Optimized leaf storage and photosynthetic nitrogen trade-off promote synergistic increases in photosynthetic rate and photosynthetic nitrogen use efficiency.

A coordinated increase in the photosynthetic rate (A) and photosynthetic nitrogen use efficiency (PNUE) is an effective strategy for improving crop yield and nitrogen (N) utilization efficiency. PNUE tends to decrease with increasing N levels, but there are natural variations. Consequently, leaf functional N partitioning in Brassica napus genotypes under different N rates was measured to explore the optimized N allocation model for synchronously increasing A and PNUE values. The results showed that genotypes whose PNUE increased with increasing N supply (PNUE-I) produced an approximate A value with a relatively low leaf N content, owing to reduced storage N (Nstore ) and close photosynthetic N (Npsn ) content. Partial least squares path modeling showed that A was dominated by the Npsn content, and PNUE was directly influenced by A and Nstore . The A value increased with the Npsn content until the Npsn content exceeded the threshold value. The boundary line of PNUE varied with the Npsn and Nstore proportions, indicating that the optimum Npsn and Nstore proportions were 51.6% and 40.3%, respectively. The Nstore proportion of PNUE-I was closer to the thresholds and benefited from lower increments in Rubisco content and nonprotein form storage N content with improved N supply. Optimized Nstore and Npsn trade-off by regulating increments in Nstore content with increased N supply, thereby promoting coordinated increases in A and PNUE.

Metabolic Profiles Reveal Changes in the Leaves and Roots of Rapeseed (Brassica napus L.) Seedlings under Nitrogen Deficiency.

Rapeseed (Brassica napus L.) is an important oil crop species and plays a crucial role in supplying edible oil worldwide. However, rapeseed production in the field is often severely inhibited due to nitrogen (N) deficiency. Metabolites play key roles in plant growth and resistance to environmental stress, but little is known about the differential synthesis and accumulation of metabolites underlying rapeseed adaptation to N deficiency. Here, we studied the phenotypic response and used LC-electrospray ionization (ESI), ESI-MS/MS, and widely untargeted metabolomic approaches to detect differences in rapeseed under normal N (HN) and N-deficient (LN) conditions. The results showed that N deficiency severely inhibited rapeseed shoot growth and promoted rapeseed root architectural changes under LN conditions. In total, 574 metabolites were detected, and there were 175 and 166 differentially accumulated metabolites in the leaves and roots between the HN and LN conditions, respectively. The significantly differentially accumulated metabolites were involved in four primary metabolic pathways, namely, sucrose, phenylalanine, amino acid, and tricarboxylic acid cycle metabolism. Notably, we found that plant hormones have distinct accumulation patterns in rapeseed and coordinate to play crucial roles in both maintaining growth and protecting against damage from plant disease under HN and LN conditions. Moreover, our results indicated that flavonoid compounds, especially anthocyanins and rutin, may play important roles in increasing root cell resistance to oxidative damage and soil pathogen infections. Overall, this work provides valuable information for understanding the overall metabolite changes in rapeseed under N deficiency conditions, which may be beneficial for improving and producing new varieties of rapeseed capable of high yields under low N conditions.

Reclassification of the biocontrol agents Bacillus subtilis BY-2 and Tu-100 as Bacillus velezensis and insights into the genomic and specialized metabolite diversity of the species.

The genomes of two historical Bacillus species strains isolated from the roots of oilseed rape and used routinely in PR China as biocontrol agents to suppress Sclerotinia disease were sequenced. Average nucleotide identity (ANI) and digital DNA-DNA hybridization analyses demonstrated that they were originally misclassified as Bacillus subtilis and now belong to the bacterial species Bacillus velezensis. A broader ANI analysis of available Bacillus genomes identified 292 B. velezensis genomes that were then subjected to core gene analysis and phylogenomics. Prediction and dereplication of specialized metabolite biosynthetic gene clusters (BGCs) defined the prevalence of multiple antimicrobial-associated BGCs and highlighted the natural product potential of B. velezensis. By defining the core and accessory antimicrobial biosynthetic capacity of the species, we offer an in-depth understanding of B. velezensis natural product capacity to facilitate the selection and testing of B. velezensis strains for use as biological control agents.

Genome-wide identification and analysis of high-affinity nitrate transporter 2 (NRT2) family genes in rapeseed (Brassica napus L.) and their responses to various stresses.

BACKGROUND: High-affinity nitrate transporter 2 (NRT2) genes have been implicated in nitrate absorption and remobilization under nitrogen (N) starvation stress in many plant species, yet little is known about this gene family respond to various stresses often occurs in the production of rapeseed (Brassica napus L.). RESULTS: This report details identification of 17 NRT2 gene family members in rapeseed, as well as, assessment of their expression profiles using RNA-seq analysis and qRT-PCR assays. In this study, all BnNRT2.1 members, BnNRT2.2a and BnNRT2.4a were specifically expressed in root tissues, while BnNRT2.7a and BnNRT2.7b were mainly expressed in aerial parts, including as the predominantly expressed NRT2 genes detected in seeds. This pattern of shoot NRT expression, along with homology to an Arabidopsis NRT expressed in seeds, strongly suggests that both BnNRT2.7 genes play roles in seed nitrate accumulation. Another rapeseed NRT, BnNRT2.5 s, exhibited intermediate expression, with transcripts detected in both shoot and root tissues. Functionality of BnNRT2s genes was further outlined by testing for adaptive responses in expression to exposure to a series of environmental stresses, including N, phosphorus (P) or potassium (K) deficiency, waterlogging and drought. In these tests, most NRT2 gene members were up-regulated by N starvation and restricted by the other stresses tested herein. In contrast to this overall trend, transcription of BnNRT2.1a was up-regulated under waterlogging and K deficiency stress, and BnNRT2.5 s was up-regulated in roots subjected to waterlogging. Furthermore, the mRNA levels of BnNRT2.7 s were enhanced under both waterlogging stress and P or K deficiency conditions. These results suggest that these three BnNRT2 genes might participate in crosstalk among different stress response pathways. CONCLUSIONS: The results presented here outline a diverse set of NRT2 genes present in the rapeseed genome that collectively carry out specific functions throughout rapeseed development, while also responding not just to N deficiency, but also to several other stresses. Targeting of individual BnNRT2 members that coordinate rapeseed nitrate uptake and transport in response to cues from multiple stress response pathways could significantly expand the genetic resources available for improving rapeseed resistance to environmental stresses.

Adaption of Roots to Nitrogen Deficiency Revealed by 3D Quantification and Proteomic Analysis.

Rapeseed (Brassica napus) is an important oil crop worldwide. However, severe inhibition of rapeseed production often occurs in the field due to nitrogen (N) deficiency. The root system is the main organ to acquire N for plant growth, but little is known about the mechanisms underlying rapeseed root adaptions to N deficiency. Here, dynamic changes in root architectural traits of N-deficient rapeseed plants were evaluated by 3D in situ quantification. Root proteome responses to N deficiency were analyzed by the tandem mass tag-based proteomics method, and related proteins were characterized further. Under N deficiency, rapeseed roots become longer, with denser cells in the meristematic zone and larger cells in the elongation zone of root tips, and also become softer with reduced solidity. A total of 171 and 755 differentially expressed proteins were identified in short- and long-term N-deficient roots, respectively. The abundance of proteins involved in cell wall organization or biogenesis was highly enhanced, but most identified peroxidases were reduced in the N-deficient roots. Notably, peroxidase activities also were decreased, which might promote root elongation while lowering the solidity of N-deficient roots. These results were consistent with the cell wall components measured in the N-deficient roots. Further functional analysis using transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrated that the two root-related differentially expressed proteins contribute to the enhanced root growth under N deficiency conditions. These results provide insights into the global changes of rapeseed root responses to N deficiency and may facilitate the development of rapeseed cultivars with high N use efficiency through root-based genetic improvements.

Characterization of mechanisms underlying degradation of sclerotia of Sclerotinia sclerotiorum by Aspergillus aculeatus Asp-4 using a combined qRT-PCR and proteomic approach.

BACKGROUND: The biological control agent Aspergillus aculeatus Asp-4 colonizes and degrades sclerotia of Sclerotinia sclerotiorum resulting in reduced germination and disease caused by this important plant pathogen. Molecular mechanisms of mycoparasites underlying colonization, degradation, and reduction of germination of sclerotia of this and other important plant pathogens remain poorly understood. RESULTS: An RNA-Seq screen of Asp-4 growing on autoclaved, ground sclerotia of S. sclerotiorum for 48 h identified 997 up-regulated and 777 down-regulated genes relative to this mycoparasite growing on potato dextrose agar (PDA) for 48 h. qRT-PCR time course experiments characterized expression dynamics of select genes encoding enzymes functioning in degradation of sclerotial components and management of environmental conditions, including environmental stress. This analysis suggested co-temporal up-regulation of genes functioning in these two processes. Proteomic analysis of Asp-4 growing on this sclerotial material for 48 h identified 26 up-regulated and 6 down-regulated proteins relative to the PDA control. Certain proteins with increased abundance had putative functions in degradation of polymeric components of sclerotia and the mitigation of environmental stress. CONCLUSIONS: Our results suggest co-temporal up-regulation of genes involved in degradation of sclerotial compounds and mitigation of environmental stress. This study furthers the analysis of mycoparasitism of sclerotial pathogens by providing the basis for molecular characterization of a previously uncharacterized mycoparasite-sclerotial interaction.

Components of a Rice-Oilseed Rape Production System Augmented with Trichoderma sp. Tri-1 Control Sclerotinia sclerotiorum on Oilseed Rape.

Sclerotinia sclerotiorum causes serious yield losses on many crops throughout the world. A multicomponent treatment that consisted of the residual rice straw remaining after rice harvest and Trichoderma sp. Tri-1 (Tri-1) formulated with the oilseed rape seedcake fertilizer was used in field soil infested with S. sclerotiorum. This treatment resulted in oilseed rape seed yield that was significantly greater than the nontreated control or when the fungicide carbendizem was used in the presence of this pathogen in field trials. Yield data suggested that the rice straw, oilseed rape seedcake, and Tri-1 components of this treatment all contributed incrementally. Similar treatment results were obtained regarding reduction in disease incidence. Slight improvements in yield and disease incidence were detected when this multicomponent treatment was combined with a fungicide spray. Inhibition of sclerotial germination by this multicomponent treatment trended greater than the nontreated control at 90, 120, and 150 days in field studies but was not significantly different from this control. This multicomponent treatment resulted in increased yield relative to the nontreated control in the absence of pathogen in a greenhouse pot study, while the straw alone and the straw plus oilseed rape seedcake treatments did not; suggesting that Tri-1 was capable of promoting growth. Experiments reported here indicate that a treatment containing components of a rice-oilseed rape production system augmented with Tri-1 can control S. sclerotiorum on oilseed rape, be used in integrated strategies containing fungicide sprays for control of this pathogen, and promote plant growth.

Effects of oilseed rape green manure on phosphorus availability of red soil and rice yield in rice-green manure rotation system.

Improving the nutrient content of red soils in southern China is a priority for efficient rice production there. To assess the effectiveness of oilseed rape as green manure for the improvement of soil phosphorus nutrient supply and rice yield in red soil areas, a long-term field plot experiment was conducted comparing two species of rape, Brassica napus (BN) and Brassica juncea (BJ). The effects of returning oilseed rape on soil phosphorus availability, phosphorus absorption, and yield of subsequent rice under rice-green manure rotation mode were analyzed, using data from the seasons of 2020 to 2021. The study found that compared with winter fallow treatment (WT) and no-tillage treatment (NT), the soil available phosphorus content of BN was increased, and that of BJ was significantly increased. The content of water-soluble inorganic phosphorus of BJ increased, and that of BN increased substantially. Compared with the WT, the soil organic matter content and soil total phosphorus content of BN significantly increased, as did the soil available potassium content of BJ, and the soil total phosphorus content of BJ was significantly increased compared with NT. The soil particulate phosphorus content of BJ and BN was significantly increased by 14.00% and 16.00%, respectively. Compared with the WT, the phosphorus activation coefficient of BJ was significantly increased by 11.41%. The rice plant tiller number under the green manure returning treatment was significantly increased by 43.16% compared with the winter fallow treatment. The green manure returning measures increased rice grain yield by promoting rice tiller numbers; BN increased rice grain yield by 9.91% and BJ by 11.68%. Based on these results, returning oilseed rape green manure could augment the phosphorus nutrients of red soil and promote phosphorus availability. Rice-oilseed rape green manure rotation could increase rice grain yield.

Development of a biologically based fertilizer, incorporating Bacillus megaterium A6, for improved phosphorus nutrition of oilseed rape.

Sustainable methods with diminished impact on the environment need to be developed for the production of oilseed rape in China and other regions of the world. A biological fertilizer consisting of Bacillus megaterium A6 cultured on oilseed rape meal improved oilseed rape seed yield (P < 0.0001) relative to the nontreated control in 2 greenhouse pot experiments using natural soil. This treatment resulted in slightly greater yield than oilseed rape meal without strain A6 in 1 of 2 experiments, suggesting a role for strain A6 in improving yield. Strain A6 was capable of solubilizing phosphorus from rock phosphate in liquid culture and produced enzymes capable of mineralizing organic phosphorus (acid phosphatase, phytase) in liquid culture and in the biological fertilizer. The biologically based fertilizer, containing strain A6, improved plant phosphorus nutrition in greenhouse pot experiments resulting in significantly greater available phosphorus in natural soil and in significantly greater plant phosphorus content relative to the nontreated control. Seed yield and available phosphorus in natural soil were significantly greater with a synthetic chemical fertilizer treatment, reduced in phosphorus content, than the biological fertilizer treatment, but a treatment containing the biological fertilizer combined with the synthetic fertilizer provided the significantly greatest seed yield, available phosphorus in natural soil, and plant phosphorus content. These results suggest that the biological fertilizer was capable of improving oilseed rape seed yield, at least in part, through the phosphorus-solubilizing activity of B. megaterium A6.

Genome-Wide Identification and Characterization of Ammonium Transporter (AMT) Genes in Rapeseed (Brassica napus L.).

Ammonium transporters (AMTs) are plasma membrane proteins mediating ammonium uptake and transport. As such, AMTs play vital roles in ammonium acquisition and mobilization, plant growth and development, and stress and pathogen defense responses. Identification of favorable AMT genotypes is a prime target for crop improvement. However, to date, systematic identification and expression analysis of AMT gene family members has not yet been reported for rapeseed (Brassica napus L.). In this study, 20 AMT genes were identified in a comprehensive search of the B. napus genome, 14 members of AMT1 and 6 members of AMT2. Tissue expression analyses revealed that the 14 AMT genes were primarily expressed in vegetative organs, suggesting that different BnaAMT genes might function in specific tissues at the different development stages. Meanwhile, qRT-PCR analysis found that several BnaAMTs strongly respond to the exogenous N conditions, implying the functional roles of AMT genes in ammonium absorption in rapeseed. Moreover, the rapeseed AMT genes were found to be differentially regulated by N, P, and K deficiency, indicating that crosstalk might exist in response to different stresses. Additionally, the subcellular localization of several BnaAMT proteins was confirmed in Arabidopsis protoplasts, and their functions were studied in detail by heterologous expression in yeast. In summary, our studies revealed the potential roles of BnaAMT genes in N acquisition or transportation and abiotic stress response and could provide valuable resources for revealing the functionality of AMTs in rapeseed.

Green Manure Amendment Can Reduce Nitrogen Fertilizer Application Rates for Oilseed Rape in Maize-Oilseed Rape Rotation.

Excessive use of chemical fertilizers has led to a reduction in the quality of arable land and environmental pollution. Using green manure to replace chemical fertilizers is one of the most effective solutions. To study the effect of green manure on the requirement for nitrogen fertilizer in oilseed rape, a field experiment with maize-oilseed rape rotation was conducted. Green manure was intercropped between rows of maize and returned after the maize harvest, with no green manure intercropped as control. Different nitrogen fertilizer treatments (0, 65%, 75% and 100% N rates, respectively) were applied during the oilseed rape season. The results showed that with a 35% reduction in nitrogen application rate, the rapeseed grain yield was significantly higher with the maize intercropping with green manure returned to the field than with the maize monocropping treatment at the same nitrogen level. Under conditions of intercropping and return of green manure, compared with the full standard rate of nitrogen fertilizer treatment, a reduction in nitrogen application of 25-30% in the rape season had no significant effect on rape yield. The agronomic efficiency of nitrogen fertilizer on oilseed rape increased significantly, by 47.61-121%, with green manure incorporation. In addition, green manure incorporation significantly increased the soil organic matter content and the soil-available nitrogen content when chemical nitrogen fertilization was abandoned. Benefit analysis showed that a 25-35% reduction in chemical nitrogen fertilizer applied to oilseed rape crops could be achieved by intercropping green manure in the maize season before the sowing of rapeseed in the experimental area. In the long-term, this measure would increase nitrogen utility, reduce production costs, and have concomitant environmental benefits of improving the quality of cultivated land.

[Application of ICP-MS to detection of mineral elements in double-low and double-high rapeseed].

The object of the present study was to investigate ten necessary mineral elements in different varieties of rapeseed by ICP-MS. Thirty three samples of double-low (low in both glucosinolates and erucic acid) rapeseed (DLR) and 12 samples of double-high rapeseed (DHR) in the Yangtze River Valley were collected. The results showed that rapeseed contained many kinds of necessary elements, and there was a difference between DLR and DHR The average concentrations of P, K, Ca, Mg, S, Fe, Mn, Cu, Zn and B in DLR were 6.26 mg x g(-1), 10.17 mg x g(-1), 6.75 mg x g(-1), 4.82 mg x g(-1), 4.52 mg x g(-1), 96.20 microg x g(-1), 37.10 microg x g(-1), 3.84 microg x g(-1), 41.61 microg x g(-1) and 12.16 microg x g(-1), respectively, while for the mineral elements in DHR, the average values were 5.97 mg x g(-1), 10. 14 mg x g(-1), 6.31 mg x g(-1), 4.50 mg x g(-1), 9.06 mg x g(-1), 77.03 microg x g(-1), 49.86 microg x g(-1), 3.72 microg x g(-1), 40.58 microg x g(-1) and 11.79 microg x g(-1). The contents of most elements in DLR were higher than those in DHR, such as P, K, Ca, Mg, Fe, Cu, Zn and B. Furthermore, the differences in Ca, Mg and Fe contents between the two varieties were significant. It was concluded that the difference in concentrations of mineral elements can be regarded as a necessary factor for popularizing double-low rapeseed in the Yangtze River Valley.

Soil Enzyme Activity in Soils Subjected to Flooding and the Effect on Nitrogen and Phosphorus Uptake by Oilseed Rape.

Waterlogging presents one of the greatest constraints for agricultural crops. In order to elucidate the influences of waterlogging stress on the growth of oilseed rape, a pot experiment was performed investigating the impact of waterlogging on nitrogen (N) and phosphorus (P) accumulation in oilseed rape, and mineral N and available P profiles and enzyme activities of soils. The experiment included waterlogging treatments lasting 3 (I), 6 (II), and 9 (III) days, and a control treatment without waterlogging (CK). Results showed that waterlogging lasting 3 or more days significantly depressed the growth of oilseed rape, and prolonged the recovery time of plant growth with the period of flooding. Waterlogging notably influenced the N and P concentrations in plant tissues, and also affected mineral N, available P profiles, and activities of enzymes (including urease, phosphatase, invertase, and catalase) in the soils. With the exception of catalase, flooding suppressed the activity of urease, phosphatase, and invertase to varying degrees, and the longer the flooding time, the greater the suppression. The effect of waterlogging on mineral N and P profiles resulted from the altered proportions of NH4 +-N and NO3 --N, and the decreased available P concentrations in these soils, respectively. The effect on P was more significant than on N in both soil nutrient profile and plant utilization.

Genome-Wide Identification and Expression Analysis of NRAMP Family Genes in Soybean (Glycine Max L.).

The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis.

A new method for the preservation of axenic fungal cultures.

Microbiological work requires a reliable source of cultures that are not only well defined and taxonomically determined, but are also adequately preserved without changes in their morphological, physiological and genetic traits. Here we describe an easy, cost effective and rapid method for reliably preserving filamentous fungi on cellophane pieces at -80°C for use in laboratory culture collections.