CRISPR/Cas9-Mediated Knockout of GmFATB1 Significantly Reduced the Amount of Saturated Fatty Acids in Soybean Seeds

Soybean (Glycine max) oil is one of the most widely used vegetable oils across the world. Breeding of soybean to reduce the saturated fatty acid (FA) content, which is linked to cardiovascular disease, would be of great significance for nutritional improvement. Acyl-acyl carrier protein thioesterases (FATs) can release free FAs and acyl-ACP, which ultimately affects the FA profile. In this study, we identified a pair of soybean FATB coding genes, GmFATB1a and GmFATB1b. Mutants that knock out either or both of the GmFATB1 genes were obtained via CRISPR/Cas9. Single mutants, fatb1a and fatb1b, showed a decrease in leaf palmitic and stearic acid contents, ranging from 11% to 21%. The double mutant, fatb1a:1b, had a 42% and 35% decrease in palmitic and stearic acid content, displayed growth defects, and were male sterility. Analysis of the seed oil profile revealed that fatb1a and fatb1b had significant lower palmitic and stearic acid contents, 39–53% and 17–37%, respectively, while that of the unsaturated FAs were the same. The relative content of the beneficial FA, linoleic acid, was increased by 1.3–3.6%. The oil profile changes in these mutants were confirmed for four generations. Overall, our data illustrate that GmFATB1 knockout mutants have great potential in improving the soybean oil quality for human health.     

Functional Redundancy of FLOWERING LOCUS T 3b in Soybean Flowering Time Regulation

Photoperiodic flowering is an important agronomic trait that determines adaptability and yield in soybean and is strongly influenced by FLOWERING LOCUS T (FT) genes. Due to the presence of multiple FT homologs in the genome, their functions in soybean are not fully understood. Here, we show that GmFT3b exhibits functional redundancy in regulating soybean photoperiodic flowering. Bioinformatic analysis revealed that GmFT3b is a typical floral inducer FT homolog and that the protein is localized to the nucleus. Moreover, GmFT3b expression was induced by photoperiod and circadian rhythm and was more responsive to long-day (LD) conditions. We generated a homozygous ft3b knockout and three GmFT3b-overexpressing soybean lines for evaluation under different photoperiods. There were no significant differences in flowering time between the wild-type, the GmFT3b overexpressors, and the ft3b knockouts under natural long-day, short-day, or LD conditions. Although the downstream flowering-related genes GmFUL1 (a, b), GmAP1d, and GmLFY1 were slightly down-regulated in ft3b plants, the floral inducers GmFT5a and GmFT5b were highly expressed, indicating potential compensation for the loss of GmFT3b. We suggest that GmFT3b acts redundantly in flowering time regulation and may be compensated by other FT homologs in soybean.     

Identification and Characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage (Brassica rapa L.)

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9–16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2–4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.     

Compensation Mechanism of the Photosynthetic Apparatus in Arabidopsis thaliana ch1 Mutants

The origin of chlorophyll b deficiency is a mutation (ch1) in chlorophyllide a oxygenase (CAO), the enzyme responsible for Chl b synthesis. Regulation of Chl b synthesis is essential for understanding the mechanism of plant acclimation to various conditions. Therefore, the main aim of this study was to find the strategy in plants for compensation of low chlorophyll content by characterizing and comparing the performance and spectral properties of the photosynthetic apparatus related to the lipid and protein composition in four selected Arabidopsis ch1 mutants and two Arabidopsis ecotypes. Mutation in different loci of the CAO gene, viz., NW41, ch1.1, ch1.2 and ch1.3, manifested itself in a distinct chlorina phenotype, pigment and photosynthetic protein composition. Changes in the CAO mRNA levels and chlorophyllide a (Chlide a) content in ecotypes and ch1 mutants indicated their significant role in the adjustment mechanism of the photosynthetic apparatus to low-light conditions. Exposure of mutants with a lower chlorophyll b content to short-term (1LL) and long-term low-light stress (10LL) enabled showing a shift in the structure of the PSI and PSII complexes via spectral analysis and the thylakoid composition studies. We demonstrated that both ecotypes, Col-1 and Ler-0, reacted to high-light (HL) conditions in a way remarkably resembling the response of ch1 mutants to normal (NL) conditions. We also presented possible ways of regulating the conversion of chlorophyll a to b depending on the type of light stress conditions.     

Constitutive Photomorphogenic 1 Enhances ER Stress Tolerance in Arabidopsis

Interaction between light signaling and stress response has been recently reported in plants. Here, we investigated the role of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a key regulator of light signaling, in endoplasmic reticulum (ER) stress response in Arabidopsis. The cop1-4 mutant Arabidopsis plants were highly sensitive to ER stress induced by treatment with tunicarmycin (Tm). Interestingly, the abundance of nuclear-localized COP1 increased under ER stress conditions. Complementation of cop1-4 mutant plants with the wild-type or variant types of COP1 revealed that the nuclear localization and dimerization of COP1 are essential for its function in plant ER stress response. Moreover, the protein amount of ELONGATED HYPOCOTYL 5 (HY5), which inhibits bZIP28 to activate the unfolded protein response (UPR), decreased under ER stress conditions in a COP1-dependent manner. Accordingly, the binding of bZIP28 to the BIP3 promoter was reduced in cop1-4 plants and increased in hy5 plants compared with the wild type. Furthermore, introduction of the hy5 mutant locus into the cop1-4 mutant background rescued its ER stress-sensitive phenotype. Altogether, our results suggest that COP1, a negative regulator of light signaling, positively controls ER stress response by partially degrading HY5 in the nucleus.     

Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana

Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5′-RACE (RNA ligase-mediated rapid amplification of 5′ cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean.     

Genetic Dissection of Light-Regulated Adventitious Root Induction in Arabidopsis thaliana Hypocotyls

Photomorphogenic responses of etiolated seedlings include the inhibition of hypocotyl elongation and opening of the apical hook. In addition, dark-grown seedlings respond to light by the formation of adventitious roots (AR) on the hypocotyl. How light signaling controls adventitious rooting is less well understood. Hereto, we analyzed adventitious rooting under different light conditions in wild type and photomorphogenesis mutants in Arabidopsis thaliana. Etiolation was not essential for AR formation but raised the competence to form AR under white and blue light. The blue light receptors CRY1 and PHOT1/PHOT2 are key elements contributing to the induction of AR formation in response to light. Furthermore, etiolation-controlled competence for AR formation depended on the COP9 signalosome, E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC (COP1), the COP1 interacting SUPPRESSOR OF PHYA-105 (SPA) kinase family members (SPA1,2 and 3) and Phytochrome-Interacting Factors (PIF). In contrast, ELONGATED HYPOCOTYL5 (HY5), suppressed AR formation. These findings provide a genetic framework that explains the high and low AR competence of Arabidopsis thaliana hypocotyls that were treated with dark, and light, respectively. We propose that light-induced auxin signal dissipation generates a transient auxin maximum that explains AR induction by a dark to light switch.     

CpMAX1a,a Cytochrome P450 Monooxygenase Gene of Chimonanthus praecox Regulates Shoot Branching in Arabidopsis

Strigolactones (SLs) are a class of important hormones in the regulation of plant branching. In the model plant Arabidopsis, AtMAX1 encodes a cytochrome P450 protein and is a crucial gene in the strigolactone synthesis pathway. Yet, the regulatory mechanism of MAX1 in the shoot branching of wintersweet (Chimonanthus praecox) remains unclear. Here we identified and isolated three MAX1 homologous genes, namely CpMAX1a, CpMAX1b, and CpMAX1c. Quantitative real-time PCR (qRT-PCR) revealed the expression of CpMAX1a in all tissues, being highest in leaves, whereas CpMAX1b was only expressed in stems, while CpMAX1c was expressed in both roots and stem tips. However, CpMAX1a’s expression decreased significantly after decapitation; hence, we verified its gene function. CpMAX1a was located in Arabidopsis chloroplasts. Overexpressing CpMAX1a restored the phenotype of the branching mutant max1–3, and reduced the rosette branch number, but resulted in no significant phenotypic differences from the wild type. Additionally, expression of AtBRC1 was significantly upregulated in transgenic lines, indicating that the CpMAX1a gene has a function similar to the homologous gene of Arabidopsis. In conclusion, our study shows that CpMAX1a plays a conserved role in regulating the branch development of wintersweet. This work provides a molecular and theoretical basis for better understanding the branch development of wintersweet.     

Pax3 and Pax7 Exhibit Distinct and Overlapping Functions in Marking Muscle Satellite Cells and Muscle Repair in a Marine Teleost,Sebastes schlegelii

Pax3 and Pax7 are members of the Pax gene family which are essential for embryo and organ development. Both genes have been proved to be markers of muscle satellite cells and play key roles in the process of muscle growth and repair. Here, we identified two Pax3 genes (SsPax3a and SsPax3b) and two Pax7 genes (SsPax7a and SsPax7b) in a marine teleost, black rockfish (Sebastes schlegelii). Our results showed SsPax3 and SsPax7 marked distinct populations of muscle satellite cells, which originated from the multi-cell stage and somite stage, respectively. In addition, we constructed a muscle injury model to explore the function of these four genes during muscle repair. Hematoxylin–eosin (H–E) of injured muscle sections showed new-formed myofibers occurred at 16 days post-injury (dpi). ISH (in situ hybridization) analysis demonstrated that the expression level of SsPax3a and two SsPax7 genes increased gradually during 0–16 dpi and peaked at 16 dpi. Interestingly, SsPax3b showed no significant differences during the injury repair process, indicating that the satellite cells labeled by SsPax3b were not involved in muscle repair. These results imply that the muscle stem cell populations in teleosts are more complicated than in mammals. This lays the foundation for future studies on the molecular mechanism of indeterminant growth and muscle repair of large fish species.     

Synthesis and palladium-mediated cross-coupling reaction of cyclic (kyklo-) and open-chain (kentro-) telechelic precursors

A series of uniform-size, cyclic poly (THF)s having a bromophenyl (1a), a pentynoyl (1b) and a phenylboronate (1c) group, together with their open-chain, center-functional counterparts having the relevant functional group (2a and 2b), have been prepared in high yields through the esterification of a hydroxyl group of a cyclic (kyklo-) and an open-chain (kentro-) telechelic precursors. They were subsequently subjected to palladium-mediated, Sonogashira and Suzuki coupling reactions, i.e., 1a and 1b as well as 2a and 2b for the former, and 1a and 1c for the latter, respectively. SEC showed that the Sonogashira process could produce effectively the corresponding cross-coupling products, i.e, an 8-shaped and a 4-armed star polymer, respectively. The Suzuki process, on the other hand, failed to proceed under examined conditions.     

GsRSS3L,a Candidate Gene Underlying Soybean Resistance to Seedcoat Mottling Derived from Wild Soybean (Glycine soja Sieb. and Zucc)

Soybeans are a major crop that produce the best vegetable oil and protein for use in food and beverage products worldwide. However, one of the most well-known viral infections affecting soybeans is the Soybean Mosaic Virus (SMV), a member of the Potyviridae family. A crucial method for preventing SMV damage is the breeding of resistant soybean cultivars. Adult resistance and resistance of seedcoat mottling are two types of resistance to SMV. Most studies have focused on adult-plant resistance but not on the resistance to seedcoat mottling. In this study, chromosome segment-substituted lines derived from a cross between Suinong14 (cultivated soybean) and ZYD00006 (wild soybean) were used to identify the chromosome region and candidate genes underlying soybean resistance to seed coat mottling. Herein, two quantitative trait loci (QTLs) were found on chromosome 17, and eighteen genes were found in the QTL region. RNA-seq was used to evaluate the differentially expressed genes (DEGs) among the eighteen genes located in the QTLs. According to the obtained data, variations were observed in the expression of five genes following SMV infection. Furthermore, Nicotiana benthamiana was subjected to an Agrobacterium-mediated transient expression assay to investigate the role of the five candidate genes in SMV resistance. It has also been revealed that Glyma.17g238900 encoding a RICE SALT SENSITIVE 3-like protein (RSS3L) can inhibit the multiplication of SMV in N. benthamiana. Moreover, two nonsynonymous single-nucleotide polymorphisms (SNPs) were found in the coding sequence of Glyma.17g238900 derived from the wild soybean ZYD00006 (GsRSS3L), and the two amino acid mutants may be associated with SMV resistance. Hence, it has been suggested that GsRSS3L confers seedcoat mottling resistance, shedding light on the mechanism of soybean resistance to SMV.     

Roles of AGD2a in Plant Development and Microbial Interactions of Lotus japonicus

Arabidopsis AGD2 (Aberrant Growth and Death2) and its close homolog ALD1 (AGD2-like defense response protein 1) have divergent roles in plant defense. We previously reported that modulation of salicylic acid (SA) contents by ALD1 affects numbers of nodules produced by Lotus japonicus, but AGD2′s role in leguminous plants remains unclear. A combination of enzymatic analysis and biological characterization of genetic materials was used to study the function of AGD2 (LjAGD2a and LjAGD2b) in L. japonicus. Both LjAGD2a and LjAGD2b could complement dapD and dapE mutants of Escherichia coli and had aminotransferase activity in vitro. ljagd2 plants, with insertional mutations of LjAGD2, had delayed flowering times and reduced seed weights. In contrast, overexpression of LjAGD2a in L. japonicus induced early flowering, with increases in seed and flower sizes, but reductions in pollen fertility and seed setting rates. Additionally, ljagd2a mutation resulted in increased expression of nodulin genes and corresponding increases in infection threads and nodule numbers following inoculation with Rhizobium. Changes in expression of LjAGD2a in L. japonicus also affected endogenous SA contents and hence resistance to pathogens. Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase and plays important roles in plant development. Moreover, LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume–microbe interaction.     

Extrusion drawn amorphous poly(ethylene terephthalate): 4. Irreversible spontaneous elongation

Uniaxially-oriented poly(ethylene terephthalate) (PET) films prepared by solid-state co-extrusion exhibit irreversible spontaneous elongation (rather than shrinkage) under specific conditions. Results for these conditions show that marked elongation (up to 20%) can occur during annealing of unconstrained samples. This phenomenon, which is not commonly observed for polymers, depends strongly on the prior conditions of extrusion draw. There is significant increase in length for films prepared with extrusion draw ratio (EDR) at 2.0 in the extrusion draw temperature (T_ext) range 80–100°C. The extrusion rate is also significant. Lower extrusion rates favour spontaneous elongation on subsequent heating. In addition, the annealing temperature (T_a) also affects elongation. Samples extruded at T_ext=80°C to EDR of 2.0 show maximum elongation at T_a=180–190°C. However at higher temperatures, e.g. at 10°C below the melting temperature and higher, shrinkage occurs instead. Moreover, annealing at T_a=90°C for different periods of time (t_a) shows that prior to the elongation a moderate amount of shrinkage occurs (t_a ? 30 s). The results suggest a correlation between spontaneous elongation and crystallization during anealing.     

TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing⁺ (TbyS⁺), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS⁺ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS⁺, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.