B. subtilis CNBG-PGPR-1 induces methionine to regulate ethylene pathway and ROS scavenging for improving salt tolerance of tomato.

Soil salinity severely threatens plant growth and crop yields. The utilization of PGPR is an effective strategy for enhancing plant salt tolerance, but the mechanisms involved in this process have rarely been reported. In this study, we investigated the effects of Bacillus subtilis CNBG-PGPR-1 on improving plant salt tolerance and elucidated the molecular pathways involved. The results showed that CNBG-PGPR-1 significantly improved the cellular homeostasis and photosynthetic efficiency of leaves and reduced ion toxicity and osmotic stress caused by salt in tomato. Transcriptome analysis uncovered that CNBG-PGPR-1 enhanced plant salt tolerance through the activation of complex molecular pathways, with plant hormone signal transduction playing an important role. Comparative analysis and pharmacological experiments confirmed that the ethylene pathway was closely related to the beneficial effect of CNBG-PGPR-1 on improving plant salt tolerance. Furthermore, we found that methionine, a precursor of ethylene synthesis, significantly accumulated in response to CNBG-PGPR-1 in tomato. Exogenous L-methionine largely mimicked the beneficial effects of CNBG-PGPR-1 and activated the expression of ethylene pathway-related genes, indicating CNBG-PGPR-1 induces methionine accumulation to regulate the ethylene pathway in tomato. Finally, CNBG-PGPR-1 reduced salt-induced ROS by activating ROS scavenger-encoding genes, mainly involved in GSH metabolism and POD-related genes, which were also closely linked to methionine metabolism. Overall, our studies demonstrate that CNBG-PGPR-1-induced methionine is a key regulator in enhancing plant salt tolerance through the ethylene pathway and ROS scavenging, providing a novel understanding of the mechanism by which beneficial microbes improve plant salt tolerance.

A Critical Review of Conventional and Modern Approaches to Develop Herbicide-Resistance in Rice.

Together with rice, weeds strive for nutrients and space in farmland, resulting in reduced rice yield and quality. Planting herbicide-resistant rice varieties is one of the effective ways to control weeds. In recent years, a series of breakthroughs have been made to generate herbicide-resistant germplasm, especially the emergence of biotechnological tools such as gene editing, which provides an inherent advantage for the knock-out or knock-in of the desired genes. In order to develop herbicide-resistant rice germplasm resources, gene manipulation has been conducted to enhance the herbicide tolerance of rice varieties through the utilization of techniques such as physical and chemical mutagenesis, as well as genome editing. Based on the current research and persisting problems in rice paddy fields, research on the generation of herbicide-resistant rice still needs to explore genetic mechanisms, stacking multiple resistant genes in a single genotype, and transgene-free genome editing using the CRISPR system. Current rapidly developing gene editing technologies can be used to mutate herbicide target genes, enabling targeted genes to maintain their biological functions, and reducing the binding ability of target gene encoded proteins to corresponding herbicides, ultimately resulting in herbicide-resistant crops. In this review article, we have summarized the utilization of conventional and modern approaches to develop herbicide-resistant cultivars in rice as an effective strategy for weed control in paddy fields, and discussed the technology and research directions for creating herbicide-resistant rice in the future.

Organelle genome composition and candidate gene identification for Nsa cytoplasmic male sterility in Brassica napus.

BACKGROUND: Nsa cytoplasmic male sterility (CMS) is a novel alloplasmic male sterility system derived from somatic hybridization between Brassica napus and Sinapis arvensis. Identification of the CMS-associated gene is a prerequisite for a better understanding of the origin and molecular mechanism of this CMS. With the development of genome sequencing technology, organelle genomes of Nsa CMS line and its maintainer line were sequenced by pyro-sequencing technology, and comparative analysis of the organelle genomes was carried out to characterize the organelle genome composition of Nsa CMS as well as to identify the candidate Nsa CMS-associated genes. RESULTS: Nsa CMS mitochondrial genome showed a higher collinearity with that of S. arvensis than B. napus, indicating that Nsa CMS mitochondrial genome was mainly derived from S. arvensis. However, mitochondrial genome recombination of parental lines was clearly detected. In contrast, the chloroplast genome of Nsa CMS was highly collinear with its B. napus parent, without any evidence of recombination of the two parental chloroplast genomes or integration from S. arvensis. There were 16 open reading frames (ORFs) specifically existed in Nsa CMS mitochondrial genome, which could not be identified in the maintainer line. Among them, three ORFs (orf224, orf309, orf346) possessing chimeric and transmembrane structure are most likely to be the candidate CMS genes. Sequences of all three candidate CMS genes in Nsa CMS line were found to be 100% identical with those from S. arvensis mitochondrial genome. Phylogenetic and homologous analysis showed that all the mitochondrial genes were highly conserved during evolution. CONCLUSIONS: Nsa CMS contains a recombined mitochondrial genome of its two parental species with the majority form S. arvensis. Three candidate Nsa CMS genes were identified and proven to be derived from S. arvensis other than recombination of its two parental species. Further functional study of the candidate genes will help to identify the gene responsible for the CMS and the underlying molecular mechanism.

Transcriptome and Hormone Comparison of Three Cytoplasmic Male Sterile Systems in Brassica napus.

The interaction between plant mitochondria and the nucleus markedly influences stress responses and morphological features, including growth and development. An important example of this interaction is cytoplasmic male sterility (CMS), which results in plants producing non-functional pollen. In current research work, we compared the phenotypic differences in floral buds of different Brassica napus CMS (Polima, Ogura, Nsa) lines with their corresponding maintainer lines. By comparing anther developmental stages between CMS and maintainer lines, we identified that in the Nsa CMS line abnormality occurred at the tetrad stage of pollen development. Phytohormone assays demonstrated that IAA content decreased in sterile lines as compared to maintainer lines, while the total hormone content was increased two-fold in the S2 stage compared with the S1 stage. ABA content was higher in the S1 stage and exhibited a two-fold decreasing trend in S2 stage. Sterile lines however, had increased ABA content at both stages compared with the corresponding maintainer lines. Through transcriptome sequencing, we compared differentially expressed unigenes in sterile and maintainer lines at both (S1 and S2) developmental stages. We also explored the co-expressed genes of the three sterile lines in the two stages and classified these genes by gene function. By analyzing transcriptome data and validating by RT-PCR, it was shown that some transcription factors (TFs) and hormone-related genes were weakly or not expressed in the sterile lines. This research work provides preliminary identification of the pollen abortion stage in Nsa CMS line. Our focus on genes specifically expressed in sterile lines may be useful to understand the regulation of CMS.

Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus.

Oilseed rape (Brassica napus L.) is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR)-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

RNA HELICASE 32 is essential for female gametophyte development in Arabidopsis.

The normal progression of mitotic cycles and synchronized development within female reproductive organs are pivotal for sexual reproduction in plants. Nevertheless, our understanding of the genetic regulation governing mitotic cycles during the haploid phase of higher plants remains limited. In this study, we characterized RNA HELICASE 32 (RH32), which plays an essential role in female gametogenesis in Arabidopsis. The rh32 heterozygous mutant was semi-sterile, whereas the homozygous mutant was nonviable. The rh32 mutant allele could be transmitted through the male gametophyte, but not the female gametophyte. Phenotypic analysis revealed impaired mitotic progression, synchronization, and cell specification in rh32 female gametophytes, causing the arrest of embryo sacs. In the delayed pollination test, none of the retarded embryo sacs developed into functional female gametophytes, and the vast majority of rh32 female gametophytes were defective in the formation of the large central vacuole. RH32 is strongly expressed in the embryo sac. Knock-down of RH32 resulted in the accumulation of unprocessed 18S pre-rRNA, implying that RH32 is involved in ribosome synthesis. Based on these findings, we propose that RH32 plays a role in ribosome synthesis, which is critical for multiple processes in female gametophyte development.

Computational Tools and Resources for CRISPR/Cas Genome Editing.

The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants, as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genome editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization. Lastly, we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.

Establishment of new convenient two-line system for hybrid production by targeting mutation of OPR3 in allopolyploid Brassica napus.

The two-line pollination control system, which usually depends on the utilization of thermosensitive or photoperiod genic male-sterile lines, has been widely used in various crops. However, this system is susceptible to instability issues caused by uncontrollable weather fluctuations. A stable and handy two-line pollination control system is highly desirable in many crop species for heterosis exploitation. Oxophytodienoic acid reductase 3 (OPR3) was proven to be involved in jasmonate biosynthesis. In the present study, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat) was utilized to mutate two OPR3 homologs in Brassica napus. After two OPR3 homologs were simultaneously mutated, mutants exhibited complete male sterility, and fertility could be easily restored by exogenous MeJA treatment. Hybrids produced from crosses between the opr3 sterile lines and normal varieties exhibited heterosis. This new two-line system based on OPR3 mutation provides higher stability and convenience than traditional systems. By using exogenous MeJA treatment to restore fertility, the system enables more precise control of male fertility transition, which has great potential to significantly contribute to the maneuverable production of hybrid seeds in rapeseed as well as other Brassica species crops.

Jasmonic acid pretreatment improves salt tolerance of wheat by regulating hormones biosynthesis and antioxidant capacity.

Salt stress is a severe environmental factor that detrimentally affects wheat growth and production worldwide. Previous studies illustrate that exogenous jasmonic acid (JA) significantly improved salt tolerance in plants. However, little is known about the underlying molecular mechanisms of JA induced physiochemical changes in wheat seedlings under salt stress conditions. In this study, biophysiochemical and transcriptome analysis was conducted to explore the mechanisms of exogenous JA induced salt tolerance in wheat. Exogenous JA increased salt tolerance of wheat seedlings by alleviating membrane lipid oxidation, improving root morphology, enhancing the contents of ABA, JA and SA and increasing relative water content. In the RNA-seq profiles, we identified a total of 54,263 unigenes and 1,407 unigenes showed differentially expressed patterns in JA pretreated wheat seedlings exposed to salt stress comparing to those with salt stress alone. Subsequently, gene ontology (GO) and KEGG pathway enrichment analysis characterized that DEGs involved in linoleic acid metabolism and plant hormone signal transduction pathways were up-regulated predominantly in JA pretreated wheat seedlings exposed to salt stress. We noticed that genes that involved in antioxidative defense system and that encoding transcription factors were mainly up- or down-regulated. Moreover, SOD, POD, CAT and APX activities were increased in JA pretreated wheat seedlings exposed to salt stress, which is in accordance with the transcript profiles of the relevant genes. Taken together, our results demonstrate that the genes and enzymes involved in physiological and biochemical processes of antioxidant system, plant hormones and transcriptional regulation contributed to JA-mediated enhancement of salt tolerance in wheat. These findings will facilitate the elucidation of the potential molecular mechanisms associated with JA-dependent amelioration of salt stress in wheat and lay theoretical foundations for future studies concerning the improvement of plant tolerance to abiotic environmental stresses.

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum-valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.