Enhancing the efficiency of photosynthesis represents a promising strategy to improve crop yields, with keeping the steady state of PSII being key to determining the photosynthetic performance. However, the mechanisms whereby the stability of PSII is maintained in oxygenic organisms remain to be explored. Here, we report that the Psb28 protein functions in regulating the homeostasis of PSII under different light conditions in Arabidopsis thaliana. The psb28 mutant is much smaller than the wild-type plants under normal growth light, which is due to its significantly reduced PSII activity. Similar defects were seen under low light and became more pronounced under photoinhibitory light. Notably, the amounts of PSII core complexes and core subunits are specifically decreased in psb28, whereas the abundance of other representative components of photosynthetic complexes remains largely unaltered. Although the PSII activity of psb28 was severely reduced when subjected to high light, its recovery from photoinactivation was not affected. By contrast, the degradation of PSII core protein subunits is dramatically accelerated in the presence of lincomycin. These results indicate that psb28 is defective in the photoprotection of PSII, which is consistent with the observation that the overall NPQ is much lower in psb28 compared to the wild type. Moreover, the Psb28 protein is associated with PSII core complexes and interacts mainly with the CP47 subunit of PSII core. Taken together, these findings reveal an important role for Psb28 in the protection and stabilization of PSII core in response to changes in light environments.

Drought is one of the most important abiotic stresses, and seriously threatens plant development and productivity. Increasing evidence indicates that chromatin remodelers are pivotal for plant drought response. However, molecular mechanisms of chromatin remodelers-mediated plant drought responses remain obscure. In this study, we found a novel interactor of BRM called BRM-associated protein involved in drought response (BAPID), which interacted with SWI/SNF chromatin remodeler BRM and drought-induced transcription factor Di19. Our findings demonstrated that BAPID acted as a positive drought regulator since drought tolerance was increased in BAPID-overexpressing plants, but decreased in BAPID-deficient plants, and physically bound to PR1, PR2, and PR5 promoters to mediate expression of PR genes to defend against dehydration stress. Genetic approaches demonstrated that BRM acted epistatically to BAPID and Di19 in drought response in Arabidopsis. Furthermore, the BAPID protein-inhibited interaction between BRM and Di19, and suppressed the inhibition of BRM on the Di19-PR module by mediating the H3K27me3 deposition at PR loci, thus changing nucleosome accessibility of Di19 and activating transcription of PR genes in response to drought. Our results shed light on the molecular mechanism whereby the BAPID-BRM-Di19-PRs pathway mediates plant drought responses. We provide data improving our understanding of chromatin remodeler-mediated plant drought regulation network.

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.

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.

Vasculature anatomy and density are major determinants of plant solute fluxes, long-distance signalling and photosynthetic capabilities, but the underlying mechanisms remain largely unknown. Here, we discover that frequency of cellular autophagosome formation is intimately interconnected with leaf vasculature differentiation in that elevated expression of key autophagy facilitators drives vasculature development, while increasing expression of transcriptional regulators of vasculature differentiation also enhances autophagy. We identify VDof1 as bi-functional regulator that uniquely combines transcription factor domains with interaction-motifs for association with autophagy components and thereby functions in vasculature formation and autophagy at two levels. In nuclei, VDof1 associates with key regulators of vascular anatomy including ANT1, SCR and SHR and also activates transcription of the autophagy facilitator Atg8. In chloroplasts, VDof1 directly assembles with several key components of autophagy and chloroplast degradation. Collectively, these findings uncover a hitherto undescribed role of autophagy as regulatory pathway for modulating leaf vascular development.

为便于体外研究甘蓝型油菜的胚胎发育和种子油脂积累规律,以春油菜862的胚胎为材料,构建胚胎离体培养体系,通过综合比对基础培养基类型、蔗糖浓度、琼脂浓度,确定含蔗糖8％和琼脂0.6％的B5培养基作为胚胎离体培养和胚胎胚性生长的最佳体外培养体系,并用于研究种子油脂的积累和变化.在体系中分别加入甘蓝型油菜的角果皮和叶片提取物,调查对胚胎油脂积累的影响,发现含有角果皮提取物和叶片提取物的培养基均能提高胚胎油脂积累效率,且角果皮提取物对胚胎油脂积累的效率显著高于叶片提取物,源于高油材料的角果皮、叶片提取物对胚胎油脂的积累效率显著高于低油材料.

创制突变体库是甘蓝型油菜品种选育和重要性状遗传解析的重要基础.春性甘蓝型油菜开花不需漫长的春化过程,生育期短,在创制突变体库方面具有独特优势.本研究采用甲基磺酸乙酯(EMS)诱变春性油菜862种子,以构建春性油菜EMS诱变突变体资源库.通过比较8种不同浓度EMS(0～1.4％,间隔0.2％)对发芽率的影响,最终确定使用0.8％的EMS作为最佳处理浓度,发芽率为37.2％.田间种植筛选与表型统计初步鉴定了M2代1823个单株的形态学性状,突变频率为3.91％,主要包括叶色、叶形、株高、株型、角果长短、种粒大小、种皮颜色、种子含油量以及其它性状变异等,突变类型十分丰富.该突变体库的构建为油菜功能基因组学研究和分子育种提供表型多样的种质资源.

Nitric oxide (NO) is a key signaling molecule regulating several plant developmental and stress responses. Here, we report that NO plays an important role in seed oil content and fatty acid composition. RNAi silencing of Arabidopsis S-nitrosoglutathione reductase 1 (GSNOR1) led to reduced seed oil content. In contrast, nitrate reductase double mutant nia1nia2 had increased seed oil content, compared with wild-type plants. Moreover, the concentrations of palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3) were higher, whereas those of stearic acid (C18:0), oleic acid (C18:1), and arachidonic acid (C20:1) were lower, in seeds of GSNOR1 RNAi lines. Similar results were obtained with rapeseed embryos cultured in vitro with the NO donor sodium nitroprusside (SNP), and the NO inhibitor NG-Nitro-L-arginine Methyl Ester (L-NAME). Compared with non-treated embryos, the oil content decreased in SNP-treated embryos, and increased in L-NAME-treated embryos. Relative concentrations of C16:0, C18:2 and C18:3 were higher, whereas C18:1 concentration decreased in rapeseed embryos treated with SNP. Proteomics and transcriptome analysis revealed that three S-nitrosated proteins and some key genes involved in oil synthesis, were differentially regulated in SNP-treated embryos. Therefore, regulating NO content could be a novel approach to increasing seed oil content in cultivated oil crops.

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.

In plants, green non-foliar organs are able to perform photosynthesis just as leaves do, and the seed-enclosing pod acts as an essential photosynthetic organ in legume and Brassica species. To date, the contribution of pod photosynthesis to seed yield and related components still remains largely unexplored, and in Arabidopsis thaliana, the photosynthetic activity of the silique (pod) is unknown. In this study, an Arabidopsis glk1/glk2 mutant defective in both leaf and silique photosynthesis was used to create tissue-specific functional complementation lines. These lines were used to analyze the contribution of silique wall photosynthesis to seed yield and related traits, and to permit the comparison of this contribution with that of leaf photosynthesis. Our results showed that, together with leaves, the photosynthetic assimilation of the silique wall greatly contributed to total seed yield per plant. As for individual components of yield traits, leaf photosynthesis alone contributed to the seed number per silique and silique length, while silique wall photosynthesis alone contributed to thousand-seed weight. In addition, enhancing the photosynthetic capacity of the silique wall by overexpressing the photosynthesis-related RCA gene in this tissue resulted in significantly increased seed weight and oil content in the wild-type (WT) background. These results reveal that silique wall photosynthesis plays an important role in seed-related traits, and that enhancing silique photosynthesis in WT plants can further improve seed yield-related traits and oil production. This finding may have significant implications for improving the seed yield and oil production of oilseed crops and other species with pod-like organs.

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.

我国油菜含油量低,严重影响了国产菜籽的市场竞争力,提高含油量是油菜育种的当务之急.拟南芥与油菜同属十字花科,利用拟南芥筛选油脂合成及调控相关的新基因并将其应用于油菜,是快速选育高油油菜品种的有效手段.本研究采用密度梯度离心方法及核磁共振检测油菜种子的含油量,从6 868份拟南芥突变体库资源中筛选得到9个种子含油量较野生型呈显著性差异的株系.通过T-DNA插入突变的特点,筛选得到6个调控种子油脂合成的候选基因:AT2G19450、AT3G26790、AT3G54320、AT5G06270、AT2G21840和AT3G47450,其中AT5G06270,AT2G21840和AT3G47450属于未知功能基因,为深入研究种子含油量和其调控机理提供了一定的参考.

Seed weight (SW), which is one of the three major factors influencing grain yield, has been widely accepted as a complex trait that is controlled by polygenes, particularly in polyploid crops. Brassica napus L., which is the second leading crop source for vegetable oil around the world, is a tetraploid (4×) species. In the present study, we identified a major quantitative trait locus (QTL) on chromosome A9 of rapeseed in which the genes for SW and silique length (SL) were colocated. By fine mapping and association analysis, we uncovered a 165-bp deletion in the auxin-response factor 18 (ARF18) gene associated with increased SW and SL. ARF18 encodes an auxin-response factor and shows inhibitory activity on downstream auxin genes. This 55-aa deletion prevents ARF18 from forming homodimers, in turn resulting in the loss of binding activity. Furthermore, reciprocal crossing has shown that this QTL affects SW by maternal effects. Transcription analysis has shown that ARF18 regulates cell growth in the silique wall by acting via an auxin-response pathway. Together, our results suggest that ARF18 regulates silique wall development and determines SW via maternal regulation. In addition, our study reveals the first (to our knowledge) QTL in rapeseed and may provide insights into gene cloning involving polyploid crops.

Selection of reference genes in Brassica napus, a tetraploid (4×) species, is a very difficult task without information on genome and transcriptome. By now, only several traditional reference genes which show significant expression differentiation under different conditions are used in B. napus. In the present study, based on genome and transcriptome data of the rapeseed Zhongshuang-11 cultivar, 14 candidate reference genes were screened for investigation in different tissues, cultivars, and treated conditions of B. napus. These genes were as follows: ELF5, ENTH, F-BOX7, F-BOX2, FYPP1, GDI1, GYF, MCP2d, OTP80, PPR, SPOC, Unknown1, Unknown2 and UBA. Among them, excluding GYF and FYPP1, another 12 genes, were identified to perform better than traditional reference genes ACTIN7 and GAPDH. To further validate the accuracy of the newly developed reference genes in normalization, expression levels of BnCAT1 (B. napus catalase 1) in different rapeseed tissues and seedlings under stress conditions were normalized by the three most stable reference genes PPR, GDI1, and ENTH and little difference existed in normalization results. To the best of our knowledge, this is the first time B. napus reference genes have been provided with the help of complete genome and transcriptome information. The new reference genes provided in this study are more accurate than previously reported reference genes in quantifying expression levels of B. napus genes.

Cosuppression of an Arabidopsis Rubisco small subunit gene RBCS3B at Arabidopsis resulted in albino or pale green phenotypes which were caused by ROS accumulation

Background Rapeseed (Brassica napus L.) is an important oil crop in the world, and increasing its oil content is a major breeding goal. The studies on seed structure and characteristics of different oil content rapeseed could help us to understand the biological mechanism of lipid accumulation, and be helpful for rapeseed breeding. Methodology/Principal Findings Here we report on the seed ultrastructure of an ultrahigh oil content rapeseed line YN171, whose oil content is 64.8%, and compared with other high and low oil content rapeseed lines. The results indicated that the cytoplasms of cotyledon, radicle, and aleuronic cells were completely filled with oil and protein bodies, and YN171 had a high oil body organelle to cell area ratio for all cell types. In the cotyledon cells, oil body organelles comprised 81% of the total cell area in YN171, but only 53 to 58% in three high oil content lines and 33 to 38% in three low oil content lines. The high oil body organelle to cotyledon cell area ratio and the cotyledon ratio in seed were the main reasons for the ultrahigh oil content of YN171. The correlation analysis indicated that oil content is significantly negatively correlated with protein content, but is not correlated with fatty acid composition. Conclusions/Significance Our results indicate that the oil content of YN171 could be enhanced by increasing the oil body organelle to cell ratio for some cell types. The oil body organelle to seed ratio significantly highly positively correlates with oil content, and could be used to predict seed oil content. Based on the structural analysis of different oil content rapeseed lines, we estimate the maximum of rapeseed oil content could reach 75%. Our results will help us to screen and identify high oil content lines in rapeseed breeding.

Background Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay. Results A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%. Conclusions Despite the polyploid nature of B. napus , nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.