Oxidase activity analysis during rooting periods after cutting propagation
POD is a highly active oxidase that widely exists in plants, plays an important role in cell differentiation and has high activity. At the initial stage of cutting (Figure 1), the POD activity of the cuttings increased rapidly to the highest level. We speculate that PPO was beneficial to the formation of calli and ARs and that it could synthesize root-promoting substances, which is beneficial to the rooting of cuttings. At the initial stage of cutting, the activity of PPO reached the maximum and decreased gradually with the induction of ARs; therefore, the activity of PPO may be related to the emergence of calli. IAAO can decompose IAA and regulate the level of IAA in plants. Thus, the activity of the IAAO enzyme affects the formation of cutting root primordia. The decrease in IAAO activity in the early stage of stem cuttings helps to reduce the IAA in the decomposition body, which is beneficial to the rooting of cuttings. After AR formation, IAA synthesis decreased, and IAAO activity increased. The dynamic changes in POD, IAAO and PPO in the three clones were consistent, and the magnitude of the changes in the s1 period was proportional to the degree of promotion of AR formation.
Analysis of sequencing and assembly results
A total of 101.20 Gb of clean data was obtained from the library composed of 15 larch samples, and the clean data for each sample was 5.74 Gb, with the percentage of Q30 bases being 93.75% and above. A total of 43,246 unigenes were obtained after assembly; the N50 values of the transcripts and unigenes were 1,891 and 1,799, respectively, and the integrity of the assembly was high. There were 17,220 unigenes with lengths greater than 1 kb.
Functional annotation and classification of differentially expressed genes
The BLAST parameter E-value ≤ 1e-5 and the HMMER parameter E-value ≤ 1e- 10 were selected. Finally, 31,234 unigenes with annotation information were obtained, and the most annotated databases were NR (29,944, 95.87%), GO (23,210, 74.31%) and Pfam (22,221, 71.14%).
Enrichment analysis of differentially expressed genes by Gene Ontology (GO)
There are three categories in the Gene Ontology database: Molecular Function, Cellular Component and Biological Process. In the four AR formation stages, 57 GO terms were enriched (Figure 2). Differentially expressed genes in the four comparison groups were mainly enriched in metabolic process, cellular process and single organism process categories in Biological Process; cell, cell part, organelle and membrane categories in Molecular Function; and binding, catalytic activity and transporter activity categories in Cellular Component.
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs
The KEGG database systematically analyses the metabolic pathways of gene products in cells and the function of these gene products. (Figure 3.) Plant hormone signal transduction, phenylpropanoid biosynthesis and photosynthesis are the common metabolic pathways that were significantly enriched. Carbon metabolism is a unique significantly enriched pathway in s0 vs. s2. Carbon metabolism is an important process in organisms that can provide energy for plant growth and the structural components of cells and cell walls15. Plant–pathogen interaction and plant hormone signal transduction are the metabolic pathways with the largest amount of upregulated expression in the four stages, and the number of upregulated genes in the plant–pathogen interaction pathway was higher than that of downregulated genes. More DEGs in the plant hormone signal transduction pathway in the s0 vs. s2 and s0 vs. s3 stages were downregulated than upregulated. Photosynthesis-antenna proteins were highly enriched and downregulated in all four stages. Photosynthesis-antenna proteins are the most important part of the light-harvesting complex in photosynthesis and the most important part for obtaining light energy. The reason for the downregulation of photosynthesis-antenna protein expression may be that the stem cuttings were undergoing normal photosynthesis before cutting but as there is no reactant to induce the photosynthetic capacity after cutting because the root system is not developed and cannot absorb water. In addition, the MAPK signalling pathway-plant pathway was significantly enriched in s0 vs. s1, s0 vs. s3 and s0 vs. s4, and the DEGs were more often upregulated than downregulated. The MAPK signalling pathway is a signal transduction system that can promote the development of ARs in larch through extracellular signals that mediate the intracellular response of eukaryotic cells.
Screening of genes related to phytohormone signal transduction pathways
Auxin biosynthesis, transport and signal transduction play an important role in the occurrence and development of ARs; therefore, the expression of plant hormone-induced genes and related molecules has been widely studied. Based on GO and KEGG annotation analyses of all the DEGs, all four types of materials were found to be enriched in the plant hormone transduction pathway. Larch cutting propagation mostly belongs to the callus rooting type, so it can be considered that the production of calli is a very important stage. This process is not only a prerequisite for root primordium differentiation but also protects against the invasion of viruses and bacteria, restrains wound decay, preserves nutrients in the process of differentiation, and creates good conditions for stem cuttings to take root. Thus, we list the DEGs involved in hormone metabolism in the callus stage and AR induction stage (Table 1.); 36 genes were upregulated, and 33 were downregulated. With the emergence of calli and the induction of ARs, key genes within the auxin, gibberellin, brassinosteroid, jasmonic and salicylic pathways were upregulated at all stages, such as LAX1 (Auxin transporter-like protein 1), GID2 (Gibberellin insensitive dwarf 2), PYR1 (Pyrabactin resistance 1), CRK1 (Cysteine-rich receptor-like protein kinase 1), TIFY10B (Protein TIFY 10b) and TGA10 (bZIP transcription factor TGA10), while other genes showed consistently downregulated expression, such as ARF4 (Auxin response Factor 4), AHK3 (Histidine kinase 3), NSP2 (Protein nodulation signalling pathway 2), BEH4(BES1/BZR1 homologue protein 4) and DUF547 (Protein of unknown function 547).
Table 1
Hormone-related common DEGs (FPKM) of larch in the two stages
Hormone type
|
Gene number
|
s0 vs. s1
|
s0 vs. s2
|
Annotation
|
Database
|
Auxin
|
c141104.graph_c0
|
4.29
|
4.83
|
AUX1
|
KEGG/Swissprot
|
|
c135836.graph_c0
|
-1.29
|
-1.78
|
ARF
|
KEGG/nr/Swissprot
|
Cytokinin
|
c158645.graph_c0
|
-3.87
|
-1.75
|
B-ARR
|
KEGG
|
|
c147469.graph_c0
|
-3.42
|
-2.45
|
A-ARR
|
KEGG
|
|
c157788.graph_c0
|
-1.38
|
-2.83
|
A-ARR
|
KEGG
|
|
c158000.graph_c0
|
-2.62
|
-1.83
|
A-ARR
|
KEGG
|
Gibberellin
|
c155744.graph_c1
|
2.62
|
2.77
|
GID1
|
nr/Swissprot
|
|
c121654.graph_c0
|
2.23
|
2.28
|
GID2
|
KEGG/Swissprot
|
|
c126205.graph_c0
|
2.26
|
2.04
|
GID2
|
KEGG/Swissprot
|
|
c152078.graph_c0
|
2.48
|
1.65
|
GID2
|
KEGG/Swissprot
|
|
c129325.graph_c0
|
-2.99
|
-3.87
|
DELLA
|
KEGG
|
|
c141744.graph_c0
|
-3.83
|
-3.42
|
TF
|
KEGG
|
|
c158009.graph_c0
|
-1.69
|
-1.38
|
GID1
|
KEGG
|
Abscisic
|
c141172.graph_c0
|
5.35
|
6.13
|
PYR/PYL
|
KEGG
|
Brassinosteroid
|
c153133.graph_c1
|
3.38
|
4.74
|
BAK1
|
KEGG
|
|
c158615.graph_c0
|
2.03
|
1.52
|
BAK1
|
KEGG
|
|
c153361.graph_c0
|
-1.82
|
-2.16
|
BKI1
|
KEGG
|
|
c150449.graph_c0
|
-2.38
|
-2.98
|
BZR1/2
|
KEGG
|
Jasmonic Acid
|
c142639.graph_c0
|
1.20
|
1.14
|
COI1
|
KEGG
|
|
c154380.graph_c0
|
3.91
|
3.18
|
TIFY
|
Swissprot
|
|
c158387.graph_c0
|
5.45
|
4.96
|
TIFY
|
Swissprot
|
|
c156747.graph_c0
|
2.56
|
2.07
|
BHLH
|
Swissprot
|
|
c147063.graph_c0
|
-1.86
|
-2.28
|
JAR1
|
KEGG
|
Salicylic Acid
|
c156773.graph_c0
|
4.35
|
4.35
|
TGA
|
KEGG/Swissprot
|
|
c147358.graph_c0
|
5.54
|
5.54
|
PR-1
|
KEGG
|
|
c148550.graph_c0
|
4.02
|
4.02
|
PR-2
|
KEGG
|
Screening of genes related to rooting
According to the results of physiological and biochemical indices, the s1 period may be an important period in the rooting process of larch stem cuttings. To better identify the related genes that can promote rooting and formation, we selected the differential genes from the s0 vs. s1 and s0 vs. s2 comparisons for GO term enrichment analysis (Figure 4) in metabolic pathways. Five metabolic pathways that may be related to rooting and formation were found: organ boundary specification between lateral organs and the meristem, gibberellic acid-mediated signalling pathway, ethylene-activated signalling pathway, regulation of plant organ morphogenesis, and cell wall modification involved in multidimensional cell growth. To identify the genes that are not in these five metabolic pathways but may regulate these metabolic pathways, we singled out all the differentially expressed transcription factors (TFs) and constructed a gene interaction network map (we deleted the differentially expressed TFs that were not found to regulate genes in the pathway) (Figure 5). Pink represents the genes within the five pathways, red represents the genes outside the five pathways, and grey represents the functional proteins. The higher the connectivity of the gene is, the more likely it is in the hub position in the network, which means that it has a more important role. After consulting the relevant literature, we identified 11 genes most likely related to rooting from the LBD, AP2/ERF, MYB, NAC and bHLH families and used them in the following experiments.
DEGs of transcription factors
TFs as trans-acting factors regulating basal expression, have important roles in plant growth and development role, through specific interaction with cis-acting elements in the promoter region of the gene, the expression of downstream related genes is initiated16. Studies have shown that the TFs that affect the growth and development of plant roots are AUX/IAA, NAC, WRKY, AP2/ERF, LBD and etc, they participate in signal transduction and regulate the expression of functional genes, their overexpression can activate the coordinated expression of functional genes related to root formation, which can affect the adventure root formation of plants. AP2/ERF transcription factors are a large class of transcription factors that exist mainly in plants. They can regulate various processes of plant development and play an important role in hormone regulation and stress responses17, For example, controlling flower growth and development18, root initiation and development19-12, fruit development and ripening22, LOB gene is the first gene in the LBD family. The LBD gene family can be classified into two categories, the first is the complete leucine-like zipper motif, and the second is the incomplete leucine-like zipper motif23. There are many TFs involved in the differential genes common to the four stages of this study, which will not only affect the adventure root formation of larch, but also help us to mine the genes related to rooting of larch.
Quantification and validation of gene expression levels
Ten DEGs were randomly selected for qRT–PCR to verify the reliability of the RNA-Seq data. The qRT–PCR assay results showed strong similarity with the RNA-Seq data (Figure 6), which suggested that the RNA-Seq results were reliable.