Comparative root transcriptome analysis of two soybean cultivars with different cadmium sensitivities reveals the underlying tolerant mechanisms

The soybean can provide rich protein and fat and has great economic value worldwide. Cadmium (Cd) is a toxic heavy metal to organisms. It can accumulate in plants and be transmitted to the human body via food chain. Cd is a serious threat to soybean development, especially to root growth. Some soybean cultivars present tolerant symptoms under Cd stress; however, the potential mechanisms are not fully understood. Here, we optimized RNA-seq to identify the differentially expressed genes (DEGs) in Cd-sensitive (KUAI) and Cd-tolerant (KAIYU) soybean roots and compared the DEGs between KAIYU and KUAI. A total of 1,506 and 1,870 DEGs were identified in the roots of KUAI and KAIYU, respectively. Through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene function analyses, we found that genes related to antioxidants and sequestration were responsible for Cd tolerance in KAIYU. In addition, overexpression of Glyma11g02661, which encodes a heavy metal transporting ATPase, significantly improved Cd tolerance in transgenic hairy roots. These results provide a preliminary understanding of the tolerance mechanisms in response to Cd stress in soybean root development and are of great importance in developing Cd-resistant soybean cultivars by using the identified DEGs through genetic modification.

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.

A Root Tip-Specific Expressing Anthocyanin Marker for Direct Identification of Transgenic Tissues by the Naked Eye in Symbiotic Studies

The Agrobacterium rhizogenes hairy root transformation system is widely used in symbiotic studies of model legumes. It typically relies on fluorescent reporters, such as DsRed, for identification of transgenic roots. The MtLAP1 transcription factor has been utilized as a reporter system in Medicago truncatula based on production of anthocyanin pigment. Here, we describe a version of this reporter driven by a root-cap specific promoter for direct observation of anthocyanin accumulation in root tips, which allows the identification of transgenic hairy roots by the naked eye. Results from our analysis suggest that the reporter had no significant effects on nodulation of M. truncatula. This approach, by virtue of its strong and specific expression in root cap cells, greatly reduces false positives and false negatives, and its use of an easily scored visible pigment should allow greater versatility and efficiency in root biology studies.

Transcriptome profiling reveals histone deacetylase 1 gene overexpression improves flavonoid, isoflavonoid, and phenylpropanoid metabolism in Arachis hypogaea hairy roots

Background The peanut (Arachis hypogaea) is a crop plant of high economic importance, but the epigenetic regulation of its root growth and development has not received sufficient attention. Research on Arabidopsis thaliana has shown that histone deacetylases (HDACs) are involved in cell growth, cell differentiation, and stress response. Few studies have focused on the role of HDACs in the root development of other plants, particularly crop plants. In earlier studies, we found large accumulations of A. hypogaea histone deacetylase 1 (AhHDA1) mRNA in peanut roots. However, we did not explore the role of AhHDA1 in peanut root development. Methods In this paper, we investigated the role of the peanut AhHDA1 gene and focused on the effect of altered AhHDA1 expression in hairy roots at both the phenotypic and transcriptional levels. We analyzed the transformation of A. hypogaea hairy roots using Agrobacterium rhizogenes and RNA sequencing to identify differentially expressed genes that were assigned to specific metabolic pathways. Transgenic hairy roots were used as experimental material to analyze the downstream genes expression and histone acetylation levels. To thoroughly understand AhHDA1 function, we also simultaneously screened the AhHDA1-interacting proteins using a yeast two-hybrid system. Results AhHDA1-overexpressing hairy roots were growth-retarded after 20 d in vitro cultivation, and they had a greater accumulation of superoxide anions and hydrogen peroxide than the control and RNAi groups. AhHDA1 overexpression in hairy roots accelerated flux through various secondary synthetic metabolic pathways, as well as inhibited the primary metabolism process. AhHDA1 overexpression also caused a significant upregulation of genes encoding the critical enzyme chalcone synthase (Araip.B8TJ0, CHS) in the flavonoid biosynthesis pathway, hydroxyisoflavanone synthase (Araip.0P3RJ) in the isoflavonoid biosynthesis pathway, and caffeoyl-CoA O-methyltransferase (Aradu.M62BY, CCoAOMT) in the phenylpropanoid biosynthesis pathway. In contrast, ferredoxin 1 (Araip.327XS), the polypeptide of the oxygen-evolving complex of photosystem II (Araip.N6ZTJ), and ribulose bisphosphate carboxylase (Aradu.5IY98) in the photosynthetic pathway were significantly downregulated by AhHDA1 overexpression. The expression levels of these genes had a positive correlation with histone acetylation levels. Conclusion Our results revealed that the relationship between altered gene metabolism activities and AhHDA1 overexpression was mainly reflected in flavonoid, isoflavonoid, and phenylpropanoid metabolism. AhHDA1 overexpression retarded the growth of transgenic hairy roots and may be associated with cell metabolism status. Future studies should focus on the function of AhHDA1-interacting proteins and their effect on root development.

Genome-Wide Analysis of the C2 Domain Family in Soybean and Identification of a Putative Abiotic Stress Response Gene GmC2-148

Plant C2 domain proteins play essential biological functions in numerous plants. In this study, 180 soybean C2 domain genes were identified by screening. Phylogenetic relationship analysis revealed that C2 domain genes fell into three distinct groups with diverged gene structure and conserved functional domain. Chromosomal location analysis indicated that C2 domain genes mapped to 20 chromosomes. The transcript profiles based on RNA-seq data showed that GmC2-58, GmC2-88, and GmC2-148 had higher levels of expression under salt, drought, and abscisic acid (ABA) treatments. GmC2-148, encoding a cell membrane-localized protein, had the highest level of response to various treatments according to real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Under salt and drought stresses, the soybean plants with GmC2-148 transgenic hairy roots showed delayed leaf rolling, a higher content of proline (Pro), and lower contents of H2O2, O2– and malondialdehyde (MDA) compared to those of the empty vector (EV) plants. The results of transgenic Arabidopsis in salt and drought treatments were consistent with those in soybean treatments. In addition, the soybean plants with GmC2-148 transgenic hairy roots increased transcript levels of several abiotic stress-related marker genes, including COR47, NCDE3, NAC11, WRKY13, DREB2A, MYB84, bZIP44, and KIN1 which resulted in enhanced abiotic stress tolerance in soybean. These results indicate that C2 domain genes are involved in response to salt and drought stresses, and this study provides a genome-wide analysis of the C2 domain family in soybean.

Enhanced Cadmium Accumulation and Tolerance in Transgenic Hairy Roots of Solanum nigrum L. Expressing Iron-Regulated Transporter Gene IRT1

Solanum nigrum L., a hyperaccumulator of cadmium (Cd), is regarded as a promising candidate for phytoremediation of heavy metal pollution. In the present study, the hairy roots of Solanum nigrum L. were selected as a model plant system to study the potential application of Iron-regulated Transporter Gene (IRT1) for the efficient phytoremediation of Cd pollution. The transgenic hairy roots of Solanum nigrum L. expressing the IRT1 gene from Arabidopsis thaliana were successfully obtained via the Agrobacterium tumegaciens-mediated method. Expression of IRT1 reduced Cd stress-induced phytotoxic effects. Significantly superior root growth, increased antioxidant enzyme activities, decreased reactive oxygen species (ROS) levels, and less cell apoptosis were observed in the transgenic hairy roots of Solanum nigrum L. compared to the wild-type lines under Cd stress. Enhanced Cd accumulation was also carried out in the transgenic hairy roots compared to the control (886.8 μg/g vs. 745.0 μg/g). These results provide an important understanding of the Cd tolerance mechanism of transgenic IRT1 hairy roots of Solanum nigrum L., and are of particular importance to the development of a transgenic candidate for efficient phytoremediation process.

GmNAC06, a NAC domain transcription factor enhances salt stress tolerance in soybean

Key message We found GmNAC06 plays an important role in salt stress responses through the phenotypic, physiological and molecular analyses of OE, VC, and Mutant composite soybean. Abstract Salinization affects 20% of all cultivated land worldwide because of the high salinity of irrigation water and the excessive use of water, and this amount is increasing daily. NAC (NAM, ATAF, and CUC) have been found to be involved in salt stress. In this study, a soybean NAC gene, GmNAC06 (Glyma06g21020.1), was cloned and functionally characterized. The results of expression analysis suggested that salt stress could influence the expression level of GmNAC06. The subcellular localization analysis results suggested that GmNAC06 may function as a transcription factor. Under salt stress, the overexpression technology combined with CRISPR-Cas9 system found that GmNAC06 could cause the accumulation of proline and glycine betaine to alleviate or avoid the negative effects of ROS; similarly, it could control the Na+/K+ ratios in hairy roots to maintain ionic homeostasis. The fresh weight of the transgenic hairy roots and the histochemical ROS staining of wild leaves suggested that transgenic hairy roots influence the function of wild leaves under salt stress conditions. Moreover, the expression levels of GmUBC2 and GmHKT1 were higher in the GmNAC06 hairy roots than in the control. Thus, the overexpression of GmNAC06 in hairy roots notably causes an entire composite plant to exhibit salt tolerance. The phenotype of composite soybean plants and transgenic Arabidopsis plants suggest that GmNAC06 plays a role in response to salt stress and could be useful in generating salt tolerant transgenic crops.

Isoflavone malonyl-CoA acyltransferase GmMaT2 is involved in nodulation of soybean (Glycine max) by modifying synthesis and secretion of isoflavones

Malonyl-CoA:flavonoid acyltransferases (MaT) modify isoflavones, but only a few have been characterized for activity and assigned to specific physiological processes. Legume roots exude isoflavone malonates into the rhizosphere, where they are hydrolyzed into isoflavone aglycones. Soybean GmMaT2 was highly expressed in seeds, root hairs, and nodules. GmMaT2 and GmMaT4 recombinant enzymes used isoflavone 7-O-glucosides as acceptors and malonyl-CoA as an acyl donor to generate isoflavone glucoside malonates. GmMaT2 had higher activity towards isoflavone glucosides than GmMaT4. Overexpression (OE) in hairy roots of GmMaT2 and 4 produced more malonyldaidzin, malonylgenistin, and malonylglycitin, and resulted in more nodules than control. However, only GmMaT2 knockdown (KD) hairy roots showed reduced levels of malonyldaidzin, malonylgenistin, and malonylglycitin, and likewise, reduced nodule numbers. These were consistent with the up-regulation of only GmMaT2 by rhizobial infection, and higher expression levels of early nodulation genes in GmMaT2- and 4-OE, but lower only in GmMaT2-KD roots compared to control roots. Higher malonyl isoflavonoid levels in transgenic hairy roots were associated with higher levels of isoflavones in root exudates and more nodules, and vice versa. We posit that GmMaT2 participates in soybean nodulation by catalyzing isoflavone malonylation and affecting malonyl isoflavone secretion for activation of Nod factor and nodulation.

Identification of loci and candidate gene GmSPX-RING1 responsible for phosphorus efficiency in soybean via genome-wide association analysis

Background Phosphorus (P) is an essential element in maintaining high biomass and yield in crops. Soybean [Glycine max (L.) Merr.] requires a large amount of P during growth and development. Improvement of P efficiency and identification of P efficiency genes are important strategies for increasing soybean yield. Results Genome-wide association analysis (GWAS) with NJAU 355 K SoySNP array was performed to identify single nucleotide polymorphisms (SNPs) significantly associated with three shoot P efficiency-related traits of a natural population of 211 cultivated soybeans and relative values of these traits under normal P (+P) condition and P deficiency (−P) condition. A total of 155 SNPs were identified significantly associated with P efficiency-related traits. SNPs that were significantly associated with shoot dry weight formed a SNP cluster on chromosome 11, while SNPs that were significantly associated with shoot P concentration formed a SNP cluster on chromosome 10. Thirteen haplotypes were identified based on 12 SNPs, and Hap9 was considered as the optimal haplotype. Four SNPs (AX-93636685, AX-93636692, AX-93932863, and AX-93932874) located on chromosome 10 were identified to be significantly associated with shoot P concentration under +P condition in two hydroponic experiments. Among these four SNPs, two of them (AX-93636685 and AX-93932874) were also significantly associated with the relative values of shoot P concentration under two P conditions. One SNP AX-93932874 was detected within 5′-untranslated region of Glyma.10 g018800, which contained SPX and RING domains and was named as GmSPX-RING1. Furthermore, the function research of GmSPX-RING1 was carried out in soybean hairy root transformation. Compared with their respective controls, P concentration in GmSPX-RING1 overexpressing transgenic hairy roots was significantly reduced by 32.75% under +P condition; In contrast, P concentration in RNA interference of GmSPX-RING1 transgenic hairy roots was increased by 38.90 and 14.51% under +P and -P conditions, respectively. Conclusions This study shows that the candidate gene GmSPX-RING1 affects soybean phosphorus efficiency by negatively regulating soybean phosphorus concentration in soybean hairy roots. The SNPs and candidate genes identified should be potential for improvement of P efficiency in future soybean breeding programs.