Hydrogen peroxide sulfenylates and inhibits the photorespiratory enzyme PGLP1 to modulate plant thermotolerance.

Climate change is resulting in more frequent and rapidly changing temperatures at both extremes that severely affect the growth and production of plants, particularly crops. Oxidative stress caused by high temperatures is one of the most damaging factors for plants. However, the role of hydrogen peroxide (H2O2) in modulating plant thermotolerance is largely unknown, and the regulation of photorespiration essential for C3 species remains to be fully clarified. Here, we report that heat stress promotes H2O2 accumulation in chloroplasts and that H2O2 stimulates sulfenylation of the chloroplast-localized photorespiratory enzyme 2-phosphoglycolate phosphatase 1 (PGLP1) at cysteine 86, inhibiting its activity and promoting the accumulation of the toxic metabolite 2-phosphoglycolate. We also demonstrate that PGLP1 has a positive function in plant thermotolerance, as PGLP1 antisense lines have greater heat sensitivity and PGLP1-overexpressing plants have higher heat-stress tolerance than the wild type. Together, our results demonstrate that heat-induced H2O2 in chloroplasts sulfenylates and inhibits PGLP1 to modulate plant thermotolerance. Furthermore, targeting CATALASE2 to chloroplasts can largely prevent the heat-induced overaccumulation of H2O2 and the sulfenylation of PGLP1, thus conferring thermotolerance without a plant growth penalty. These findings reveal that heat-induced H2O2 in chloroplasts is important for heat-caused plant damage.

Proteome-wide identification of methylglyoxalated proteins in rapeseed (Brassica napus L.).

Methylglyoxal (MG), a highly reactive cellular metabolite, is crucial for plant growth and environmental responses. MG may function by modifying its target proteins, but little is known about MG-modified proteins in plants. Here, MG-modified proteins were pulled down by an antibody against methylglyoxalated proteins and detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We identified 543 candidate proteins which are involved in multiple enzymatic activities and metabolic processes. A great number of candidate proteins were predicted to localize to cytoplasm, chloroplast, and nucleus, consistent with the known subcellular compartmentalization of MG. By further analyzing the raw LC-MS/MS data, we obtained 42 methylglyoxalated peptides in 35 proteins and identified 10 methylglyoxalated lysine residues in a myrosinase-binding protein (BnaC06G0061400ZS). In addition, we demonstrated that MG modifies the glycolate oxidase and ß-glucosidase to enhance and inhibit the enzymatic activity, respectively. Together, our study contributes to the investigation of the MG-modified proteins and their potential roles in rapeseed.

DELAYED GREENING 409 encodes a dual-localized pentatricopeptide repeat protein required for chloroplast and mitochondrial development.

In flowering plants, hundreds of RNA editing events occur in the chloroplasts and mitochondria during posttranscriptional processes. Although several pentatricopeptide repeat (PPR) proteins have been shown to form the editosome core, the precise interactions between the different editing factors are still obscure. Here, we isolated an Arabidopsis (Arabidopsis thaliana) PPR protein, designated DELAYED GREENING 409 (DG409), that was dually targeted to chloroplasts and mitochondria. This protein consists of 409 amino acids with 7 PPR motifs but lacks a C-terminal E, E+, or DYW domain. A mild dg409 knockdown mutant displays a sickly phenotype. In this mutant, the young leaves are pale green and turn green at maturity, and the development of chloroplasts and mitochondria is severely disrupted. Complete loss of DG409 function results in defective embryos. Transcriptomic analysis of the dg409 knockdown plants showed some editing defects in genes from both organelles, including CASEINOLYTIC PROTEASE P (clpP)-559, RNA POLYMERASE SUBUNIT ALPHA (rpoA)-200, ACETYL-COA CARBOXYLASE CARBOXYL TRANSFERASE SUBUNIT BETA (accD)-1568, NADH DEHYDROGENASE SUBUNIT 7 (nad7)-1505, and RIBOSOMAL PROTEIN S3 (rps3)-1344. RNA immunoprecipitation showed that DG409 was associated with the targeted transcripts in vivo. Interaction assays revealed that DG409 directly interacted with 2 DYW-type PPR proteins (EARLY CHLOROPLAST BIOGENESIS2 [AtECB2] and DYW DOMAIN PROTEIN2 [DYW2]) and 3 multiple organellar RNA editing factors (MORF2, MORF8, and MORF9). These results indicate that DG409 is involved in RNA editing via protein complexes and is therefore essential for chloroplast and mitochondrial development.

An intrinsically disordered region-containing protein mitigates the drought-growth trade-off to boost yields.

Drought stress poses a serious threat to global agricultural productivity and food security. Plant resistance to drought is typically accompanied by a growth deficit and yield penalty. Herein, we report a previously uncharacterized, dicotyledon-specific gene, Stress and Growth Interconnector (SGI), that promotes growth during drought in the oil crop rapeseed (Brassica napus) and the model plant Arabidopsis (Arabidopsis thaliana). Overexpression of SGI conferred enhanced biomass and yield under water-deficient conditions, whereas corresponding CRISPR SGI mutants exhibited the opposite effects. These attributes were achieved by mediating reactive oxygen species (ROS) homeostasis while maintaining photosynthetic efficiency to increase plant fitness under water-limiting environments. Further spatial-temporal transcriptome profiling revealed dynamic reprogramming of pathways for photosynthesis and stress responses during drought and the subsequent recovery. Mechanistically, SGI represents an intrinsically disordered region-containing protein that interacts with itself, catalase isoforms, dehydrins, and other drought-responsive positive factors, restraining ROS generation. These multifaceted interactions stabilize catalases in response to drought and facilitate their ROS-scavenging activities. Taken altogether, these findings provide insights into currently underexplored mechanisms to circumvent trade-offs between plant growth and stress tolerance that will inform strategies to breed climate-resilient, higher yielding crops for sustainable agriculture.

BnGF14-2c Positively Regulates Flowering via the Vernalization Pathway in Semi-Winter Rapeseed.

14-3-3s are general regulatory factors (GF14s or GRFs) involved in a variety of physiological regulations in plants, including the control of flowering time. However, there are poorly relevant reports in rapeseed so far. In this study, we identified a homologous 14-3-3 gene BnGF14-2c (AtGRF2_Like in Brassica napus) in rapeseed based on bioinformatic analysis by using the sequences of the flowering-related 14-3-3s in other plant species. Then, we found that overexpression of BnGF14-2c in the semi-winter rapeseed "93275" promoted flowering without vernalization. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation analysis indicated that BnGF14-2c may interact with two vernalization-related flowering regulators BnFT.A02 and BnFLC.A10., respectively. qPCR analysis showed that the expression of BnFT (AtFT_Like) was increased and the expression of two selected vernalization-related genes were reduced in the overexpression transgenic plants. Further investigation on subcellular localization demonstrated that BnGF14-2c localized in the nucleus and cytoplasm. The results of RNA-seq analysis and GUS staining indicated that BnGF14-2c is ubiquitously expressed except for mature seed coat. In general, the interaction of 14-3-3 and FLC was firstly documented in this study, indicating BnGF14-2c may act as a positive regulator of flowering in rapeseed, which is worthy for more in-depth exploration.

An integrated omics analysis reveals the gene expression profiles of maize, castor bean, and rapeseed for seed oil biosynthesis.

BACKGROUND: Seed storage lipids are valuable for human diet and for the sustainable development of mankind. In recent decades, many lipid metabolism genes and pathways have been identified, but the molecular mechanisms that underlie differences in seed oil biosynthesis in species with developed embryo and endosperm are not fully understood. RESULTS: We performed comparative genome and transcriptome analyses of castor bean and rapeseed, which have high seed oil contents, and maize, which has a low seed oil content. These results revealed the molecular underpinnings of the low seed oil content in maize. First of all, transcriptome analyses showed that more than 61% of the lipid- and carbohydrate-related genes were regulated in castor bean and rapeseed, but only 20.1% of the lipid-related genes and 22.5% of the carbohydrate-related genes were regulated in maize. Then, compared to castor bean and rapeseed, fewer lipid biosynthesis genes but more lipid metabolism genes were regulated in the maize embryo. More importantly, most maize genes encoding lipid-related transcription factors, triacylglycerol (TAG) biosynthetic enzymes, pentose phosphate pathway (PPP) and Calvin Cycle proteins were not regulated during seed oil synthesis, despite the presence of many homologs in the maize genome. Additionally, we observed differential regulation of vital oil biosynthetic enzymes and extremely high expression levels of oil biosynthetic genes in castor bean, which were consistent with the rapid accumulation of oil in castor bean developing seeds. CONCLUSIONS: Compared to high-oil seeds (castor bean and rapeseed), less oil biosynthetic genes were regulated during the seed development in low-oil seed (maize). These results shed light on molecular mechanisms of lipid biosynthesis in maize, castor bean, and rapeseed. They can provide information on key target genes that may be useful for future experimental manipulation of oil production in oil plants.

Fine-mapping and transcriptome analysis of a candidate gene controlling plant height in Brassica napus L.

BACKGROUND: Brassica napus provides approximately 13-16% of global vegetable oil for human consumption and biodiesel production. Plant height (PH) is a key trait that affects plant architecture, seed yield and harvest index. However, the genetic mechanism of PH in B. napus is poorly understood. RESULTS: A dwarf mutant df59 was isolated from a large-scale screening of an ethyl methanesulphonate-mutagenized rapeseed variety Ningyou 18. A genetic analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped on C9 chromosome by quantitative trait loci sequencing analysis and designated as BnaDwf.C9. To fine-map BnaDwf.C9, two F2 populations were constructed from crosses between conventional rapeseed cultivars (Zhongshuang 11 and Holly) and df59. BnaDwf.C9 was fine-mapped to the region between single-nucleotide polymorphism (SNP) markers M14 and M4, corresponding to a 120.87-kb interval of the B. napus 'Darmor-bzh' genome. Within this interval, seven, eight and nine annotated or predicted genes were identified in "Darmor-bzh", "Ningyou 7" and "Zhongshuang 11" reference genomes, respectively. In addition, a comparative transcriptome analysis was performed using stem tips from Ningyou 18 and df59 at the stem elongation stage. In total, 3995 differentially expressed genes (DEGs) were identified. Among them, 118 DEGs were clustered in plant hormone-related signal transduction pathways, including 81 DEGs were enriched in auxin signal transduction. Combining the results of fine-mapping and transcriptome analyses, BnaC09g20450D was considered a candidate gene for BnaDwf.C9, which contains a SNP that co-segregated in 4746 individuals. Finally, a PCR-based marker was developed based on the SNP in BnaC09g20450D. CONCLUSIONS: The combination of quantitative trait loci sequencing, fine-mapping and genome-wide transcriptomic analysis revealed one candidate gene located within the confidence interval of 120.87-kb region. This study provides a new genetic resource for semi-dwarf breeding and new insights into understanding the genetic architecture of PH in B. napus.

Systematic Analysis of the DNA Methylase and Demethylase Gene Families in Rapeseed (Brassica napus L.) and Their Expression Variations After Salt and Heat stresses.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.).

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%-67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi-allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene-free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off-target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi-dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.

Overexpression of BnaAOX1b Confers Tolerance to Osmotic and Salt Stress in Rapeseed.

Alternative oxidases (AOXs) are the terminal oxidase in the cyanide-resistant respiration pathway in plant mitochondria, which play an important role in abiotic stress and are proposed as a functional marker for high tolerant breeding. In this study, ten AOX genes (BnaAOXs) were identified, and CysI and CysII of AOX isoforms were highly conserved in rapeseed. Among them, Bna.AOX1b was mainly expressed in the ovule and displayed varying expression between rapeseed cultivars which showed different salt resistance in seed germination. We identified its mitochondrial localization of this gene. To investigate the function of BnaAOX1b in rapeseed, transgenic rapeseed lines with overexpressed BnaAOX1b were created and seed germination and seedling establishment assays were performed under osmotic, salt, and ABA treatment. The results indicated that overexpression of BnaAOX1b significantly improved seed germination under osmotic and salt stress and weakened ABA sensitivity. In addition, post-germination seedling growth was improved under high salt condition, but showed hypersensitivity to ABA. RNA-sequencing analysis indicated that the genes involved in electron transport or energy pathway were induced and a number of gene responses to salt stress and ABA were regulated in Bna.AOX1b overexpressing seeds. Taken together, our results imply that Bna.AOX1b confers tolerance to osmotic and salt stress in terms of seed germination and seedling establishment by regulating stress responsive genes and the response to ABA, and could be utilized as a candidate gene in transgenic breeding.

Three BnaIAA7 homologs are involved in auxin/brassinosteroid-mediated plant morphogenesis in rapeseed (Brassica napus L.).

KEY MESSAGE: BnaIAA7 crosstalk with BR signaling is mediated by the interaction between BnaARF8 and BnaBZR1 to regulate rapeseed plant morphogenesis. Auxin (indole-3-acetic acid, IAA) and brassinosteroids (BRs) are essential regulators of plant morphogenesis. However, their roles in rapeseed have not been reported. Here, we identified an extremely dwarf1 (ed1) mutant of rapeseed that displays reduced stature, short hypocotyls, as well as wavy and curled leaves. We isolated ED1 by map-based cloning, and found that it encodes a protein homologous to AtIAA7. ED1 acts as a repressor of IAA signaling, and IAA induces its degradation through its degron motif. A genomic-synteny analysis revealed that ED1 has four homologs in rapeseed, but two were not expressed. Analyses of transcriptomes and of various mutant BnaIAA7s in transgenic plants revealed that the three expressed BnaIAA7 homologs had diverse expression patterns. ED1/BnaC05.IAA7 predominantly functioned in stem elongation, BnaA05.IAA7 was essential for reproduction, while BnaA03.IAA7 had the potential to reduce plant height. Physical interaction assays revealed that the three BnaIAA7 homologs interacted in different ways with BnaTIRs/AFBs and BnaARFs, which may regulate the development of specific organs. Furthermore, BnaARF8 could directly interact with the BnaIAA7s and BnaBZR1. We propose that BnaIAA7s interact with BR signaling via BnaARF8 and BnaBZR1 to regulate stem elongation in rapeseed.

A Novel Chimeric Mitochondrial Gene Confers Cytoplasmic Effects on Seed Oil Content in Polyploid Rapeseed (Brassica napus).

Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.

Genome-wide screening and analysis of imprinted genes in rapeseed (Brassica napus L.) endosperm.

Species-specific genomic imprinting is an epigenetic phenomenon leading to parent-of-origin-specific differential expression of maternally and paternally inherited alleles. To date, no studies of imprinting have been reported in rapeseed, a tetraploid species. Here, we analysed global patterns of allelic gene expression in developing rapeseed endosperms from reciprocal crosses between inbred lines YN171 and 93275. A total of 183 imprinted genes, consisting of 167 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs), were identified from 14,394 genes found to harbour diagnostic SNPs between the parental lines. Some imprinted genes were validated in different endosperm stages and other parental combinations by RT-PCR analysis. A clear clustering of imprinted genes throughout the rapeseed genome was identified, which was different from most other plants. Methylation analysis of 104 out of the 183 imprinted genes showed that 11 genes (7 MEGs and 4 PEGs) harboured differentially methylated regions (DMRs). Unexpectedly, only 1 MEG out of these 11 genes had a DMR that exhibited high CG methylation rate in paternal allele and had big difference between parent alleles. These results extend our understanding of gene imprinting in plants and provide potential avenues for further research in imprinted genes.

Overexpression of CHMP7 from rapeseed and Arabidopsis causes dwarfism and premature senescence in Arabidopsis.

Endosomal sorting complexes required for transport (ESCRT) are well known in mammalians and yeast and plays an essential role in the formation of multi-vesicular bodies. Accumulating evidence has shown that ESCRT proteins contribute to proper plant development. CHMP7 (charged multi-vesicular body protein 7) is an ESCRT-III-related protein and functions in the endosomal sorting pathway in humans. However, its function in plants has not been explored in detail. In this study, we isolate the putative homolog of CHMP7 from rapeseed, BnCHMP7, which contains eight exons and encodes a protein consisting of 423 amino acid residues. Compared with the wild-type, overexpression of BnCHMP7 in Arabidopsis disturbs plant growth and decreases seed yield. Moreover, the transgenic plants also display early leaf senescence and hypersensitivity to dark treatment due to defects in autophagic degradation. Further study showed that BnCHMP7 is highly expressed in leaves and that YFP-BnCHMP7 is predominantly localized in endosome. Compared with human CHMP7, we found that BnCHMP7 not only interacts with ESCRT-III subunits SNF7.2 (CHMP4B), but also with VPS2.2 and CHMP1B. As expected, microarray analysis revealed that the expression of ESCRT transport genes is significantly affected. Additionally, the expression of some genes that are involved in senescence, protein synthesis and protein degradation is also altered in BnCHMP7-overexpressing plants. Taken together, BnCHMP7 encodes an endosome-localized protein, which causes dwarfism and leaf senescence as an ESCRT-III-related component.

Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.

BACKGROUND: In conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously. RESULTS: First, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately. CONCLUSION: A simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.