Effects of IAA on microspore embryogenesis
Compared with the control group, the incidence of isolated microspore embryogenesis in Wucai treated with IAA was significantly increased (Fig. 1). Statistical analysis showed that IAA enhanced the occurrence of embryoid bodies in a dose-dependent manner (Table. 1). Surprisingly, the lower IAA dose had no or a negative effect on the incidence of microspore embryogenesis in Wucai. However, higher doses of IAA may inhibit embryogenesis. Among these, treatment with 100 mg. L − 1 IAA had the most significant effect, and the embryo yield reached 16.4 embryos. bud − 1, 3.15 times higher than that in the control group. At the same time, 150 mg. L− 1 inhibited the development of isolated microspore embryos. These results indicate that treatment with an appropriate IAA concentration is conducive to the embryogenesis of isolated microspores in Wucai.
Cytological observation of microspore embryonic development
According to the first division of the dedifferentiation-initiated microspore, the embryonic development pathways were divided into two types: Route A (unequal division) and Route B (equal division). Route A can be further subdivided according to the second division and subsequent situations. Studies have shown that there are two pathways, A and B, for embryonic development in isolated microspore cultures of non-heading Chinese cabbage, and pathway A is more common [39].
In this study, the microspore mother cells underwent meiosis to form a tetrad (Fig. 2), and four parenchyma cells were surrounded by a thick layer of callose (Fig. 2A, a). The tetrad was separated to form isolated microspores. The microspore just separated from the tetrad was in the mononuclear metaphase, when the nuclear to cytoplasm ratio was large (Fig. 2B, b). Subsequently, the nucleus was pushed aside by vacuoles, i.e., the mononuclear phase (Fig. 2C, c), the most sensitive stage for the induction of microspore embryogenesis. The microspore undergoes mitosis and forms two cells, one large and one small (Fig. 2D, d), i.e. unequal division. Therefore, we speculated that the developmental pathway of isolated microspores in Wucai was pathway A.
Compared to the control, the IAA-treated microspores had a dispersed arrangement of four microspores during the tetrad stage (Fig. 2b). At the mid-mononucleate stage, the nucleus was significantly larger and more deeply stained, and at the mononucleate leaning stage, it was triclinic with a distinct budding furrow, probably in preparation for subsequent divisions (Fig. 2c). The microspo-res underwent unequal division, forming a large trophic nucleus and a small reproductive nucleus(Fig. 2d). In addition, the multicellular masses formed were regularly rounded (Fig. 2e) and more numerous. In the control group, the early embryos were mostly torpedo-shaped (Fig. 2F, G), while in the IAA treated group, the early embryos first formed a long stalk and eventually developed into cotyledons (Fig. 2f, g). The multicellular proto embryos divided further in culture and eventually formed microspore embryos. In addition, the related research indicated that the microspore embryo would pass through the globular embryo, the heart shaped embryo, the torpedo shaped embryo and finally develop into the cotyledonary embryo. In this study, microscopic observation of microspores after 5 days of culture and statistical analysis of 10 randomly selected fields of view showed that the number of multicellular proembryos treated with IAA was significantly higher than that of the control group (clear water treatment group) (Additional file 1: Fig. s1) The initial time of microspore embryogenesis was counted after spraying with different concentrations of IAA (Additional file 2: Fig. Table s4). Compared with the control group, the time of microspore embryogenesis was 4d earlier after 100 mg. L− 1 IAA treatment.
Physiological And Biochemical Effects
Physiological indicators such as carbohydrates were determined in the flower buds of the treatment and control groups (Fig. 3). Carbo-hydrates provide energy and nutrients for the development of anthers and microspores. In this study, we determined total soluble sugars, amino acids, starch, and soluble proteins that provide carbon and nuclear nitrogen sources for microspore development. Compared with the control group, the total soluble sugar, amino acid, starch and soluble protein content in buds treated with IAA dramatically increased. This result indicates that the higher requirement of nutrients for microspore development might be responsible for the increased rate of isolated microspore embryogenesis.
Comparison Of Changes In The Content Of Endogenous Plant Hormones
After treatment with exogenous IAA, we analyzed the endogenous levels of different plant hormones, including auxin, cytokinins (CKs), abscisic acid (ABA), gibberellins (GAs), jasmonic acid (JAs), salicylic acid (SA), and strigolactones (SLs). After exogenous IAA treatment, the content of GAs in monocotyledonous leaning stage flower buds was dramatically increased, and the ABA content was slightly decreased compared to the control, while the content of other phytohorm-
Ones did not show obvious changes (Fig. 4). The change in auxin may have affected the dynamic balance between polar transport and hormones, and the GAs may have acted alone or in combination with auxin to influence microspore development and thus embryo rate by affecting vascular bundles and nutrient transport. The significant decrease in ABA may have changed the direction of pollen development from the original gametophytic pathway to the sporophytic pathway of development.
In the plant system, the physiological effects of different categories of plant hormones have a mutually reinforcing or antagonistic effect, and the regulation of plant growth and development is often the result of the combined action of multiple hormones. Therefore, the ratio of several hormones was analyzed in this experiment. The ratios of (auxin + GAs)/ABA, (auxin + GAs + CKs)/ABA, GAs/ABA, and auxin/ABA were all higher in the treatment group than in the control group (Addition File 3: Fig. S2), while the ratios of (auxin + GAs)/CKs and auxin/CKs were not obviously different from those in the control group. These results indicate that microspore development may also be related to the relative content and the dynamic balance between hormones.
Transcriptome Sequencing Data And Quality Control
To explore the molecular mechanism of exogenous IAA in enhancing the embryogenesis of isolated microspores in Wucai, we selected IAA-treated and non-treated mononuclear leaning stage buds for RNA-Seq using Illumina technology. After sequencing quality control and data analysis, 256,144,226 clean reads were obtained, and the clean data of each sample reached more than 5cGb. Clean reads accounted for 87.13% of the original reads, and the GC content ranged from 46.1 to 46.6%. The percentage of Q30 bases was 92% and above. The efficiency of the comparison between clean reads of each sample and the reference genome ranged from 86.75 to 87.43% (Table 2). In a direct comparison of the density and discrete distribution of expression levels for different samples, the sequencing quality and gene expression levels were essentially the same (Fig. 5). This indicates that the sequencing quality was good and can could be analyzed in the next step. The sequence alignment of clean reads with the reference genome was performed using HISAT2, (Table 2). The alignment rate was greater than 86%, with 86.75–87.43% for mapped reads and 1.91–2.14% for multi-mapped reads. The reads obtained from sequencing had a high matching rate and better assembly, which ensured the accuracy of the data.
Table 1
Effect of exogenous IAA on embryo rate of isolated microspores
IAA Concentration (mg. L− 1)
|
Frequency of microspore-derived embryo
(No.of embryos per bud, Mean ± SD)
|
0
|
5.20 ± 0.53d
|
50
|
6.10 ± 0.12d
|
75
|
11.10 ± 0.28c
|
100
|
16.40 ± 0.53a
|
125
|
13.48 ± 0.23b
|
150
|
4.76 ± 0.13e
|
In Duncan's multiple comparison test, different small letters in the same column of data indicated significant differences (P < 0.05) |
Table 2
Illumina sequencing data and results of de novo assembly
Sample
|
Total Reads
|
Clean Base(G)
|
Q30(%)
|
GC-content (%)
|
Reads- mapped
|
Unique-mapped
|
Multi-mapped
|
Cont-1
|
56,593,044
|
8.49
|
94.1
|
46.41
|
49,432,181 (87.35%)
|
85.40%
|
1.95%
|
Cont-2
|
38,553,118
|
5.78
|
92.1
|
46.24
|
33,444,692 (86.75%)
|
84.84%
|
1.91%
|
Cont-3
|
39,350,566
|
5.9
|
92.35
|
46.55
|
34,297,010 (87.16%)
|
85.19%
|
1.97%
|
IAA-1
|
39,273,240
|
5.89
|
92.35
|
46.16
|
34,309,235 (87.36%)
|
85.22%
|
2.14%
|
IAA-2
|
39,439,958
|
5.92
|
92.57
|
46.19
|
34,243,135 (86.82%)
|
84.71%
|
2.11%
|
IAA-3
|
42,934,300
|
6.44
|
92.39
|
46.59
|
37,536,097 (87.43%)
|
85.46%
|
1.97%
|
Identification Of Differentially Expressed Genes
To better understand the expression patterns of DEGs, DESeq was used for differential expression analysis. The screening conditions of differential genes were as follows: absolute value of log2(FC) greater than or equal to 1, and the p-value less than 0.05. A total of 2004 DEGs were identified, comprising 1002 upregulated genes and 1002 downregulated genes (Additional File 4; Fig. s2). The number of DEGs upregulated and downregulated DEGs was equivalent. In addition, 345 new genes were identified, 52 of which were not annotated to any database. The function of these new genes remains to be determined.
Functional Annotation By Gene Ontology
To further understand the function of the DEGs, GO enrichment analysis was performed on the sequenced DEGs, which were divided into three parts: Molecular Function (MF), Biological Process (BP), and Cellular Component (CC). Each part was annotated with GO entries of 14, 15, and 2 subcategorie. The GO items annotated to BPs mainly included metabolic processes (706 genes), cellular processes (958 genes), multicellular BPs (268 genes), and developmental processes (267 genes). The GO items annotated to cell components were mainly cellular anatomical entity (1323 genes). The GO items annotated to MFs mainly included the GO classification statistics of DEGs in catalytic activity (666 genes) and binding (839 genes), as shown in Fig. 6. These results indicate that a variety of complex metabolic pathways are involved in microspore development.
Pathway mapping by Kyoto Encyclopedia of Genes and Genomes
Different gene products in organisms perform biological functions through interactions. Analysis of the pathway annotation of DEGs contributes to a further understanding of gene function. According to the experimental results (Additional file5, Fig s3), 1334 differential genes were enriched in 121 metabolic pathways. They were divided into five categories: Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, and Organizational Systems.
The 50 pathways with the most significant enrichment were selected for display, as shown in Fig. 7. The metabolic pathways were annotated with 268 genes, plant-pathogen interaction, with 61 genes annotated; pentose and glucuronide interconversions, with 44 genes; MAPK signaling pathway-plant with 32 genes; protein processing in endoplasmic reticulum with 26 genes; phenylpropanoid biosynthesis with 22 genes; and galactose metabolism with 18 genes. These results indicate that genes related to secondary metabolism, plant-pathogen interaction, polysaccharides, signal transduction, and amino acids play important roles in the microspore development of Wucai.
Pollen And Embryo Development-related Genes
Pollen development plays an important role in microspore embryogenesis and involves a variety of complex BPs. Many genes related to pollen and embryonic development have been identified in the model plant Arabidopsis thaliana [40, 41]. A total of 79 DEGs were identified as being associated with pollen and embryonic development, which 46 were upregulated and 23 were downregulated (Additional file 6; Table s2). These comprise pollen development-related genes, embryonic development related genes, GDSL lipase genes, BHLH transcription factor family genes, AP2/ERF transcription factor family genes, pectinesterase genes, WUS-related genes, GST (glutathione S-transferase)-related genes, and RALF(protein RALF-like) genes. These results indicate that these DEGs participate in pollen and embryonic development, as well as reproducetive development, and play a certain role in promoting the subsequent embryogen-esis of isolated microspores.
Transcriptional And Metabolic Regulatory Networks For Plant Hormone Synthesis And Signal Transduction
To explore the role of the regulatory network of genes and metabolites in plant hormone synthesis and signal transduction (Fig. 8A), we mapped all detected DEGs and metabolites the relevant pathways (Fig. 8B).
In the process of gibberellin synthesis, non-bioactive GA24 is converted into active GA4 and GA9 under the catalysis of the GA20OX enzyme. GA2OX, as a key enzyme in the degradation process of GAs in plants, oxidizes bioactive GA4 to inactive GA8 and GA34, and oxidizes GA9, the precursor of GA4, to inactive GA51 and GA29, thereby maintaining the dynamic balance between bioactive GAs and their intermediates in plants. During phytohormone signaling, the transcription factors PIF3 and PIF4 are activated to mediate the gibberellin signal response.
During auxin biosynthesis, tryptophan is catalyzed by enzymes to synthesize indole 3 acetamide (IAM) and indole 3 acetaldehyde (IAAld), prerequisite substances of IAA. IAA1d is synthesized into indoleacetic acid through the action of aldehyde dehydrogenase. In this study, CYP71AB was downregulated and DAO was upregulated. There were four genes encoding aldehyde dehydrogenase (ALDH), of which one was upregulated and three were downregulated. A total of 12 DEGs were detected in association with growth hormone signaling, of which seven were upregulated and five were downregulated. Changes in these DEGs resulted in the accumulation of Auxin.
During ABA synthesis, β-cyclohydroxylase (LUT5), β-carotene isomerase (DWARF27), and abscisic β-glucosyltransferase (AOG) were all downregulated, while abscisic acid 8’-hydroxylase (CYP707A) was upregulated. leading to a decrease in ABA accumulation in vivo. Both the abscisic acid receptor (PYL) and protein phosphatase (PP2C) were downregulat-ed during signal transduction.The content of endogenous hormones IAA (Fig. 8C), GA4 (Fig. 8E), and GA9 (Fig. 8F) increased dramatically and the ABA (Fig. 8D) content decreased greatly after IAA treatment .
Oxidative Phosphorylation Pathway
Normal microspore development requires an adequate energy supply, and the energy produced by mitochondrial oxidative phosphorylation in plants supplies the needs for plant growth and breeding (Fig. 9A). The DEGs were mainly enriched in NADH dehydrogenase, succinate dehydrogenase, and ATP synthase (complex V) (Fig .9B). Under the action of dehydrogenase, NADH stimulates the transmission of the electron transfer chain and pumps out H+. Succinic acid forms fumaric acid under the action of succinic dehydrogenase (SDH) and accelerates electron transfer. PPi generates ADP and Pi through pyrophosphate (PPA). Under the driving force of the electrochemical potential gradient, H + enters the mitochondrial inner membrane along the proton channel through the catalysis of ATPase. ATP is produced by ADP and Pi under the catalysis of V-type proton ATPase (VHA). A total of 17 DEGs were detected in the oxidative phosphorylation pathway, of which 14 were upregulated and 3 were downregulated.
Plant mitochondria are also the main site of ROS production, so the material substances that affect ROS production and accumulation were measured. Compared with the control group, H2O2 (Fig. 9C) and SOD (Fig .9E) contents decreased, and CAT (Fig. 9D) and POD (Fig .9F) contents increased in the IAA treated monocotyledonous leaning buds, which maintained the balance of ROS levels during microspore development and prevented ROS accumulation, leading to membrane damage.
Pentose And Glucuronate Interconversions Pathway
Pectin is a class of polysaccharide substances in plant cell walls that performs important functions in plant growth and development, including promoting cell adhesion, providing structural support, providing oligosaccharides for plant growth and defense reactions, and maintaining the liquidity of the cell wall. Pectin is mainly composed of polysaccharides rich in galacturonic acid, and PME plays an important role in the processes of microsporogenesis, cell wall expansion, and cell wall adhesion. More DEGs were enriched in the pentose and glucuronate interconversion pathway accord-ing to the 20 most significant KEGG enrichment pathways (Fig. 10). In the pentose and glucuronate interconversion pathway, we detected 44 DEGs, of which 38 were upregulated and six were downregulated (Fig. 10C). Pectin was catalyzed by PME to form pectate, which formed D-galacturonate under the action of PGA. D-galacturonate was an important component of primary cell wall in plants. UDP-D-glucuronate plays an important role in plant growth and development as well as in the removal of exogenous and endogenous toxins (Fig. 10A). The key enzymes in pectin metabolism, PME and PGA, were upregulated. Two of the three genes encoding polygalactur-onases (GSVIVT) were upregulated. There were two genes encoding UDP-sugar pyrophosphorylase (USP), of which one upregulated and one downregulated (Fig. 10C). The pectin content was significantly lower than that of the control (Fig. 10B). We speculated that exogenous IAA treatment may promote isolated microspore embryogenesis by affecting cell wall development.
Real-time Qpcr Validation Of Gene Expression Patterns
We selected 16 DEGs from the important metabolic pathways for qRT-PCR to verify the reliability of RNA-Seq (Fig. 11), including those encoding auxin-responsive protein IAA26-like (BraA01g035920.3C, IAA26), beta carotene isomerase D27,chloroplastic like (BraA09g013410.3C, D27), succinatedehydro-genase (BraA07g020250.3C, SDHB), 2-oxoglutarate-dependent dioxygenase DAO-like (BraA06g010270.3C, DAO), gibberellin 2-beta-dioxygenase 2-lik (BraA08g023600.3C, GA2OX),pectinesterase5 (BraA05g000800.3C, PME5), cytochrome P450 71A14-like (BraA06g032060.3C,CYP71AB), exopolyga-lacturonase clone GBGE184-like (BraA08g035590.3C, PGLR1), probable pectate lyase 7 (BraA03g030560.3C, PLY7), abscisic acid 8'-hydroxylase 3-like (BraA06g042600.3C, ABAH3), catalase-3 (BraA08g027750.3C, CAT3), cytokinin hydroxylase isoform X1 (BraA02g017430.3C, C7352), indole-3-acetic acid-amido synthetase GH3.2 (BraA01g001510.3C, GH3.3), ATPase-9,plasma membrane-type (BraA07g042850.3C, PMA9), pectinesterase1 (BraA06g001110.3C, PME1), and probable pectinesterase inhibitor (BraA09g051660.3C, BP19). The gene expression patterns obtained from qRT-PCR and RNA-Seq data showed similar trends, which confirmed the accuracy of the RNA-Seq results obtained in this study.