Soybean Protoplasts Research Articles

Targeted mutagenesis in soybean using the CRISPR-Cas9 system

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2–9.7% using the pCas9-AtU6-sgRNA vector and 14.7–20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.

Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.

Soybean (Glycine max) is a globally important crop, and its growth and yield are severely reduced by abiotic stresses, such as drought, heat, and cold. The cis-acting element DRE (dehydration-responsive element)/CRT plays an important role in activating gene expression in response to these stresses. The Arabidopsis DREB1/CBF genes that encode DRE-binding proteins function as transcriptional activators in the cold stress responsive gene expression. In this study, we identified 14 DREB1-type transcription factors (GmDREB1s) from a soybean genome database. The expression of most GmDREB1 genes in soybean was strongly induced by a variety of abiotic stresses, such as cold, drought, high salt, and heat. The GmDREB1 proteins activated transcription via DREs (dehydration-responsive element) in Arabidopsis and soybean protoplasts. Transcriptome analyses using transgenic Arabidopsis plants overexpressing GmDREB1s indicated that many of the downstream genes are cold-inducible and overlap with those of Arabidopsis DREB1A. We then comprehensively analyzed the downstream genes of GmDREB1B;1, which is closely related to DREB1A, using a transient expression system in soybean protoplasts. The expression of numerous genes induced by various abiotic stresses were increased by overexpressing GmDREB1B;1 in soybean, and DREs were the most conserved element in the promoters of these genes. The downstream genes of GmDREB1B;1 included numerous soybean-specific stress-inducible genes that encode an ABA receptor family protein, GmPYL21, and translation-related genes, such as ribosomal proteins. We confirmed that GmDREB1B;1 directly activates GmPYL21 expression and enhances ABRE-mediated gene expression in an ABA-independent manner. These results suggest that GmDREB1 proteins activate the expression of numerous soybean-specific stress-responsive genes under diverse abiotic stress conditions.

GmDREB2A;2, a canonical DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2-type transcription factor in soybean, is posttranslationally regulated and mediates dehydration-responsive element-dependent gene expression.

Soybean (Glycine max) is an important crop around the world. Abiotic stress conditions, such as drought and heat, adversely affect its survival, growth, and production. The DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2 (DREB2) group includes transcription factors that contribute to drought and heat stress tolerance by activating transcription through the cis-element dehydration-responsive element (DRE) in response to these stress stimuli. Two modes of regulation, transcriptional and posttranslational, are important for the activation of gene expression by DREB2A in Arabidopsis (Arabidopsis thaliana). However, the regulatory system of DREB2 in soybean is not clear. We identified a new soybean DREB2 gene, GmDREB2A;2, that was highly induced not only by dehydration and heat but also by low temperature. GmDREB2A;2 exhibited a high transactivation activity via DRE and has a serine/threonine-rich region, which corresponds to a negative regulatory domain of DREB2A that is involved in its posttranslational regulation, including destabilization. Despite the partial similarity between these sequences, the activity and stability of the GmDREB2A;2 protein were enhanced by removal of the serine/threonine-rich region in both Arabidopsis and soybean protoplasts, suggestive of a conserved regulatory mechanism that involves the recognition of serine/threonine-rich sequences with a specific pattern. The heterologous expression of GmDREB2A;2 in Arabidopsis induced DRE-regulated stress-inducible genes and improved stress tolerance. However, there were variations in the growth phenotypes of the transgenic Arabidopsis, the induced genes, and their induction ratios between GmDREB2A;2 and DREB2A. Therefore, the basic function and regulatory machinery of DREB2 have been maintained between Arabidopsis and soybean, although differentiation has also occurred.

A Novel Transcription Factor, ERD15 (Early Responsive to Dehydration 15), Connects Endoplasmic Reticulum Stress with an Osmotic Stress-induced Cell Death Signal

As in all other eukaryotic organisms, endoplasmic reticulum (ER) stress triggers the evolutionarily conserved unfolded protein response in soybean, but it also communicates with other adaptive signaling responses, such as osmotic stress-induced and ER stress-induced programmed cell death. These two signaling pathways converge at the level of gene transcription to activate an integrated cascade that is mediated by N-rich proteins (NRPs). Here, we describe a novel transcription factor, GmERD15 (Glycine max Early Responsive to Dehydration 15), which is induced by ER stress and osmotic stress to activate the expression of NRP genes. GmERD15 was isolated because of its capacity to stably associate with the NRP-B promoter in yeast. It specifically binds to a 187-bp fragment of the NRP-B promoter in vitro and activates the transcription of a reporter gene in yeast. Furthermore, GmERD15 was found in both the cytoplasm and the nucleus, and a ChIP assay revealed that it binds to the NRP-B promoter in vivo. Expression of GmERD15 in soybean protoplasts activated the NRP-B promoter and induced expression of the NRP-B gene. Collectively, these results support the interpretation that GmERD15 functions as an upstream component of stress-induced NRP-B-mediated signaling to connect stress in the ER to an osmotic stress-induced cell death signal.

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch for wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid format gave the best results in terms of low-background expression levels in the absence of ligand and high-induced expression levels of the reporter gene in the presence of nanomolar concentrations of the methoxyfenozide ligand. The performance of CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments, the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR CH9 as a partner in inducing reporter gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants.

Development of a methoxyfenozide‐responsive gene switch for applications in plants

The ecdysone receptor (EcR) has been used to develop gene switches for conditional regulation of transgene expression in plants and humans. All EcR-based gene switches developed to date for use in plants are monopartate and require micromolar concentrations of ligand for activation of the transgene; this has limited the use of these gene switches. We have developed a Choristoneura fumiferana ecdysone receptor (CfEcR)-based two-hybrid gene switch that works through the formation of a functional heterodimer between EcR and the retinoid X receptor (RXR) upon application of the chemical ligand methoxyfenozide. Methoxyfenozide is already registered for field use with an excellent safety profile, and it has potential as a gene switch ligand for applications in the field. The receptor constructs were prepared by fusing DEF domains (hinge region plus ligand-binding domain) of CfEcR to the GAL4 DNA-binding domain and EF domains (ligand-binding domain) of ultraspiracle from Choristoneura fumiferana (CfUSP) or RXR from Locusta migratoria (LmRXR), Mus musculus (MmRXR) or Homo sapiens (HsRXR) to the VP16 activation domain. These receptor constructs were tested for their ability to induce expression of the luciferase gene placed under the control of 5x GAL4 response elements and -46 35S minimal promoter in tobacco, corn and soybean protoplasts and in transgenic Arabidopsis and tobacco plants. By adopting the two-hybrid format, the sensitivity of the CfEcR gene switch has been improved from micromolar to nanomolar concentrations of methoxyfenozide. The sensitivity of the CfEcR + LmRXR two-hybrid switch was 25 to 625 times greater than the monopartate gene switch, depending on the plant species tested.

Long Term Stability of Electromanipulated Protoplasts of Glycine max var. Acme and a Catharanthus roseus Mutant

Protoplasts of soybean and an indole-alkaloid-free Madagascar periwinkle (vinca) were fused by electromanipulation. Callus cultures of these protoplasts were maintained in hormone-free semi-solid medium for nearly thirteen years. The stability of these cultures was tested by analysis of nuclear ribosomal DNA internal transcribed spacer (ITS) region. One fusion, out of the five finally tested, was regarded as exhibiting the characteristics of both the genomes from the original fused protoplasts while the others presented dominantly the ITS region of soybean.

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress.

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.

Effects of Electro- and Chemical Fusion Treatment on Cell Wall Formation and Cell Division of Plant Protoplasts.

The effects of electro- and chemical fusion procedures on cell wall formation and cell division of lettuce and soybean protoplasts were studied. Judging from Calcofluor White staining, cell wall formation of both lettuce and soybean protoplasts was stimulated by electro-fusion procedure 3 h after application. On the other hand, the cell wall formation of lettuce protoplasts was not affected by the chemical fusion treatment using polyethylene glycol (PEG). However, cell wall formation was inhibited with a very dilute concentration of PEG when lettuce protoplasts were mixed with PEG for 3 h followed by Calcofluor White staining. The enhancement of cell division of soybean protoplasts was observed by the electro-fusion treatment 3 days of culture. By transmission electron microscopy, it was also confirmed that the cell wall was detected on the surface of the electrofusion-treated protoplasts 1 day after culture. A cell wall was not observed on the surface of nontreated protoplasts. Moreover, the endoplasmic reticulum in the treated soybean protoplasts increased compared with nontreated ones.

Ultrastructural Analysis of Cell Wall Formation and Cell Division of Soybean Protoplasts.

Protoplasts of soybean (Glycine max (L.) Merr.) were isolated from suspension cultures and morphological changes during culture were observed using transmission electron microscopy. The regeneration of cell walls on the surface of the protoplasts was accompanied by the release of smooth vesicles from cytoplasm to the outside plasma membrane. The vesicles were presumed to carry some wall materials from cytoplasm for cell wall formation. With the elapse of time, the wall materials were gradually deposited and a complex cell wall layer was formed 2-3 d after culture. The fine structure of cellulose microfibrils was clearly observed about 3 d after culture using a polysaccharide staining procedure. During the cell wall formation, numerous smooth vesicles were always found between the plasma membrane and newly synthesized cell wall. Nuclear division and subsequent cytokinesis were observed about 1 d after culture. From the results obtained by the present and previous researches, it may be concluded that cell wall formation usually starts earlier than cell division and then both reactions proceed concurrently and that smooth vesicles play an important role in cell wall formation.

PEG mediated DNA uptake and transient GUS expression in carrot, rapeseed and soybean protoplasts

Transient expression of PEG mediated DNA uptake was monitored in protoplasts of carrot ( Daucus carota L. cv. Apollo, Risø and Navona), rapeseed ( Brassica napus L. cv. Line) and soybean ( Glycine max L. cv. Mandarin) by measuring the GUS activity of a chimeric CaMV 35S-GUS reporter gene inserted in a 5.6-kb pBI 221.1 plasmid. Identity of the GUS enzyme (EC 3.2.1.31) was confirmed by isoelectric focusing. A time course study revealed that expression of the GUS gene could be detected 1.5 h after DNA uptake. Peak activity was obtained in rapeseed after 27 h and in carrot and soybean after 66–72 h. Optimal experimental conditions for protoplast density, PEG concentration and the order of DNA-PEG addition were analysed. GUS activity in transfected protoplasts was determined quantitatively by a spectrofluorimetric assay and qualitatively by X-Gluc staining. A comparison between the spectrofluorimetric assay and the number of blue cells per millilitre was conducted.

An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts

Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.

Regeneration of fertile plants from protoplasts of soybean (Glycine max L. Merr.): genotypic differences in culture response

Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1-2 mm diameter colonies were formed in 5-6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l(-1) each of 2,4-D, BA and KN and 500 mg l(-1) CH for further growth. Once the colonies had become green, compact and nodular, and were 8-10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8-12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.

Protoplasts as tools in cell biology

Studies of plant protoplasts using both fluorescent dyes and electron dense probes have demonstrated endocytosis in plants. Ultrastructural work with soybean protoplasts using cationized ferritin (CF) revealed an endocytotic pathway from coated pits at the plasma membrane to coated vesicles, the partially coated reticulum, Golgi bodies, multivesicular bodies and finally the vacuole. Endocytosis may be responsible for membrane retrieval from the cell surface or degradation of elicitors or toxins during host‐pathogen interactions. Immunofluorescence studies of dividing plant protoplasts have provided new information about the preprophase band (PPB) of microtubules and the shape of spindles. Studies of PPBs in soybean protoplast cultures permitted detailed examination of PPB development and an assessment of the usefulness of the PPB index for identifying morphogenic cultures. In multinucleate protoplasts the size and number of PPBs were apparently not controlled by nuclear number. Research with conifer protoplasts resulted in the discovery of new features of gymnosperm spindles.

Studies on Cell Wall Regeneration and Cell Division in Soybean Protoplasts Using Fluorescence and Scanning Electron Microscopy.

Protoplasts were enzymatically isolated from soybean suspension cells and were cultured in a liquid medium. The reappearance of cell wall at the surface of naked protoplasts was studied by fluorescence and scanning electron microscopy. Using fluorescence microscopy (Calcofluor White staining method), the cultured protoplasts regenerated new cell walls only a few minutes after isolation and protoplasts were completely covered with new wall material after 9-7 12 h. The first division of the protoplasts was found 15 h after culture and often observed after 1-2 d culture. These regenerated cell walls could be digested with cellulase but not pectinase. By scanning electron microscopy, the wall materials deposited on the surface of protoplast were observed. After 9 h culture, short fibrils attached to projections on the plasma membrane. After that, long fibrils were formed and spread over the surface. After 1 d culture, the fibrous materials which had already deposited on the surface transformed to complex wall structure. The dividing protoplast was also observed after 12 h culture.

Analysis of large DNA from soybean (Glycine max L. Merr.) by pulsed-field gel electrophoresis.

The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.

DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase‐α. When DNA replication was partially inhibited by 10 mg l‐1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l‐1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l‐1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.

DNA replication and the development of preprophase bands in soybean protoplast cultures

DNA replication and the development of preprophase bands in soybean protoplast cultures

Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter.

The chalcone synthase (EC 2.3.1.74) gene promoter from the bean Phaseolus vulgaris L. contains a silencer element between positions -140 and -326 fro the transcription start site that is functional in electroporated soybean protoplasts. This element contains three binding sites for a bean nuclear factor (SBF-1) with DNA sequence recognition properties that are very similar to those of nuclear factor GT-1. By using a synthetic tetramer of one of the binding sites as probe, we have purified sequence-specific SBF-1 activity approximately 1750-fold from suspension-cell nuclei, by using a combination of ammonium sulfate precipitation, gel filtration, heparin-agarose chromatography, and sequence-specific DNA affinity chromatography. The factor exhibited an apparent molecular weight of 160,000-200,000 on the basis of gel filtration. A subunit molecular weight of approximately 95,000 was determined from SDS/polyacrylamide gel electrophoretic analysis of purified fractions, followed by Southwestern blot analysis (a protein blot probed with oligonucleotide probes), and from UV-cross-linking experiments. The factor lost DNA-binding activity on treatment with alkaline phosphatase. We discuss the properties of SBF-1 in relation to the functionality of GT-1 binding sequences in plant genes.