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Transcriptome analysis identifies differentially expressed genes in the progenies of a cross between two low phytic acid soybean mutants.

ABSTRACT: Phytic acid (PA) is a major antinutrient that cannot be digested by monogastric animals, but it can decrease the bioavailability of micronutrients (e.g., Zn and Fe). Lowering the PA content of crop seeds will lead to enhanced nutritional traits. Low-PA mutant crop lines carrying more than one mutated gene (lpa) have lower PA contents than mutants with a single lpa mutant gene. However, little is known about the link between PA pathway intermediates and downstream regulatory activities following the mutation of these genes in soybean. Consequently, we performed a comparative transcriptome analysis using an advanced generation recombinant inbred line with low PA levels [2mlpa (mips1/ipk1)] and a sibling line with homozygous non-mutant alleles and normal PA contents [2MWT (MIPS1/IPK1)]. An RNA sequencing analysis of five seed developmental stages revealed 7945 differentially expressed genes (DEGs) between the 2mlpa and 2MWT seeds. Moreover, 3316 DEGs were associated with 128 metabolic and signal transduction pathways and 4980 DEGs were annotated with 345 Gene Ontology terms related to biological processes. Genes associated with PA metabolism, photosynthesis, starch and sucrose metabolism, and defense mechanisms were among the DEGs in 2mlpa. Of these genes, 36 contributed to PA metabolism, including 22 genes possibly mediating the low-PA phenotype of 2mlpa. The expression of most of the genes associated with photosynthesis (81 of 117) was down-regulated in 2mlpa at the late seed developmental stage. In contrast, the expression of three genes involved in sucrose metabolism was up-regulated at the late seed developmental stage, which might explain the high sucrose content of 2mlpa soybeans. Furthermore, 604 genes related to defense mechanisms were differentially expressed between 2mlpa and 2MWT. In this study, we detected a low PA content as well as changes to multiple metabolites in the 2mlpa mutant. These results may help elucidate the regulation of metabolic events in 2mlpa. Many genes involved in PA metabolism may contribute to the substantial decrease in the PA content and the moderate accumulation of InsP3-InsP5 in the 2mlpa mutant. The other regulated genes related to photosynthesis, starch and sucrose metabolism, and defense mechanisms may provide additional insights into the nutritional and agronomic performance of 2mlpa seeds.

SUBMITTER: Jin H

PROVIDER: S-EPMC8062490 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Low phytic acid (lpa) crops are potentially eco-friendly alternative to conventional normal phytic acid (PA) crops, improving mineral bioavailability in monogastric animals as well as decreasing phosphate pollution. The lpa crops developed to date carry mutations that are directly or indirectly associated with PA biosynthesis and accumulation during seed development. These lpa crops typically exhibit altered carbohydrate profiles, increased free phosphate, and lower seedling emergence, the latter of which reduces overall crop yield, hence limiting their large-scale cultivation. Improving lpa crop yield requires an understanding of the downstream effects of the lpa genotype on seed development. Towards that end, we present a comprehensive comparison of gene-expression profiles between lpa and normal PA soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. The lpa line used in this study carries single point mutations in a myo-inositol phosphate synthase gene along with two multidrug-resistance protein ABC transporter genes.RNA sequencing data of lpa and normal PA soybean lines from five seed-developmental stages (total of 30 libraries) were used for differential expression and functional enrichment analyses. A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. The results suggest that lpa-causing mutations play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively.This study provides a global perspective of transcriptomal changes during soybean seed development in an lpa mutant. The mutants are characterized by earlier expression of genes associated with cell wall biosynthesis and a decrease in photosynthetic genes in late stages. The biological processes and transcription factors identified in this study are signatures of lpa-causing mutations.

Project description:BackgroundSeed germination is important to soybean (Glycine max) growth and development, ultimately affecting soybean yield. A lower seed field emergence has been the main hindrance for breeding soybeans low in phytate. Although this reduction could be overcome by additional breeding and selection, the mechanisms of seed germination in different low phytate mutants remain unknown. In this study, we performed a comparative transcript analysis of two low phytate soybean mutants (TW-1 and TW-1-M), which have the same mutation, a 2 bp deletion in GmMIPS1, but show a significant difference in seed field emergence, TW-1-M was higher than that of TW-1 .ResultsNumerous genes analyzed by RNA-Seq showed markedly different expression levels between TW-1-M and TW-1 mutants. Approximately 30,000-35,000 read-mapped genes and ~21000-25000 expressed genes were identified for each library. There were ~3900-9200 differentially expressed genes (DEGs) in each contrast library, the number of up-regulated genes was similar with down-regulated genes in the mutant TW-1and TW-1-M. Gene ontology functional categories of DEGs indicated that the ethylene-mediated signaling pathway, the abscisic acid-mediated signaling pathway, response to hormone, ethylene biosynthetic process, ethylene metabolic process, regulation of hormone levels, and oxidation-reduction process, regulation of flavonoid biosynthetic process and regulation of abscisic acid-activated signaling pathway had high correlations with seed germination. In total, 2457 DEGs involved in the above functional categories were identified. Twenty-two genes with 20 biological functions were the most highly up/down- regulated (absolute value Log2FC >5) in the high field emergence mutant TW-1-M and were related to metabolic or signaling pathways. Fifty-seven genes with 36 biological functions had the greatest expression abundance (FRPM >100) in germination-related pathways.ConclusionsSeed germination in the soybean low phytate mutants is a very complex process, which involves a series of physiological, morphological and transcriptional changes. Compared with TW-1, TW-1-M had a very different gene expression profile, which included genes related to plant hormones, antioxidation, anti-stress and energy metabolism processes. Our research provides a molecular basis for understanding germination mechanisms, and is also an important resource for the genetic analysis of germination in low phytate crops. Plant hormone- and antioxidation-related genes might strongly contribute to the high germination rate in the TW-1-M mutant.

Project description:BackgroundSeed germination is essential to crop growth and development, and ultimately affects its harvest. It is difficult to breed soybeans low in phytic acid with a higher seed field emergence. Although additional management and selection could overcome the phytate reduction, the mechanisms of seed germination remain unknown.ResultsA comparative proteomic analysis was conducted between two low phytic acid (LPA) soybean mutants (TW-1-M and TW-1), both of which had a deletion of 2 bp in the GmMIPS1 gene. However, the TW-1 seeds showed a significantly lower field emergence compared to the TW-1-M. There were 282 differentially accumulated proteins (DAPs) identified between two mutants at the three stages. Among these DAPs, 80 were down-accumulated and 202 were up-accumulated. Bioinformatic analysis showed that the identified proteins were related to functional categories of oxidation reduction, response to stimulus and stress, dormancy and germination processes and catalytic activity. KEGG analysis showed that these DAPs were mainly involved in energy metabolism and anti-stress pathways. Based upon the conjoint analysis of DAPs with the differentially expressed genes (DEGs) previously published among three germination stages in two LPA mutants, 30 shared DAPs/DEGs were identified with different patterns, including plant seed protein, beta-amylase, protein disulfide-isomerase, disease resistance protein, pyrophosphate-fructose 6-phosphate 1-phosphotransferase, cysteine proteinase inhibitor, non-specific lipid-transfer protein, phosphoenolpyruvate carboxylase and acyl-coenzyme A oxidase.ConclusionsSeed germination is a very complex process in LPA soybean mutants. The TW-1-M and TW-1 showed many DAPs involved in seed germination. The differential accumulation of these proteins could result in the difference of seed field emergence between the two mutants. The high germination rate in the TW-1-M might be strongly attributed to reactive oxygen species-related and plant hormone-related genes. All these findings would help us further explore the germination mechanisms in LPA crops.

Project description:Transcriptomics reveals the existence of transcripts of different coding potential and strand orientation. Alternative splicing (AS) can yield proteins with altered number and types of functional domains, suggesting the global occurrence of transcriptional and post-transcriptional events. Many biological processes, including seed maturation and desiccation, are regulated post-transcriptionally (e.g., by AS), leading to the production of more than one coding or noncoding sense transcript from a single locus.We present an integrated computational framework to predict isoform-specific functions of plant transcripts. This framework includes a novel plant-specific weighted support vector machine classifier called CodeWise, which predicts the coding potential of transcripts with over 96 % accuracy, and several other tools enabling global sequence similarity, functional domain, and co-expression network analyses. First, this framework was applied to all detected transcripts (103,106), out of which 13 % was predicted by CodeWise to be noncoding RNAs in developing soybean embryos. Second, to investigate the role of AS during soybean embryo development, a population of 2,938 alternatively spliced and differentially expressed splice variants was analyzed and mined with respect to timing of expression. Conserved domain analyses revealed that AS resulted in global changes in the number, types, and extent of truncation of functional domains in protein variants. Isoform-specific co-expression network analysis using ArrayMining and clustering analyses revealed specific sub-networks and potential interactions among the components of selected signaling pathways related to seed maturation and the acquisition of desiccation tolerance. These signaling pathways involved abscisic acid- and FUSCA3-related transcripts, several of which were classified as noncoding and/or antisense transcripts and were co-expressed with corresponding coding transcripts. Noncoding and antisense transcripts likely play important regulatory roles in seed maturation- and desiccation-related signaling in soybean.This work demonstrates how our integrated framework can be implemented to make experimentally testable predictions regarding the coding potential, co-expression, co-regulation, and function of transcripts and proteins related to a biological process of interest.

Project description:The low phytic acid (lpa) trait in soybeans can be conferred by loss-of-function mutations in genes encoding myo-inositol phosphate synthase and two epistatically interacting genes encoding multidrug-resistance protein ATP-binding cassette (ABC) transporters. However, perturbations in phytic acid biosynthesis are associated with poor seed vigor. Since the benefits of the lpa trait, in terms of end-use quality and sustainability, far outweigh the negatives associated with poor seed performance, a fuller understanding of the molecular basis behind the negatives will assist crop breeders and engineers in producing variates with lpa and better germination rate. The gene regulatory network (GRN) for developing low and normal phytic acid soybean seeds was previously constructed, with genes modulating a variety of processes pertinent to phytic acid metabolism and seed viability being identified. In this study, a comparative time series analysis of low and normal phytic acid soybeans was carried out to investigate the transcriptional regulatory elements governing the transitional dynamics from dry seed to germinated seed. GRNs were reverse engineered from time series transcriptomic data of three distinct genotypic subsets composed of lpa soybean lines and their normal phytic acid sibling lines. Using a robust unsupervised network inference scheme, putative regulatory interactions were inferred for each subset of genotypes. These interactions were further validated by published regulatory interactions found in Arabidopsis thaliana and motif sequence analysis. Results indicate that lpa seeds have increased sensitivity to stress, which could be due to changes in phytic acid levels, disrupted inositol phosphate signaling, disrupted phosphate ion (Pi) homeostasis, and altered myo-inositol metabolism. Putative regulatory interactions were identified for the latter two processes. Changes in abscisic acid (ABA) signaling candidate transcription factors (TFs) putatively regulating genes in this process were identified as well. Analysis of the GRNs reveal altered regulation in processes that may be affecting the germination of lpa soybean seeds. Therefore, this work contributes to the ongoing effort to elucidate molecular mechanisms underlying altered seed viability, germination and field emergence of lpa crops, understanding of which is necessary in order to mitigate these problems.

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Transcriptome analysis identifies differentially expressed genes in the progenies of a cross between two low phytic acid soybean mutants.

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