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De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

ABSTRACT: Plant regeneration via somatic embryogenesis (SE) is the key step for genetic improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, but the molecular mechanisms underlying SE in cotton is still unclear. Here, RNA-Sequencing was used to analyze the genes expressed during SE and their expression dynamics using RNAs isolated from non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryos (SEs). A total of 101, 670 unigenes were de novo assembled. The genes differentially expressed (DEGs) amongst NEC, EC and SEs were identified, annotated and classified. More DEGs were found between SEs and EC than between EC and NEC. A significant number of DEGs were related to hormone homeostasis, stress and ROS responses, and metabolism of polyamines. To confirm the expression dynamics of selected DEGs involved in various pathways, experiments were set up to investigate the effects of hormones (Indole-3-butytric acid, IBA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results showed that exogenous application of IBA and KT positively regulated the development of EC and SEs, and that polyamines and H2O2 promoted the conversion of EC into SEs. Furthermore, we found that low and moderate stress is beneficial for proliferation of EC and SEs formation. Together, our global analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress response synergistically regulating SE in cotton.

SUBMITTER: Cheng WH

PROVIDER: S-EPMC5040755 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

Cheng Wen-Han WH Zhu Hua-Guo HG Tian Wen-Gang WG Zhu Shou-Hong SH Xiong Xian-Peng XP Sun Yu-Qiang YQ Zhu Qian-Hao QH Sun Jie J

Plant molecular biology 20160810 3

Plant regeneration via somatic embryogenesis (SE) is the key step for genetic improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, but the molecular mechanisms underlying SE in cotton is still unclear. Here, RNA-Sequencing was used to analyze the genes expressed during SE and their expression dynamics using RNAs isolated from non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryos (SEs). A total of 101, 670 unigenes were de novo as ...[more]

PMID: 27511192

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Project description:As a notable illustration of totipotency and plant regeneration, somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway, the key step for genetic engineering. Investigations examining the totipotency process are of great fundamental and practical importance in crop biotechnology. However, high-frequency regeneration of cotton via SE has been limited due to genotype-dependent response. The molecular basis deciphering SE genotype recalcitrance remains largely unexplored in cotton. In the current study, to comprehensively investigate the dynamic transcriptional profiling and gene regulatory patterns involved in SE process, a genome-wide RNA sequencing analysis was performed in two cotton genotypes with distinct embryogenic abilities, the highly embryogenic genotype Yuzao 1 (YZ) and the recalcitrant genotype Lumian 1 (LM). Three typical developmental staged cultures of early SE-hypocotyls (HY), nonembryogenic calli (NEC) and primary embryogenic calli (PEC)-were selected to establish the transcriptional profiles. Our data revealed that a total of 62,562 transcripts were present amongst different developmental stages in the two genotypes. Of these, 18,394 and 26,514 differentially expressed genes (DEGs) were identified during callus dedifferentiation (NEC-VS-HY) and embryogenic transdifferentiation (PEC-VS-NEC), respectively in the recalcitrant genotype, 21,842 and 22,343 DEGs in the highly embryogenic genotype. Furthermore, DEGs were clustered into six expression patterns during cotton SE process in the two genotypes. Moreover, functional enrichment analysis revealed that DEGs were significantly enriched in fatty acid, tryptophan and pyruvate metabolism in the highly embryogenic genotype and in DNA conformation change otherwise in the recalcitrant genotype. In addition, critical SE-associated expressed transcription factors, as well as alternative splicing events, were notably and preferentially activated during embryogenic transdifferentiation in the highly embryogenic genotype compared with the recalcitrant genotype. Taken together, by systematically comparing two genotypes with distinct embryogenic abilities, the findings in our study revealed a comprehensive overview of the dynamic gene regulatory patterns and uncharacterized complex regulatory pathways during cotton SE genotype-dependent response. Our work provides insights into the molecular basis and important gene resources for understanding the underlying genotype recalcitrance during SE process and plant regeneration, thereby holding great promise for accelerating the application of biotechnology to cotton for improving its breeding efficiency.

Project description:BackgroundHow to develop new cotton varieties possessing high yield traits of Upland cotton and superior fiber quality traits of Sea Island cotton remains a key task for cotton breeders and researchers. While multiple attempts bring in little significant progresses, the development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for aforementioned breeding purposes in upland cotton improvement. Based on the excellent fiber performance and relatively clear chromosome substitution segments information identified by Simple Sequence Repeat (SSR) markers, two CSSLs, MBI9915 and MBI9749, together with the recurrent parent CCRI36 were chosen to conduct transcriptome sequencing during the development stages of fiber elongation and Secondary Cell Wall (SCW) synthesis (from 10DPA and 28DPA), aiming at revealing the mechanism of fiber development and the potential contribution of chromosome substitution segments from Sea Island cotton to fiber development of Upland cotton.ResultsIn total, 15 RNA-seq libraries were constructed and sequenced separately, generating 705.433 million clean reads with mean GC content of 45.13% and average Q30 of 90.26%. Through multiple comparisons between libraries, 1801 differentially expressed genes (DEGs) were identified, of which the 902 up-regulated DEGs were mainly involved in cell wall organization and response to oxidative stress and auxin, while the 898 down-regulated ones participated in translation, regulation of transcription, DNA-templated and cytoplasmic translation based on GO annotation and KEGG enrichment analysis. Subsequently, STEM software was performed to explicate the temporal expression pattern of DEGs. Two peroxidases and four flavonoid pathway-related genes were identified in the "oxidation-reduction process", which could play a role in fiber development and quality formation. Finally, the reliability of RNA-seq data was validated by quantitative real-time PCR of randomly selected 20 genes.ConclusionsThe present report focuses on the similarities and differences of transcriptome profiles between the two CSSLs and the recurrent parent CCRI36 and provides novel insights into the molecular mechanism of fiber development, and into further exploration of the feasible contribution of G. barbadense substitution segments to fiber quality formation, which will lay solid foundation for simultaneously improving fiber yield and quality of upland cotton through CSSLs.

Project description:Somatic embryogenesis (S.E.) is a versatile model for understanding the mechanisms of plant embryogenesis and a useful tool for plant propagation. To decipher the intricate molecular program and potentially to control the parameters affecting the frequency of S.E., a proteomics approach based on two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF was used. A total of 149 unique differentially expressed proteins (DEPs) were identified at different stages of cotton S.E. compared with the initial control (0 h explants). The expression profile and functional annotation of these DEPs revealed that S.E. activated stress-related proteins, including several reactive oxygen species (ROS)-scavenging enzymes. Proteins implicated in metabolic, developmental, and reproductive processes were also identified. Further experiments were performed to confirm the role of ROS-scavenging enzymes, suggesting the involvement of ROS homeostasis during S.E. in cotton. Suppressing the expression of specifically identified GhAPX proteins resulted in the inhibition of dedifferentiation. Accelerated redifferentiation was observed in the suppression lines of GhAPXs or GhGSTL3 in parallel with the alteration of endogenous ascorbate metabolism and accumulation of endogenous H2O2 content. Moreover, disrupting endogenous redox homeostasis through the application of high concentrations of DPI, H2O2, BSO, or GSH inhibited the dedifferentiation of cotton explants. Mild oxidation induced through BSO treatment facilitated the transition from embryogenic calluses (ECs) to somatic embryos. Meanwhile, auxin homeostasis was altered through the perturbation of ROS homeostasis by chemical treatments or suppression of ROS-scavenging proteins, along with the activating/suppressing the transcription of genes related to auxin transportation and signaling. These results show that stress responses are activated during S.E. and may regulate the ROS homeostasis by interacting with auxin signaling.

Project description:BackgroundThe axillary bud is an important index of cotton plant-type traits, and the molecular mechanism of axillary bud development in upland cotton has not yet been reported. We obtained a mutant (designated mZ571) with a high-budding phenotype in axillary bud development from the low-budding phenotype variety G. hirsutum Z571 (CCRI 9A02), which provided ideal materials for the study of complex regulatory networks of axillary bud development. In this study, RNA sequencing was carried out to detect gene expression levels during three stages of axillary buds in Z571 (LB, low budding) and mZ571 mutant (HB, high budding).ResultsA total of 7162 DEGs were identified in the three groups (HB-E vs. LB-E, HB-G1 vs. LB-G1, HB-G2 vs. LB-G2), including 4014 downregulated and 3184 upregulated DEGs. Additionally, 221 DEGs were commonly identified in all three groups, accounting for approximately 3.09% of the total DEGs. These DEGs were identified, annotated and classified. A significant number of DEGs were related to hormone metabolism, hormone signal transduction, and starch and sucrose metabolism. In addition, 45, 22 and 9 DEGs involved in hormone metabolic pathways and 67, 22 and 19 DEGs involved in hormone signal transduction pathwayspathway were identified in HB-E vs. LB-E, HB-G1 vs. LB-G1, and HB-G2 vs. LB-G2, respectively, suggesting that endogenous hormones are the primary factors influencing cotton axillary bud growth. Hormone and soluble sugar content measurements revealed that mZ571 exhibited higher concentrations of zeatin, gibberellins and soluble sugar in all three stages, which confirmed that these hormone metabolism-, hormone signal transduction- and starch metabolism-related genes showed interaction effects contributing to the divergence of axillary bud growth between mZ571 and Z571.ConclusionsOur results confirmed the importance of endogenous hormones and sugars in the development of axillary buds, and we found that mZ571 plants, with a high-budding phenotype of axillary buds, exhibited higher endogenous hormone and sugar concentrations. Overall, we present a model for the emergence and development of cotton axillary buds that provides insights into the complexity and dynamic nature of the regulatory network during axillary bud emergence and development.

Project description:Somatic embryogenesis is a notable illustration of cell totipotency, by which somatic cells undergo dedifferentiation and then differentiate into somatic embryos. Our previous work demonstrated that pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in camphor tree. To better understand the molecular basis of somatic embryogenesis induced by osmotic stress, de novo transcriptome sequencing of three tissues of camphor tree (immature zygotic embryos, sucrose-pretreated immature zygotic embryos, and somatic embryos induced from sucrose-pretreated zygotic embryos) were conducted using Illumina Hiseq 2000 platform.A total of 30.70 G high quality clean reads were obtained from cDNA libraries of the three samples. The overall de novo assembly of cDNA sequence data generated 205592 transcripts, with an average length of 998 bp. 114229 unigenes (55.56 % of all transcripts) with an average length of 680 bp were annotated with gene descriptions, gene ontology terms or metabolic pathways based on Blastx search against Nr, Nt, Swissprot, GO, COG/KOG, and KEGG databases. CEGMA software identified 237 out of 248 ultra-conserved core proteins as 'complete' in the transcriptome assembly, showing a completeness of 95.6 %. A total of 897 genes previously annotated to be potentially involved in somatic embryogenesis were identified. Comparative transcriptome analysis showed that a total of 3335 genes were differentially expressed in the three samples. The differentially expressed genes were divided into six groups based on K-means clustering. Expression level analysis of 52 somatic embryogenesis-related genes indicated a high correlation between RNA-seq and qRT-PCR data. Gene enrichment analysis showed significantly differential expression of genes responding to stress and stimulus.The present work reported a de novo transcriptome assembly and global analysis focused on gene expression changes during initiation and formation of somatic embryos in camphor tree. Differential expression of somatic embryogenesis-related genes indicates that sucrose induced somatic embryogenesis may share or partly share the mechanisms of somatic embryogenesis induced by plant hormones. This study provides comprehensive transcript information and gene expression data for camphor tree. It could also serve as an important platform resource for further functional studies in plant embryogenesis.

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De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

Publications

De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

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