Edited by: Gerald Alan Berkowitz, University of Connecticut, United States
Reviewed by: Changai Wu, Shandong Agricultural University, China; Kun Wang, Michigan State University, United States; Daowen Wang, Institute of Genetics and Developmental Biology (CAS), China
*Correspondence: Shubo Wan
Xinguo Li
This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science
†These authors have contributed equally to this work as co-first authors.
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Calcium not only serves as a necessary nutrient for plant growth but also acts as a ubiquitous central hub in a large number of signaling pathways. Free Ca2+ deficiency in the soil may cause early embryo abortion, which eventually led to abnormal development of peanut pod during the harvest season. To understand the mechanisms of Ca2+ regulation in pod development, transcriptome analysis of peanut gynophores and pods was performed by comparing the treatments between free Ca2+ sufficiency and free Ca2+ deficiency using Illumina HiSeq™ 2000. 9,903,082,800 nt bases are generated totally. After assembly, the average length of 102,819 unigenes is 999 nt, N50 is 1,782 nt. RNA-seq based gene expression profilings showed a large number of genes at the transcriptional level changed significantly between the aerial pegs and underground swelling pods under free Ca2+ sufficienct or deficiency treatments, respectively. Genes encoding key members of Ca2+ signaling transduction pathway, enzymes for hormone metabolism, cell division and growth, transcriptional factor as well as embryo development were highlighted. This information provides useful information for our further study. The results of digital gene expression (DGE) indicated that exogenous calcium might contribute to the development of peanut pod through its signal transduction pathway, meanwhile, promote the normal transition of the gynophores to the reproductive development.
Peanut (
Ca2+, a universal second messenger, plays an important role in plant growth and development, including cell division and apoptosis, polarity formation, differentiation and senescence (Hepler,
Digital gene expression (DGE) profiling, by comparing reads with the reference genome, can comprehensively and rapidly detect the specific gene expression of a particular species and distinguish the differences in the expression of poorly expressed genes (Glazinska et al.,
In this study, RNA-Seq and digital gene profiling were combined to investigate and compare differentially expressed genes (DEGs) between aerial gynophores and underground pods grown under free-Ca2+-sufficient or deficient Our objectives were to: (1) compare the DEGs of aerial gynophores between free Ca2+ sufficiency and free Ca2+ deficiency treatments, (2) identify candidate genes related to pod swelling affected by calcium, (3) find out the main regulatory pathways of calcium to promote pod development.
The
In peanut production, 15 days after pegging (DAP) was confirmed to be the most sensitive period for the free Ca2+ content in the soil. In this study, gynophores under free Ca2+-deficient treatment 1 (GD1), free Ca2+-deficient treatment 2 (GD2), free Ca2+-sufficient treatment 1 (GS1), free Ca2+-sufficient treatment 2 (GS2), as well as pods under free Ca2+-deficient treatment 1 (PD1), free Ca2+-deficient treatment 2 (PD2), free Ca2+-sufficient treatment 1 (PS1), and free Ca2+-sufficient treatment 2 (PS2), were harvested, frozen in liquid nitrogen and then stored at −70°C. The 8 samples were used to detect global changes in gene expression. Two biological replicates were used in this study. The mixed samples were used in transcriptome sequencing.
Total RNA was isolated from the harvested materials using the total RNA extraction Kit (TaKaRa, Inc., Dalian, China) according to the manufacturer's instructions. Agilent 2100 and NanoDrop were used to detect RNA quality and purity. Only high-quality RNA samples were chosen for RNA-seq analyses. Beads with Oligo (dT) were used to enrich for mRNA and the RNA was fragmented into short fragments. First-strand cDNA was synthesized using random hexamer primers and buffer, while dNTPs, RNaseH and DNA polymerase I were added to synthesize the second strand (Wang et al.,
Differential expression profiling was conducted among the gynophores and pods under free- Ca2+-deficient or sufficient treatments. Prior to further analysis, raw data should be filtered to decrease data noise and obtain clean data (Cock et al.,
The contents of endogenous hormone indole acetic acid (IAA) and gibberellin (GA3) were determined by enzyme linked immunosorbent assay (ELISA), which was performed as a commercial service in China agricultural university.
Gynophores and pod samples under the same treatments as those in RNA-seq were analyzed using qRT-PCR. cDNAs were reverse transcribed using the PrimeScript™ first-strand cDNA synthesis kit (K1622 Thermo scientific). The PCR was amplified using SYBR Premix Ex Taq™ following the manufacturer's instructions (TaKaRa, Inc., Dalian, China) with the qRT-PCR amplification instrument (ABI 7500, USA). The target gene primers (Table
Ca2+ deficiency in soil induces early embryo abortion in peanuts, and this process produces empty pods. This phenomenon was verified by the full pod number and dry weight of pods (Table
The effects of calcium treatment on peanut phenotype.
CK | 11.67 ± 1.15a | 9.82 ± 1.21a | 9.33 ± 0.58a | 23.65 ± 1.57a |
NC12 | 16.67 ± 1.15b | 15.25 ± 1.37b | 16.67 ± 1.15b | 33.65 ± 1.68b |
Functional annotation of unigenes gives protein functional annotation, COG functional annotation and Gene Ontology (GO) functional annotation. Unigenes were annotated with the databases of NR, NT, Swiss-Prot, KEGG, COG, and GO. Then the number of annotated unigenes was counted with each database (Figure
The unigene number annotated in six public database searched.
Characteristics of similarity search of unigenes against Nr database.
We use Blast2GO program with NR annotation to obtain the GO annotation of the unigenes. GO has three ontologies: molecular function, cellular component and biological process. Every GO-term belongs to a type of ontology (Conesa et al.,
A total of 14,985 sequences, containing 18,215 potential EST-SSRs, were detected with software MicroSAtellite (MISA). The quantity statistics of SSR classification showed that tri-nucleotide (40.86%) was the most abundant type of repeat motif, followed by di-nucleotide (29.94%), mononucleotide (21.61%), hexa-nucleotide (2.67%), quad-nucleotide (2.53%), and penta-nucleotide (2.38%) repeat units (Table
Eight DGE libraries were created from four independent biological samples using gynophores and pods with free Ca2+-deficient or -sufficient treatments as described in the method section. The clean reads (in millions) obtained from the different libraries were as follows: GD1, 14.91; GD2, 15.59; GS1, 14.54; GS2, 14.34; PD1, 16.02; PD2, 16.67; PS1, 14.19; and PS2, 12.84. The saturation analysis showed that when the number of reads reached a certain amount, the growth curve of detected genes tended to flatten in all libraries, indicating that the sequencing was saturated for gene identification (Figure
GO annotation of the DEGs from two pairwise comparisons (GD/GS and PD/PS) was used to classify genes into different sub-categories belonging to the three main GO categories. In the biological process category, we identified three abundant sub-categories across both comparisons, namely, metabolic process, cellular process, and single organism process. Cell, cell part, and membrane were the main sub-categories identified in the cellular component category, while catalytic activity and binding dominated the molecular function category (Figure
GO annotation of the DEGs from the two pairwise comparisons (GD/GS, PD/PS).
KEGG enrichment analysis allowed the mapping of DEGs to different pathways. The top 20 pathways in each pairwise comparison (mentioned above) are shown in Figure
FPKM method was used to calculate the gene expression level. The reference transcripts of a progenitor of cultivated peanut (
Differentially expressed genes in GD, GS, PD, and PS.
In order to confirm the transcriptome sequencing results, 10 DEGs in GS, GD, PS, and PD were selected randomly for qRT-PCR to analyze the expression levels. The selected genes participate in calcium signal transduction and phytohormone biosynthesis pathway, material biosynthesis, transcription factor and transporter. The consistency of trends between qRT-PCR and RNA-seq data indicated the credibility of the RNA-seq data (Figure
qRT-PCR verification of expression of selected genes.
More than 20 unigenes annotated as transcription factors including NAC, WRKY, bHLH, and ethylene-responsive transcription factors were regulated in GS, while M1/AGAMOUS/DEFICIENS/SRF (MADS) transcription factor family were down-regulated in GS compared with GD. Contrary to the DEGs in pods, no significant difference in calcium-related genes between GD and GS was found, while the expression of some calcium-binding proteins (CBPs), and calcium-binding transcription factors were up-regulated in GS (Table
The expression pattern of lipoxygenases (
We identified the DEGs between PD and PS considering that calcium plays an important role in the development of peanut pods. Unigenes encoding proteins involved in calcium signaling transduction pathways such as calcium-dependent protein kinases (CDPKs), calcium-binding protein (CBP) and calmodulin (CaM)-binding protein were up-regulated under free Ca2+-sufficient conditions. In particular, the expression of Ca2+-related protein mitogen-activated protein kinase kinase kinase (MAPKKK) was also higher in PS than that in PD (Table
Two categories of plant hormone-related genes (auxin and GA) showed significantly different expression between PD and PS. The auxin-related genes including 7 auxin response factors (ARFs) and 1 indole-3-acetic acid-amido synthetase were up-regulated in PS. Two selected DEGs, namely, GA 20-oxidase and GA receptor GID1, were up-regulated, while GA 2-oxidase, which is one of the key enzymes that catalyzes the inactivation of biological GA, was down-regulated in PS. Meanwhile, the ent-kaurenoic acid oxidase (KAO), an important enzyme in GA biosynthesis (Yamaguchi and Kamiya,
Compare IAA and GA3 contents between PS and PD. *Significant difference compared PD with PS using Student's
Genes encoding storage protein oleosin and some synthetases, such as tetrahydrocannabinolic acid synthase, long chain acyl-CoA synthetase, and callose synthase, exhibited higher level (5–8-folds) under free-Ca2+-sufficient conditions. In addition, eight lipoxygenase were also up-regulated in PS. Previous studies indicated that lipoxygenase is the first key enzyme in the synthesis pathway of jasmonic acid, which plays an important role in the germination, growth, and stress resistance of plant seeds (Rahimi et al.,
Early embryonic development is an important period for the formation of peanut yield and quality. Accumulating evidence illustrated that this complex process is regulated by light, endogenous hormones and environment stimuli. Ca2+ deficiency in this process leads to early embryo abortion in peanut, resulting in unfilled pod and reduced peanut yield. This phenomenon is a long-term concern in the acidic red-yellow soil in southern China (Zhang et al.,
In recent years, with widespread application of sequencing technology, transcriptome and proteomics provide views to explore the mechanism of the development of aerial peg and swelling of underground pod (Chen et al.,
Members of the Ca2+ signaling pathway, including CaM-binding protein, CDPK, and MAPKKK, were found to be highly expressed in PS (Table
Several studies have indicated that plant hormones, such as auxin, kinetin, ABA, and ethylene, can temporally and spatially coordinate the elongation of gynophore and swelling of pod (Jacobs,
Identified DEGs from gynophores and pods under free Ca2+ sufficient treatment and their functions during early pod development.
In this study, we performed a comparative transcriptome and differential expression analysis of peanut gynophores and pods under free Ca2+ sufficient and deficient conditions, respectively. Transcription factors including WRKY, NAC, bHLH, and MADS-box transcription factors were found up-regulated in GS, while MADS transcription factor family were down-regulated in GS compared to GD. Genes involved in plant hormone biosynthesis and transporters were found to be differentially expressed in the GS and GD. Different from the aerial part of peanut, genes involved in calcium signaling transduction pathways were identified up-regulated in pods when free Ca2+ was sufficient. In addition, hormone and embryo-related genes also showed differentially expressed between PD and PS. To our best knowledge, we firstly provided mechanism of exogenous calcium on the aerial and underground parts of peanut at the early stage of seed development.
SW and XL designed the experiment and drafted the manuscript. SY and LL carried out most of the experiment, analyzed the transcriptome and digital gene expression data, wrote the methods part of the manuscript. JZ, FG, and JM performed experiments, JW, NS, and YG grew the plants and prepared the samples. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by Natural Science Foundation of Shandong Province (ZR2015YL077, BS2015SW020), Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences (2015YQN02, 2014QNM38, 2015YQN12), National Natural Science Foundation of China (31571581, 31571605), the Supporting Plan of National Science and Technology of China (2014BAD11B04), Major Projects of Science and Technology Innovation in Shandong Academy of Agricultural Sciences (2014CXZ06-6).
The Supplementary Material for this article can be found online at:
The length distribution of contigs and unigenes.
GO classification analysis of unigenes in All-unigene. GO functions is showed in X-axis. The right Y-axis shows the number of genes which have the GO function, and the left Y-axis shows the percentage.
Clusters of orthologous groups (COG) classification of Unigenes in All-Unigene.
Quantity statistics of SSR classification. The X-axis is the repeat times of repeat units. The Y-axis is the number of SSRs.
Sequence saturation analysis of eight samples. The X-axis shows the number of clean reads, units is 100 k, the Y-axis shows the number of genes, units is %.
Scatter plot of top 20 KEGG pathways of two pairwise comparisons (GD/GS, PD/PS).
Summary for the transcriptome sequencing and assembly.
Summary of EST-SSR searching results.
Gene-specific primers used in quantitative real-time PCR.
Differentially expressed genes between GD and GS.
Differentially expressed genes between PD and PS.