- Background: Maize is one of the primary crops of genetic manipulation, which provides an excellent means of promoting stress resistance and increasing yield. - By analyzing the common and specific DEGs of the four materials, we found that there were 321 upregulated genes and 386 downregulated genes identified in the high-regeneration lines (141 and DH40), whereas 611 upregulated genes and 500 downregulated genes were specifically expressed in the low-regeneration lines (ZYDH381 – 1 and DH3732). - Analysis of the DEG expression patterns indicated a sharp change at stage I in both the high- and low-regeneration lines, which suggested that stage I constitutes a crucial period for EC regeneration. - Full list of author information is available at the end of the article. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Armstrong et al. - Previous studies revealed that the geno- type is an important factor that restricts the regeneration of the maize embryonic callus [4–7, 85]. - Nishimura et al. - Phenotypic evaluation of the four inbred lines. - The EC regeneration capacities of the four lines were investi- gated in our previous study [85]. - The results of the base composition and quality analysis showed that the clean reads had a good base composition (the T curve was in accordance with the A curve, and the G curve was in accordance with the C curve), and the percentage of low-quality reads was lower than 20% (Additional file 1: Figure S1). - Finally, only 60.73 to 67.38% of the remaining reads could be uniquely mapped onto the reference genome sequence (Table 1).. - A correl- ation value between biological replicates for each of the stages was calculated according to the FPKM result. - (Additional file 2: Figure S2), indicating good repeatabil- ity of the sequencing data.. - Reliability validation of DEG expression via qRT-PCR Ten DEGs involved in different biological processes (photosynthesis, plant hormone signal transduction, and protein phosphorylation) were randomly selected for qRT-PCR to validate the reliability of the transcriptome sequencing data. - For each line, the common DEGs among all the stages were much higher than the specific DEGs de- tected by each of the stages (Fig. - 3), indicating that only a small number of the DEGs were involved in the early redifferentiation capability of EC. - patterns in each of the low-regeneration lines (ZYDH381–1 and DH3732) during the process of redif- ferentiation (Fig. - 1 Phenotypic evaluation of the four inbred lines. - a Regeneration ability of the EC of the four inbred lines. - b The growth status of the EC of maize inbred lines 141 and DH3732 at 0 d, 3 d, 6 d, and 9 d. - The expression patterns of the up- or downregulated specific common DEGs were classified into five clusters (Fig. - Interestingly, the majority of the clusters in both the high- and low-regeneration lines indi- cated sharp changes in DEGs at stage I, but more moderate changes at stage II and III, which suggested that stage I played a key role in the regeneration of EC. - GO analysis for specific common DEGs. - To obtain functional annotations of the genes involved in EC regeneration capacity, we carried out GO analysis for the specific common DEGs. - Both the upregulated and downregulated genes of the specific common DEGs of 141 and DH40 were categorized into three functions:. - 2 Correlation of the differential expression ratio between qRT-PCR and RNA-Seq in the three stages. - 3 Venn diagram of the DEGs for each inbred line. - 4 Venn diagram of the DEGs between each inbred line. - To further understand the functional role of the DEGs in the process of EC regeneration, KEGG pathway analysis of the specific common DEGs of 141 and DH40 was con- ducted (Additional file 9: Table S15). - In stage II, 91 upregulated DEGs and 87 Table 2 The most differentially expressed specific common DEGs of the two high-regeneration lines. - Photosynthesis, ribosome, and por- phyrin and chlorophyll metabolism were three of the most significantly enriched upregulated pathways and were shared by the three stages of EC regeneration, indicating the con- served and important roles of these three metabolic path- ways in the process of EC regeneration. - Moreover, plant hormone signal transduction was the most significantly enriched pathway for the downregulated DEGs shared by the three stages of EC regeneration, suggesting that the de- creased expression of the genes in this pathway positively in- fluences EC regeneration. - Pathways enriched in the specific common DEGs of ZYDH381–1 and DH3732 are indicated in Additional file 10:. - By Table 3 The most differentially expressed specific common DEGs of the two low-regeneration lines. - comparing the DEGs of the four inbred lines, we found that a number of genes were involved in processes of photosyn- thesis, hormone signaling transduction, cell cycle, embryo and meristem initiation, circadian rhythm of plant, and phenylpropanoid biosynthesis. - According to the functional annotations of the specific common DEGs of 141 and DH40, 14 upregulated genes were in- volved in the pathway of photosynthesis (Fig. - Espe- cially CRY1 and ELF3, which respond to blue light and there- fore affect the photomorphogenesis of the plant, the other DEGs respond to red light and regulate plant cell elongation and flowering (Additional file 15: Figure S8).. - and ARF ( Zm00001d014690 ) were identified in the downregulated genes of the specific common DEGs of ZYDH381–1 and DH3732. - POD protects tissues and cells from oxi- dative damage by catalyzing the reduction of H 2 O 2 , and its accumulation accelerates the browning of the callus [28]. - PAL catalyzes the nonoxidative elimination of am- monia from L-Phe to yield trans -cinnamate in the first step of the phenylpropanoid pathway, which plays an es- sential role in plant development and the stress response [29–31]. - are both members of the cyclin family, which regulates CDK activity [34]. - Cluster analysis of the relative expressions of these DEGs (Additional file 19: Figure S12) showed that inbred lines 141 and DH40 were clustered into one group, while ZYDH381 – 1 and DH3732 as well as the three stages of each lines were separately clustered together. - 6 Relative expression patterns of the DEGs involved in tissue regeneration. - Rikiishi et al. - However, there are few reports related to the specific molecular mechanism of the effect of light on the regeneration of embryo-derived EC in maize.. - Photosynthesis is the basic energy conversion process on Earth, facilitating the utilization of the energy from sunlight by living organisms [42]. - Chlorophyll, a compound of magnesium porphyrin that absorbs energy from light, is one of the most important pigments re- lated to photosynthesis [44]. - Sergio et al. - Plant circadian rhythms are associated with the syn- chrony of the plant with the light cycle of its surround- ing environment, providing the plant with information on the season, thereby informing flowering and pollin- ator attraction [49, 50]. - Therefore, the downregulated expression of the DEGs re- lated to plant circadian rhythms might be detrimental to the redifferentiation of EC (Fig. - In tissue culturing of the garlic bulb, the efficiency of shoot regeneration was improved by the addition of 1–10 μM JA in B5 basic medium [55]. - Given its negative regulation of downstream JA responsive genes, we speculated that the downregulated expression of these genes prompted the expression of downstream re- sponsive genes and therefore motivated the regeneration of the callus. - accumulation of ABA inhibited the shoot regeneration of the callus derived from immature barley embryos [57]. - Previous studies indicated that the balance of auxin and CTK is key to controlling the dedifferentiation and differentiation of plant cells, and either shoots or roots can be regenerated from a callus by adjusting the auxin–cytokinin ratios of the induction medium [58–. - Our study indicated that most of the DEGs related to auxin and CTK transduc- tion were increased in 141 and DH40 but decreased in the poor regeneration lines (Fig. - Notably, all of the DEGs were upregulated in ZYDH381–1 and DH3732 but downregulated or unchanged in 141 and DH40. - Most of the cell cycle-related genes were upregulated in ZYDH381–1 and DH3732 but downregulated or unchanged in 141 and DH40, except embryogenesis-related genes (WOX), which were upregulated in 141 and DH40 but downreg- ulated in ZYDH381–1 and DH3732. - We discovered that most of the cell cycle-related DEGs (FtsH, CDK, CCNA, CCNB) were significantly upregulated in the two poor regeneration materials but exhibited opposite trends in the two high-regeneration materials. - Accordingly, we suggested that the increased expression of these DEGs facilitated the maintenance of the condition of EC and hindered the redifferentiation of EC. - Lowe et al. - In this study, transcriptome analysis of the EC of the four maize inbred lines showed that the specific com- mon DEGs of the high-regeneration lines were mainly associated with photosynthesis, porphyrin and chloro- phyll metabolism, ribosomes, and plant hormone signal transduction, while those of the low regeneration lines were mainly related to taurine and hypotaurine metabol- ism, nitrogen metabolism, fatty acid elongation, starch and sucrose metabolism, phenylpropanoid biosynthesis, and plant circadian rhythm. - No replacement of fresh media was conducted dur- ing each of the culture phases. - The specific components of the medium were listed in Additional file 20: Table S1.. - 7 Numbers and transcription levels of DEGs of the four maize inbred lines. - b Relative expression levels of the DEGs at the three stages of EC redifferentiation for each maize inbred line. - Each box plot shows the distribution of the relative transcription level [log2 (fold-change)] of the DEGs. - The red line indicates a one-fold change relative to the transcription level of the control samples. - Hierarchical clustering of the DEGs was performed using the pheatmap package in R. - The expression level ranges of the DEGs are displayed in Fig. - For DH40, the FC of the DEGs ranged from − 6.449 to 6.488. - The expression of the DEGs in ZYDH381 – 1 ranged from − 5.214 to 5.914. - The amplification program was performed ac- cording to the standard protocol of the Applied Biosystems 7500 Real-Time PCR System: 40 cycles of 98 °C for 2 min, 98 °C for 2 s, and 59 °C for 10 s, followed by a thermal de- naturing step to generate the melting curves for amplifica- tion specificity verification. - Up specific common DEGs of the two high regeneration lines in 5 clusters. - Down specific common DEGs of the two high regeneration lines in 5 clusters. - Up specific common DEGs of the two low regeneration lines in 5 clusters. - Down specific common DEGs of the two low regeneration lines in 5 clusters. - GO analysis of specific common DEGs of 141 and DH40 (A. - GO analysis of specific common DEGs of ZYDH381 – 1 and DH3732 (A. - Genome expression profile analysis of the immature maize embryo during dedifferentiation. - Comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines A188 and A632. - Proceedings of the VIIth International Congress on Plant Tissue and Cell Culture, Amsterdam, The Netherlands, 24 – 29 June 1990[M]. - Iwase A, Mita K, Nonaka S, et al. - Sarkar AK, Luijten M, Miyashima S, et al. - The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche. - Galinha C, Hofhuis H, Luijten M, et al. - Iwase A, Harashima H, Ikeuchi M, et al. - Cheng Y, Liu H, Cao L, et al. - Kanehisa M, Araki M, et al. - Yamaguchi M, Kato H, Yoshida S, et al. - Genome-wide analysis of the cyclin family in Arabidopsis and comparative phylogenetic analysis of plant cyclin-like proteins. - Bieniossek C, Schalch T, Bumann M, et al. - The molecular architecture of the metalloprotease FtsH. - Rikiishi K, Matsuura T, Maekawa M, et al. - Chico JM, Chini A, Fonseca S, et al. - Rikiishi K, Matsuura T, Ikeda Y, et al. - Correlation of the induction of various enzymes concerned with Phenylpropanoid and lignin synthesis during differentiation of bean callus (Phaseolus vulgaris L. - Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8/9 in developing seeds and somatic embryos of the gymnosperm Picea abies.. - RNA-Seq analysis of the Arabidopsis transcriptome in pluripotent Calli. - A genetic study of the regeneration capacity of embryonic callus from the maize immature embryo culture
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