (LaCYP89A5, LaWAKL20, and LaUGT85A3) were determined to work together with LaSCL6.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Nipponbare (O. sativa L. japonica) seeds, A. thaliana ecotype Columbia seeds, Nicotiana benthamiana seeds, and immature L. kaempferi seeds were used in this study. Nipponbare embryogenic calli were obtained from seeds [44]. A. thaliana seeds were sterilized, sown on 1/2 Murashige and Skoog medium, vernalized for 3 d at 4 °C in the dark, and then transferred to a culture room (16 h light/8 h dark, 150 μmol·m−2·s−1, 22 °C). Seven-day-old uniform A. thaliana seedlings were planted in soil, with four plants in each square plastic pot, and transferred to the culture room. N. benthamiana seeds were germinated in soil and grown in a growth chamber under controlled conditions (16 h light/8 h dark, 26 °C). Six-week-old N. benthamiana plants were used in experiments. Immature L. kaempferi seeds were collected from a Dagujia seed orchard (42°22′ N, 124°51′ E) in Liaoning province, in northeast China; from these, the embryonal suspensor mass was generated according to the methods used in a previous study [32].

4.2. Vector Construction and Plant Transformation

The full coding sequences of LaSCL6-var1 (GenBank accession No. MK501379) and LaSCL6-var2 (GenBank accession No. JX280920) were cloned into the plant expression vector pCAMBIA-1305.1, which contains the cauliflower mosaic virus 35S promoter, using Nco Ⅰ and a Pml Ⅰ restriction sites to generate 35S::LaSCL6-var1 and 35S::LaSCL6-var2, respectively. All constructs were confirmed via sequencing before being introduced into the Agrobacterium tumefaciens strains GV3101 and EHA105. All the primers used are listed in Supplemental Table S5.
35S::LaSCL6-var1 and 35S::LaSCL6-var2 were separately transformed into Nipponbare by EHA105 with hygromycin resistance [44]. Transgenic calli and seedlings were screened on a medium containing 50 mg·L−1 hygromycin. 35S::LaSCL6-var1 and 35S::LaSCL6-var2 were separately transformed into the wild-type A. thaliana by GV3101 using the floral dipping method [45]. Transgenic seeds were screened on 1/2MS plates containing 50 mg·L−1 kanamycin. Twenty-one-day-old A. thaliana seedlings and thirty-day-old Nipponbare seedlings from four independent lines of the wild type, 35S::LaSCL6-var1, and 35S::LaSCL6-var2 were used for RNA-Seq analysis.

4.3. RNA Isolation, RNA-Seq Library Preparation, and Gene Expression Analysis

Total RNA was extracted using an RNAiso Plus reagent kit (TaKaRa, Shiga, Japan). RNA quantity and concentration were measured on a 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) and NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA). Totals of 12 A. thaliana and 12 Nipponbare mRNA libraries were prepared according to the Illumina RNA sequencing protocols and sequenced using paired-end sequencing with 150 bp lengths on the NovaSeq 6000 platforms (Illumina, San Diego, CA, USA). The RNA-Seq data have been uploaded to the China National Center for Bioinformation database under the designation PRJCA030648. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed on 1 October 2020) and Trimmomatic ver.0.30 [46] were used to perform the initial quality control check of the transcriptome data and produce the clean data, respectively. The clean reads were then separately aligned to the corresponding reference genomes using HISAT2 (v2.1.0) [47]. DEGseq2 was used for differential expression analysis [48]. Genes with the |log2 fold change| ≥ 1 and a p-value or corrected p-value of <0.05 [49] were termed as DEGs. Three pairwise comparisons were performed to identify the DEGs, as follows (1) 35S::LaSCL6-var1 vs. WT; (2) 35S::LaSCL6-var2 vs. WT; and (3) 35S::LaSCL6-var2 vs. 35S::LaSCL6-var1 (Figure 1).
Common DEGs from the different comparisons were obtained by intersecting the respective gene sets and visualizing using the Venn diagram drawing tool Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed on 29 October 2020). The normalized expression value for each gene was measured in fragments per kilobase of transcript per million mapped reads (FPKM) [50].

4.4. Correlation Analysis and Sequence Blast

The FPKM values of LaSCL6-var1 and LaSCL6-var2 were calculated for all the sequenced samples. The PCCs between the expression patterns of the DEGs and LaSCL6-var1 or LaSCL6-var2 were then analyzed. A PCC value of ≥0.9 or ≤−0.9 was considered to indicate correlation, and the DEG had the same or opposite expression pattern as LaSCL6-var1 or LaSCL6-var2. The best-matching protein sequence for the screened O. sativa or A. thaliana DEG from the Larix genome was identified using BLAST, with an E-value of <10−5, and this protein was considered as the potential target gene of LaSCL6. The analysis process is shown in Figure 1.

4.5. Promoter Sequence Analysis and Cloning

The promoter sequences of screened genes in O. sativa and A. thaliana were obtained from EnsemblPlants (http://plants.ensembl.org/index.html, accessed on 29 October 2020). The potential target genes were used to search the Larix genome (https://www.ncbi.nlm.nih.gov/nuccore/WOXR00000000.2, accessed on 29 October 2020) [31], and 2000 bp fragments upstream of ATG were regarded as the promoter sequences. Transcription factor binding site prediction was performed using the PlantRegMap server (http://plantregmap.gao-lab.org/binding_site_prediction.php, accessed on 29 October 2020) based on the data of four species, namely, A. thaliana, Populus trichocarpa, Solanum lycopersium, and Zea mays, with a p-value of <10−4 [51]. The GRAS motif numbers in each promoter were then analyzed.
The promoter sequences were amplified from the genomic DNA template and then sequenced. The genomic DNA was isolated with the CTAB plant genome DNA rapid extraction kit (Aidlab Biotech, Beijing China) according to the manufacturer’s protocol. The target fragments were amplified with Platinum® Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The PCR products were purified with a gel extraction kit (Tiangen, Beijing China), ligated into the pEASY®-T1 simple cloning vector (TransGen, Beijing China), and sequenced. All the primers used are listed in Supplemental Table S5.

4.6. Y1H Assay

Y1H assays were performed to verify the prediction results. The open reading frames of LaSCL6-var1 and LaSCL6-var2 were amplified by means of PCR and cloned into the pGADT7 vector, resulting in pGADT7-transcription factor plasmids. The sequence fragments of candidate gene promoters were amplified and cloned into the pHIS2 vector, resulting in the pHIS2-promoter plasmids. Next, the bait and prey constructs were co-transformed into the yeast strain Y187 using the lithium acetate method, and yeast cells were plated on SD/-Leu/-Trp media and cultured for 3–5 days. The positive clones were selected and plated separately on SD/-Leu-Trp-His media with 30, 60, or 90 mM 3-amino-1, 2, 4-triazole, and cultured for 3–5 days. Possible interactions between transcription factors and promoters were determined based on the growth status of yeast colonies. All the primers used are listed in Supplemental Table S5.

4.7. Dual-LUC Assay

To confirm the Y1H results, Dual-LUC assays were performed. The full-length coding sequences of LaSCL6-var1 and LaSCL6-var2 were individually inserted into the pGreenII 0029 62-SK vector to generate the effector constructs. The promoter fragments of candidate genes were individually cloned into pGreenII 0800-LUC vectors to generate the reporter constructs. All recombinant constructs were individually transformed into A. tumefaciens strain GV3101. N. benthamiana leaves were infected with the mixed Agrobacterium strain. Detection of fluorescence was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison WI, USA). The LUC activity was normalized to REN activity, and the relative LUC/REN ratios were used to represent the promoter activity. For each combination, LUC/REN ratios from at least three independent transformations were determined. All the primers used are listed in Supplemental Table S5.

4.8. Sequence Analysis and Subcellular Localization

Sequences of the GRAS family genes SCL6-II (At2G45160), SCL6-III (At3G60630), and SCL6-IV (At4G00150) were retrieved from the Arabidopsis TAIR10 genome release (http://www.arabidopsis.org, accessed on 15 March 2021). The protein sequences of LaSCL6-var1, LaSCL6-var2, and three A. thaliana SCL6s were aligned using ClustalX (v2.0). A phylogenetic tree was constructed using a neighbor-joining method with 1000 bootstrap replicates using MEGA (v10.0).
The full-length coding sequences of LaSCL6-var1 and LaSCL6-var2 were separately amplified and inserted at the N- or C-terminal of GFP driven by the CaMV35S promoter. LaSCL6-var1/LaSCL6-var2-GFP and GFP-LaSCL6-var1/LaSCL6-var2 were co-transformed into A. thaliana protoplasts using NLS-mKate, which was used as a nuclear marker [52]. Transient expressions of the GFP fusions in A. thaliana protoplasts were performed as previously described [53]. Finally, A. thaliana protoplasts were observed using confocal microscopy.

4.9. Y2H Screening and Identification

The cDNA library for Y2H experiments was constructed by cloning cDNA synthesized from the mRNAs of L. kaempferi embryonal-suspensor mass and stems into the prey vector pGADT7 (Takara, Shiga, Japan). The full-length coding sequences of LaSCL6-var1/LaSCL6-var2 and five truncated sequences were amplified via PCR using the indicated primers and inserted into the bait vector pGBKT7 (Takara, Shiga, Japan) using the PEG/LiAc method. These recombinant constructs were introduced into the yeast strain AH109 and tested for autoactivation and toxicity. Because of the strong autoactivation of full-length LaSCL6-var1 and LaSCL6-var2, we used the five truncated fragments for Y2H screening assays, following the user manual of the Matchmaker Gold Y2H system’s user manual (Yeast Protocols Handbook; Takara, Shiga, Japan). The positive clones’ sequencing results were then analyzed via BLAST.
Additional Y2H experiments were performed to test the interactions of the screened transcription factor and the corresponding LaSCL6 fragment. The coding sequence of the screened transcription factor was cloned into the pGADT7 vector. Next, the constructs were co-transformed into yeast strain AH109, and yeast cells were grown on SD/-Leu-Trp media for 3–5 days. The positive clones were selected and plated onto SD/-Ade/-His/-Leu/-Trp media and cultured for 3–5 days, and positive clones were then transferred onto SD/-Ade/-His/-Leu/-Trp media containing 4 mg·mL−1 X-α-Gal to test possible interactions based on the growth status and blue-color development in the yeast colonies. All the primers used are listed in Supplemental Table S5.

4.10. BiFC Assay

We used the BiFC assay to directly visualize protein–protein interactions in vivo. The coding sequences of LaSCL6 and LaAP2L2 were cloned into the pSM vector to produce the nYFP-LaSCL6 and LaAP2L2-cYFP constructs, respectively. Each construct was individually transformed into A. tumefaciens strain GV3101. Then, the mixed Agrobacterium strain was introduced into N. benthamiana leaves via agro-infiltration. After 2 days of incubation, YFP fluorescence was observed in transformed leaf epidermal cells under a laser confocal microscope (Nikon C2-ER, Nikon, Tokyo Japan). All the primers used are listed in Supplemental Table S5.

5. Conclusions

In the present study, by identifying the target genes and interacting protein of LaSCL6 in L. kaempferi, we improved the regulatory network of LaSCL6 (Figure 6), in which LaSCL6 is regulated at several levels. Analyses of this regulatory network help to increase our understanding of the molecular mechanism of miR171-LaSCL6 in larch somatic embryogenesis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13213072/s1, Supplemental Table S1. Summary of transcriptome sequencing of transgenic Oryza sativa and Arabidopsis thaliana. Supplemental Table S2. Screening and comparison of differentially expressed genes in different comparisons. Supplemental Table S3. GRAS binding sites analysis in predicted gene promoters using information of the four species. Supplemental Table S4. Accession numbers for cloned sequences. Supplemental Table S5. Primes used in the research.

Author Contributions

Q.-L.Z. carried out the study, analyzed the data, and wrote the manuscript. Z.-L.Y. helped to analyze the data. W.-F.L. designed the study, analyzed the data, and revised the manuscript. L.-W.Q. provided suggestions on the experimental design and analyses. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32271904).

Data Availability Statement

Sequence data and corresponding GenBank accession numbers can be found in Supplemental Table S4. The RNA-Seq data have been uploaded to the China National Center for Bioinformation database under the designation PRJCA030648.

Acknowledgments

We thank Danyang Wang (Northwest University) for vector construction.

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Flow chart of transcriptome analysis. WT, wild-type; DEGs, differentially expressed genes. (A) Four Larix genes were obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in in the comparison of LaSCL6-var1 vs. WT in O. sativa and A. thaliana. (B) One Larix gene was obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in the comparison of LaSCL6-var2 vs. WT in O. sativa and A. thaliana. (C) 13 Larix genes were obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in the comparison of LaSCL6-var2 vs. LaSCL6-var1 in O. sativa and A. thaliana. (D) 15 Larix genes were obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in the comparison of LaSCL6-var1/2 vs. WT in O. sativa and A. thaliana.
Figure 1. Flow chart of transcriptome analysis. WT, wild-type; DEGs, differentially expressed genes. (A) Four Larix genes were obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in in the comparison of LaSCL6-var1 vs. WT in O. sativa and A. thaliana. (B) One Larix gene was obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in the comparison of LaSCL6-var2 vs. WT in O. sativa and A. thaliana. (C) 13 Larix genes were obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in the comparison of LaSCL6-var2 vs. LaSCL6-var1 in O. sativa and A. thaliana. (D) 15 Larix genes were obtained via expression pattern analysis and sequence alignment of the differentially expressed genes in the comparison of LaSCL6-var1/2 vs. WT in O. sativa and A. thaliana.
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Figure 2. Analysis of interactions between LaSCL6-var1 and its candidate target genes. (A) Yeast one-hybrid assays show that LaSCL6-var1 binds to the promoters of LaCYP89A5, LaWAKL20, LaENODL1, LaPIC30, and LaCYP84A1. (B) Schematic diagrams of the bait and prey vectors used in yeast one-hybrid assays. (C) Schematic diagrams of the effector and reporter vectors used in dual-LUC assays. (D–H) Dual-LUC analysis was performed using transient infiltration of Nicotiana benthamiana leaves with equal concentrations of Agrobacterium GV3101 cells transformed with effectors and reporters separately. The values were obtained as a ratio of the activity of firefly luciferase (LUC) and renilla luciferase (REN). Data represent values obtained as mean ± SD of three biological duplications. Error bars represent standard errors. *** p ≤ 0.001, ** p ≤ 0.01, Student’s t-test.
Figure 2. Analysis of interactions between LaSCL6-var1 and its candidate target genes. (A) Yeast one-hybrid assays show that LaSCL6-var1 binds to the promoters of LaCYP89A5, LaWAKL20, LaENODL1, LaPIC30, and LaCYP84A1. (B) Schematic diagrams of the bait and prey vectors used in yeast one-hybrid assays. (C) Schematic diagrams of the effector and reporter vectors used in dual-LUC assays. (D–H) Dual-LUC analysis was performed using transient infiltration of Nicotiana benthamiana leaves with equal concentrations of Agrobacterium GV3101 cells transformed with effectors and reporters separately. The values were obtained as a ratio of the activity of firefly luciferase (LUC) and renilla luciferase (REN). Data represent values obtained as mean ± SD of three biological duplications. Error bars represent standard errors. *** p ≤ 0.001, ** p ≤ 0.01, Student’s t-test.
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Figure 3. Analysis of interactions between LaSCL6-var2 and its candidate target genes. (A) Yeast one-hybrid assays show that LaSCL6-var2 binds to the promoters of LaCYP89A5, LaWAKL20, LaCCA1, and LaUGT85A3. (B) Schematic diagrams of the bait and prey vectors used in yeast one-hybrid assays. (C) Schematic diagrams of the effector and reporter vectors used in dual-LUC assays. (D–G) Dual-LUC analysis was performed by means of transient infiltration of Nicotiana benthamiana leaves with equal concentrations of Agrobacterium GV3101 cells transformed with effectors and reporters, respectively. The values were obtained as a ratio of the activity of firefly luciferase (LUC) and renilla luciferase (REN). Data represent values obtained as mean ± SD of three biological duplications. Error bars represent standard errors. *** p ≤ 0.001, NS p > 0.05, Student’s t-test.
Figure 3. Analysis of interactions between LaSCL6-var2 and its candidate target genes. (A) Yeast one-hybrid assays show that LaSCL6-var2 binds to the promoters of LaCYP89A5, LaWAKL20, LaCCA1, and LaUGT85A3. (B) Schematic diagrams of the bait and prey vectors used in yeast one-hybrid assays. (C) Schematic diagrams of the effector and reporter vectors used in dual-LUC assays. (D–G) Dual-LUC analysis was performed by means of transient infiltration of Nicotiana benthamiana leaves with equal concentrations of Agrobacterium GV3101 cells transformed with effectors and reporters, respectively. The values were obtained as a ratio of the activity of firefly luciferase (LUC) and renilla luciferase (REN). Data represent values obtained as mean ± SD of three biological duplications. Error bars represent standard errors. *** p ≤ 0.001, NS p > 0.05, Student’s t-test.
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Figure 4. Subcellular localization of LaSCL6-var1 and LaSCL6-var2. (A) Alignment and evolutionary analysis of amino acid sequences. The underling amino acid sequences indicate the nuclear localization signal (NLS). * identical sequences. (B) Subcellular localization of LaSCL6-var1 and LaSCL6-var2 in Arabidopsis thaliana protoplasts. Protoplasts were transiently transformed with 35S:: LaSCL6-var1/LaSCL6-var2-GFP constructs, 35S::GFP-LaSCL6-var1/LaSCL6-var2, and 35S:: GFP vector, respectively. GFP fluorescence was observed with a fluorescence microscope. NLS-mKATE was included for nuclear localization. Images were taken in a dark field for green fluorescence, while the cell outlines were photographed in a bright field. Bars = 10 μm.
Figure 4. Subcellular localization of LaSCL6-var1 and LaSCL6-var2. (A) Alignment and evolutionary analysis of amino acid sequences. The underling amino acid sequences indicate the nuclear localization signal (NLS). * identical sequences. (B) Subcellular localization of LaSCL6-var1 and LaSCL6-var2 in Arabidopsis thaliana protoplasts. Protoplasts were transiently transformed with 35S:: LaSCL6-var1/LaSCL6-var2-GFP constructs, 35S::GFP-LaSCL6-var1/LaSCL6-var2, and 35S:: GFP vector, respectively. GFP fluorescence was observed with a fluorescence microscope. NLS-mKATE was included for nuclear localization. Images were taken in a dark field for green fluorescence, while the cell outlines were photographed in a bright field. Bars = 10 μm.
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Figure 5. Screening and identification of interaction proteins of LaSCL6-var1 and LaSCL6-var2. (A) Schematic of vector construction of yeast two-hybrid screening. (B) Statistical results of yeast two-hybrid screening. (C) Yeast two-hybrid (Y2H) assays show that LaSCL6 interacts with LaAP2L2. (D) Bimolecular fluorescence complementation (BiFC) visualization of the interaction between LaSCL6 and LaAP2L2 in tobacco leaves. YFP fluorescence in nucleus means an interaction. NLS-mKATE was included for nuclear localization. The negative controls were performed using nYFP-LaSCL6 with empty cYFP and LaAP2L2-cYFP with empty nYFP. Bars = 20 μm.
Figure 5. Screening and identification of interaction proteins of LaSCL6-var1 and LaSCL6-var2. (A) Schematic of vector construction of yeast two-hybrid screening. (B) Statistical results of yeast two-hybrid screening. (C) Yeast two-hybrid (Y2H) assays show that LaSCL6 interacts with LaAP2L2. (D) Bimolecular fluorescence complementation (BiFC) visualization of the interaction between LaSCL6 and LaAP2L2 in tobacco leaves. YFP fluorescence in nucleus means an interaction. NLS-mKATE was included for nuclear localization. The negative controls were performed using nYFP-LaSCL6 with empty cYFP and LaAP2L2-cYFP with empty nYFP. Bars = 20 μm.
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Figure 6. Regulation of LaSCL6 in larch.
Figure 6. Regulation of LaSCL6 in larch.
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Zang, Q.-L.; Ye, Z.-L.; Qi, L.-W.; Li, W.-F. Identification of Target Gene and Interacting Protein of Two LaSCL6 Alternative Splicing Variants Provides Novel Insights into Larch Somatic Embryogenesis. Plants 2024, 13, 3072. https://doi.org/10.3390/plants13213072

AMA Style

Zang Q-L, Ye Z-L, Qi L-W, Li W-F. Identification of Target Gene and Interacting Protein of Two LaSCL6 Alternative Splicing Variants Provides Novel Insights into Larch Somatic Embryogenesis. Plants. 2024; 13(21):3072. https://doi.org/10.3390/plants13213072

Chicago/Turabian Style

Zang, Qiao-Lu, Zha-Long Ye, Li-Wang Qi, and Wan-Feng Li. 2024. "Identification of Target Gene and Interacting Protein of Two LaSCL6 Alternative Splicing Variants Provides Novel Insights into Larch Somatic Embryogenesis" Plants 13, no. 21: 3072. https://doi.org/10.3390/plants13213072

APA Style

Zang, Q. -L., Ye, Z. -L., Qi, L. -W., & Li, W. -F. (2024). Identification of Target Gene and Interacting Protein of Two LaSCL6 Alternative Splicing Variants Provides Novel Insights into Larch Somatic Embryogenesis. Plants, 13(21), 3072. https://doi.org/10.3390/plants13213072

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