Creating a zero amylose barley with high soluble sugar content by genome editing.

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and ß-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and ß-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

The transporter PHO84/NtPT1 is a target of aluminum to affect phosphorus absorption in Saccharomyces cerevisiae and Nicotiana tabacum L.

The molecular mechanism of aluminum toxicity in biological systems is not completely understood. Saccharomyces cerevisiae is one of the most used model organisms in the study of environmental metal toxicity. Using an unbiased metallomic approach in yeast, we found that aluminum treatment caused phosphorus deprivation, and the lack of phosphorus increased as the pH of the environment decreased compared to the control strain. By screening the phosphate signaling and response pathway (PHO pathway) in yeast with the synthetic lethality of a new phosphorus-restricted aluminum-sensitive gene, we observed that pho84Δ mutation conferred severe growth defect to aluminum under low-phosphorus conditions, and the addition of phosphate alleviated this sensitivity. Subsequently, the data showed that PHO84 determined the intracellular aluminum-induced phosphorus deficiency, and the expression of PHO84 was positively correlated with aluminum stress, which was mediated by phosphorus through the coordinated regulation of PHO4/PHO2. Moreover, aluminum reduced phosphorus absorption and inhibited tobacco plant growth in acidic media. In addition, the high-affinity phosphate transporter NtPT1 in tobacco exhibited similar effects to PHO84, and overexpression of NtPT1 conferred aluminum resistance in yeast cells. Taken together, positive feedback regulation of the PHO pathway centered on the high-affinity phosphate transporters is a highly conservative mechanism in response to aluminum toxicity. The results may provide a basis for aluminum-resistant microorganisms or plant engineering and acidic soil treatment.

Landscape genomics reveals adaptive genetic differentiation driven by multiple environmental variables in naked barley on the Qinghai-Tibetan Plateau.

Understanding the local adaptation of crops has long been a concern of evolutionary biologists and molecular ecologists. Identifying the adaptive genetic variability in the genome is crucial not only to provide insights into the genetic mechanism of local adaptation but also to explore the adaptation potential of crops. This study aimed to identify the climatic drivers of naked barley landraces and putative adaptive loci driving local adaptation on the Qinghai-Tibetan Plateau (QTP). To this end, a total of 157 diverse naked barley accessions were genotyped using the genotyping-by-sequencing approach, which yielded 3123 high-quality SNPs for population structure analysis and partial redundancy analysis, and 37,636 SNPs for outlier analysis. The population structure analysis indicated that naked barley landraces could be divided into four groups. We found that the genomic diversity of naked barley landraces could be partly traced back to the geographical and environmental diversity of the landscape. In total, 136 signatures associated with temperature, precipitation, and ultraviolet radiation were identified, of which 13 had pleiotropic effects. We mapped 447 genes, including a known gene HvSs1. Some genes involved in cold stress and regulation of flowering time were detected near eight signatures. Taken together, these results highlight the existence of putative adaptive loci in naked barley on QTP and thus improve our current understanding of the genetic basis of local adaptation.

Corrigendum: Effects of different fertilization conditions and different geographical locations on the diversity and composition of the rhizosphere microbiota of Qingke (Hordeum vulgare L.) plants in different growth stages.

[This corrects the article DOI: 10.3389/fmicb.2023.1094034.].

Effects of different fertilization conditions and different geographical locations on the diversity and composition of the rhizosphere microbiota of Qingke (Hordeum vulgare L.) plants in different growth stages.

Introduction: The excessive use of chemical fertilizer causes increasing environmental and food security crisis. Organic fertilizer improves physical and biological activities of soil. Rhizosphere microbiota, which consist of highly diverse microorganisms, play an important role in soil quality. However, there is limited information about the effects of different fertilization conditions on the growth of Qingke plants and composition of the rhizosphere microbiota of the plants. Methods: In this study, we characterized the rhizosphere microbiota of Qingke plants grown in three main Qingke-producing areas (Tibet, Qinghai, and Gansu). In each of the three areas, seven different fertilization conditions (m1-m7, m1: Unfertilized; m2: Farmer Practice; m3: 75% Farmer Practice; m4: 75% Farmer Practice +25% Organic manure; m5: 50% Farmer Practice; m6: 50% Farmer Practice +50% Organic manure; m7: 100% Organic manure) were applied. The growth and yields of the Qingke plants were also compared under the seven fertilization conditions. Results: There were significant differences in alpha diversity indices among the three areas. In each area, differences in fertilization conditions and differences in the growth stages of Qingke plants resulted in differences in the beta diversity of the rhizosphere microbiota. Meanwhile, in each area, fertilization conditions, soil depths, and the growth stages of Qingke plants significantly affected the relative abundance of the top 10 phyla and the top 20 bacterial genera. For most of microbial pairs established through network analysis, the significance of their correlations in each of the microbial co-occurrence networks of the three experimental sites was different. Moreover, in each of the three networks, there were significant differences in relative abundance and genera among most nodes (i.e., the genera Pseudonocardia, Skermanella, Pseudonocardia, Skermanella, Aridibacter, and Illumatobacter). The soil chemical properties (i.e., TN, TP, SOM, AN, AK, CEC, Ca, and K) were positively or negatively correlated with the relative abundance of the top 30 genera derived from the three main Qingke-producing areas (p < 0.05). Fertilization conditions markedly influenced the height of a Qingke plant, the number of spikes in a Qingke plant, the number of kernels in a spike, and the fresh weight of a Qingke plant. Considering the yield, the most effective fertilization conditions for Qingke is combining application 50% chemical fertilizer and 50% organic manure. Conclusion: The results of the present study can provide theoretical basis for practice of reducing the use of chemical fertilizer in agriculture.

Population structure analysis and genome-wide association study of a hexaploid oat landrace and cultivar collection.

Introduction: Oat (Avena sativa L.) is an important cereal crop grown worldwide for grain and forage, owing to its high adaptability to diverse environments. However, the genetic and genomics research of oat is lagging behind that of other staple cereal crops. Methods: In this study, a collection of 288 oat lines originating worldwide was evaluated using 2,213 single nucleotide polymorphism (SNP) markers obtained from an oat iSelect 6K-beadchip array to study its genetic diversity, population structure, and linkage disequilibrium (LD) as well as the genotype-phenotype association for hullessness and lemma color. Results: The average gene diversity and polymorphic information content (PIC) were 0.324 and 0.262, respectively. The first three principal components (PCs) accounted for 30.33% of the genetic variation, indicating that the population structure of this panel of oat lines was stronger than that reported in most previous studies. In addition, accessions could be classified into two subpopulations using a Bayesian clustering approach, and the clustering pattern of accessions was closely associated with their region of origin. Additionally, evaluation of LD decay using 2,143 mapped markers revealed that the intrachromosomal whole-genome LD decayed rapidly to a critical r2 value of 0.156 for marker pairs separated by a genetic distance of 1.41 cM. Genome-wide association study (GWAS) detected six significant associations with the hullessness trait. Four of these six markers were located on the Mrg21 linkage group between 194.0 and 205.7 cM, while the other two significant markers mapped to Mrg05 and Mrg09. Three significant SNPs, showing strong association with lemma color, were located on linkage groups Mrg17, Mrg18, and Mrg20. Discussion: Our results discerned relevant patterns of genetic diversity, population structure, and LD among members of a worldwide collection of oat landraces and cultivars proposed to be 'typical' of the Qinghai-Tibetan Plateau. These results have important implications for further studies on association mapping and practical breeding in high-altitude oat.

Evolutionary patterns of plastome resolve multiple origins of the Ns-containing polyploid species in Triticeae.

Tracing evolutionary history proves challenging for polyploid groups that have evolved rapidly, especially if an ancestor of a polyploid is extinct. The Ns-containing polyploids are recognized as the NsXm and StHNsXm genomic constitutions in Triticeae. The Ns originated from Psathyrostachys, while the Xm represented a genome of unknown origin. Here, we use genetic information in plastome to trace the complex lineage history of the Ns-containing polyploid species by sampling 26 polyploids and 90 diploid taxa representing 23 basic genomes in Triticeae. Phylogenetic reconstruction, cluster plot of genetic distance matrix, and migration event demonstrated that (1) the Ns plastome originated from different Psathyrostachys species, and the Xm plastome may originate from an ancestral lineage of Henrardia, Agropyron, and Eremopyrum; (2) the Ns, Xm, and St genome donors separately served as the maternal parents during the speciation of the Ns-containing polyploid species, resulting in a maternal haplotype polymorphism; (3) North AmericanLeymusspecies might originate from colonization during late Miocene via the Bering land bridge and were the paternal donor of the StHNsXm genome Pascopyrum species. Our results shed new light on our understanding of the rich diversity and ecological adaptation of the Ns-containing polyploid species.

Combined RNA-seq and Phenotype Analysis Reveals a Potential Molecular Mechanism of the Difference in Grain Size of Naked Barley From the Qinghai-Tibetan Plateau.

To understand the molecular mechanism controlling the size of barley grains, a number of traits were analyzed and RNA-seq was conducted on grains of two barley materials with a significant difference in thousand-grain weight (TGW) after flowering. The trait dataset delineates the dynamic changes in grain size after flowering, and it provides an understanding of the source of the difference in TGW. By comparing the transcripts of barley grains at several stages after flowering, we identified the gene expression characteristics and significantly enriched pathways in each stage. At the early stage of grain development, genes involved in fatty acid metabolism, plant hormone signal transduction, and pathways involved in cytoskeleton formation were significantly upregulated. At the later stage of grain development, genes involved in starch synthesis, glucose metabolism, and other pathways were significantly upregulated. Further, we used weighted gene coexpression network analysis (WGCNA) and correlation analysis of trait datasets to identify the coexpressed gene modules significantly associated with traits, such as grain length (GL), grain width (GW), and dry weight (DW). After comparing the modules with the differentially expressed gene (DEG) set, 12 candidate genes were selected, and among these, four genes were homologous to genes that regulate grain size in rice and other plants. The combined analysis identified many potential key regulatory factors that may control barley grain size and yield potential, thus providing new insights into the molecular mechanism of barley grain size.

Characterization of the complete chloroplast genome of Avena chinensis (Poales: Poaceae).

Avena chinensis is recognized as one of the cereals with high nutritional value in the world. In this study, the complete chloroplast (cp) genome sequence of A. chinensis was reported. The complete cp genome of A. chinensis was 135,899 bp in length with a GC content of 38.51%, including a large single copy (LSC) region of 80,117 bp, a small single copy (SSC) region of 12,576 bp, and a pair of inverted repeated regions of 21,603 bp. The A. chinensis cp genome encoded 128 functional genes, including 82 protein-coding genes, 38 tRNAs, and eight rRNAs. The phylogenetic analysis showed that A. chinensis was closely related to Avena hybrid and Avena occidentalis.

BSC2 enhances cell resistance to AmB by inhibiting oxidative damage in Saccharomyces cerevisiae.

Amphotericin B has been the gold standard for the treatment of invasive mycosis for many years. Its resistance mechanisms are reported to be mainly related to the decrease of ergosterol content or the changes of cell wall. Previous study has shown that Saccharomyces cerevisiae strain lack of BSC2 was sensitive significantly to Amphotericin B. In the present study, the role of BSC2 on Amphotericin B resistance were investigated. We found that BSC2 enhanced the resistance of yeast cells to Amphotericin B, which was not related to cellular ergosterol content. BSC2 can maintain the permeability of mitochondrial membrane and cell membrane integrity by inhibiting the accumulation of intercellular reactive oxygen species and alleviating the production of lipid peroxidation and superoxide radical. These alterations were attributed to the enhancement of the activities of superoxide dismutase, catalase and glutathione peroxidase, and the increased glutathione content. Taken together, BSC2 inhibits oxidative damage induced by Amphotericin B through increasing activities of antioxidant enzymes and levels of GSH to alleviate the accumulation of reactive oxygen species, lipid peroxidation and superoxide radical, resulting in the maintenance of mitochondrial membrane potential and cell membrane integrity. However, Amphotericin B resistance mediated by BSC2 is independent of Yap1p, GSH1 and Hog1p. The results demonstrate for the first time that BSC2 enhances cell resistance to Amphotericin B by inhibiting oxidative damage in yeast. Our findings improve current understanding of the mechanism of Amphotericin B resistance and provide potential strategy for reducing Amphotericin B resistance.

Effect of cadmium on mRNA mistranslation in Saccharomyces cerevisiae.

Although highly accurate molecular processes and various messenger RNA (mRNA) quality control and ribosome proofreading mechanisms are used by organisms to transcribe their genes and maintain the fidelity of genetic information, errors are inherent in all biological systems. Low-level translation errors caused by an imbalance of homologous and nonhomologous amino acids caused by stress conditions are particularly common. Paradoxically, advantageous phenotypic diversity can be generated by such errors in eukaryotes through unknown molecular processes. Here, we found that the significant cadmium-resistant phenotype was correlated with an increased mistranslation rate of the mRNA in Saccharomyces cerevisiae. This phenotypic change was also related to endogenous sulfur amino acid starvation. Compared with the control, the mistranslation rate caused by cadmium was significantly increased (p < .01). With the increase of cysteine contents in medium, the mistranslation rate of WT(BY4742a) decreased significantly (p < .01). This demonstrates that cadmium treatment and sulfur amino acid starvation both can induce translation errors. Although cadmium uptake is independent of the Sul1 transporter, cadmium-induced mRNA mistranslation is dependent on the sulfate uptake of the Sul1p transporter. Furthermore, cadmium-induced translation errors depend on methionine biosynthesis. Taken together, cadmium causes endogenous sulfur starvation, leading to an increase in the mRNA mistranslation, which contributes to the resistance of yeast cells to cadmium. We provide a new pathway mediating the toxicity of cadmium, and we propose that altering mRNA mistranslation may portray a different form of environmental adaptation.

OLE1 reduces cadmium-induced oxidative damage in Saccharomyces cerevisiae.

Cadmium has been shown to be an important environmental pollutant. Our previous studies have shown that the regulation of OLE1 in the synthesis of unsaturated fatty acids may act as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage induced by cadmium. However, the involvement of OLE1 in cadmium-induced oxidative stress is still unclear. In this study, we explored the effects of OLE1 on cadmium-induced oxidative stress in yeast. Different from ascorbic acid, the overexpression of OLE1 did not affect the activities of superoxide dismutase, catalase and glutathione peroxidase. Although OLE1 overexpression induced an increase in GSH levels, the anti-oxidative mechanism of OLE1 was GSH1 independent. On the other hand, similar to ascorbic acid, OLE1 overexpression significantly reduced the levels of superoxide radical, carbonyl protein and lipid peroxidation. Additionally, overexpression of OLE1 effectively prevented cell membrane damage induced by cadmium. OLE1 could also reduce the cytotoxicities of other heavy metals stress, including copper, tri- and hexavalent chromium. Thus, our results indicate that overexpression of OLE1 reduces oxidative stress induced by cadmium possibly through the inhibition of lipid peroxidation and protection of the cytoplasmic membrane from damage in yeast cells. Furthermore, we determined that the anti-oxidative effect of OLE1 is independent of antioxidant enzymes and GSH1.

The alternative splicing of EAM8 contributes to early flowering and short-season adaptation in a landrace barley from the Qinghai-Tibetan Plateau.

KEY MESSAGE: The early flowering of Lalu was determined to be due to a novel spontaneous eam8 mutation, which resulted in intron retention and the formation of a putative truncated protein. Barley is a staple crop grown over an extensive area in the Qinghai-Tibetan Plateau. Understanding the genetic mechanism for its success in a high altitude is important for crop improvement in marginal environments. Early flowering is a critical adaptive trait that strongly influences reproductive fitness in a short growing season. Loss-of-function mutations at the circadian clock gene EARLY MATURITY 8 (EAM8) promote rapid flowering. In this study, we identified a novel, spontaneous recessive eam8 mutant with an early flowering phenotype in a Tibetan barley landrace Lalu, which is natively grown at a high altitude of approximately 4000 m asl. The co-segregation analysis in a F2 population derived from the cross Lalu (early flowering) × Diqing 1 (late flowering) confirmed that early flowering of Lalu was determined to be due to an allele at EAM8. The eam8 allele from Lalu carries an A/G alternative splicing mutation at position 3257 in intron 3, designated eam8.l; this alternative splicing event leads to intron retention and a putative truncated protein. Of the 134 sequenced barley accessions, which are primarily native to the Qinghai-Tibet Plateau, three accessions carried this mutation. The eam8.l mutation was likely to have originated in wild barley due to the presence of the Lalu haplotype in H. spontaneum from Tibet. Overall, alternative splicing has contributed to the evolution of the barley circadian clock and in the short-season adaptation of local barley germplasm. The study has also identified a novel donor of early-flowering barley which will be useful for barley improvement.

Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae.

The heavy metal cadmium is widely used and released into the environment, posing a severe threat to crops and humans. Saccharomyces cerevisiae is one of the most commonly used organisms in the investigation of environmental metal toxicity. We investigated cadmium stress and the adaptive mechanisms of yeast by screening a genome-wide essential gene overexpression library. A candidate gene, OLE1, encodes a delta-9 desaturase and was associated with high anti-cadmium-stress activity. The results demonstrated that the expression of OLE1 was positively correlated with cadmium stress tolerance and induction was independent of Mga2p and Spt23p (important regulatory factors for OLE1). Moreover, in response to cadmium stress, cellular levels of monounsaturated fatty acids were increased. The addition of exogenous unsaturated fatty acids simulated overexpression of OLE1, leading to cadmium resistance. Such regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. IMPORTANCE: A S. cerevisiae gene encoding a delta-9 desaturase, OLE1, was associated with high anti-cadmium-stress activity. The data suggest that the regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. The discovery of OLE1 involvement in membrane stability may indicate a novel defense strategy against cadmium stress.

Preparation of two flavonoid glycosides with unique structures from barley seedlings by membrane separation technology and preparative high-performance liquid chromatography.

Barley seedlings are rich in flavones that can have positive effects on people with antihypoxia and antifatigue. Lutonarin and saponarin are two major flavonoid glycosides that have unique structures in barley seedlings. This study presents a new approach for the preparation of lutonarin and saponarin from barely seedlings by membrane separation technology and preparative high-performance liquid chromatography. Preparative conditions of these two flavonoid glycosides by membrane separation technology were studied using response surface methodology. Under the optimized conditions, the total contents of these two flavonoid glycosides amounts to 17.0%.

The Arabidopsis kinesin gene AtKin-1 plays a role in the nuclear division process during megagametogenesis.

KEY MESSAGE: Atkin - 1 , the only Kinesin-1 member of Arabidopsis thaliana , plays a role during female gametogenesis through regulation of nuclear division cycles. Kinesins are microtubule-dependent motor proteins found in eukaryotic organisms. They constitute a superfamily that can be further classified into at least 14 families. In the Kinesin-1 family, members from animal and fungi play roles in long-distance transport of organelles and vesicles. Although Kinesin-1-like sequences have been identified in higher plants, little is known about their function in plant cells, other than in a recently identified Kinesin-1-like protein in a rice pollen semi-sterile mutant. In this study, the gene encoding the only Kinesin-1 member in Arabidopsis, AtKin-1 was found to be specifically expressed in ovules and anthers. AtKin-1 loss-of-function mutants showed substantially aborted ovules in siliques, and this finding was supported by complementation testing. Reciprocal crossing between mutant and wild-type plants indicated that a defect in AtKin-1 results in partially aborted megagametophytes, with no observable effects on pollen fertility. Further observation of ovule development in the mutant pistils indicated that the enlargement of the megaspore was blocked and nuclear division arrested at the one-nucleate stage during embryo sac formation. Our data suggest that AtKin-1 plays a role in the nuclear division cycles during megagametogenesis.

Oxalate decarboxylase from Agrobacterium tumefaciens C58 is translocated by a twin arginine translocation system.

Oxalate decarboxylases (OXDCs) (E.C. 4.1.1.2) are enzymes catalyzing the conversion of oxalate to formate and CO2 The OXDCs found in fungi and bacteria belong to functionally diverse protein superfamily known as the cupins. Fungi-originated OXDCs are secretory enzymes. However, most bacterial OXDCs are localized in the cytosol, and may be involved in energy metabolism. In Agrobacterium tumefaciens C58, a locus for a putative oxalate decarboxylase is present. In the study reported here, an enzyme was overexpressed in Escherichia coli and showed oxalate carboxylase activity. Computational analysis revealed the A. tumefaciens C58 OXDC contains a signal peptide mediating translocation of the enzyme into the periplasm that was supported by expression of signal-peptideless and full-length versions of the enzyme in A. tumefaciens C58. Further site-directed mutagenesis experiment demonstrated that the A. tumefaciens C58 OXDC is most likely translocated by a twin-arginine translocation (TAT) system.