high-N Conditions Research Articles

High-nitrogen-induced γ-aminobutyric acid triggers host immunity and pathogen oxidative stress tolerance in tomato and Ralstonia solanacearum interaction.

Soil nitrogen (N) significantly influences the interaction between plants and pathogens, yet its impact on host defenses and pathogen strategies via alterations in plant metabolism remains unclear. Through metabolic and genetic studies, this research demonstrates that high-N-input exacerbates tomato bacterial wilt by altering γ-aminobutyric acid (GABA) metabolism of host plants. Under high-N conditions, the nitrate sensor NIN-like protein 7 (SlNLP7) promotes the glutamate decarboxylase 2/4 (SlGAD2/4) transcription and GABA synthesis by directly binding to the promoters of SlGAD2/4. The tomato plants with enhanced GABA levels showed stronger immune responses but remained susceptible to Ralstonia solanacearum. This led to the discovery that GABA produced by the host actually heightens the pathogen's virulence. We identified the R. solanacearum LysR-type transcriptional regulator OxyR protein, which senses host-derived GABA and, upon interaction, triggers a response involving protein dimerization that enhances the pathogen's oxidative stress tolerance by activating the expression of catalase (katE/katGa). These findings reveal GABA's dual role in activating host immunity and enhancing pathogen tolerance to oxidative stress, highlighting the complex relationship between tomato plants and R. solanacearum, influenced by soil N status.

Low Nitrogen Input Mitigates Quantitative but Not Qualitative Reconfiguration of Leaf Primary Metabolism in Brassica napus L. Subjected to Drought and Rehydration.

In the context of climate change and the reduction of mineral nitrogen (N) inputs applied to the field, winter oilseed rape (WOSR) will have to cope with low-N conditions combined with water limitation periods. Since these stresses can significantly reduce seed yield and seed quality, maintaining WOSR productivity under a wide range of growth conditions represents a major goal for crop improvement. N metabolism plays a pivotal role during the metabolic acclimation to drought in Brassica species by supporting the accumulation of osmoprotective compounds and the source-to-sink remobilization of nutrients. Thus, N deficiency could have detrimental effects on the acclimation of WOSR to drought. Here, we took advantage of a previously established experiment to evaluate the metabolic acclimation of WOSR during 14 days of drought, followed by 8 days of rehydration under high- or low-N fertilization regimes. For this purpose, we selected three leaf ranks exhibiting contrasted sink/source status to perform absolute quantification of plant central metabolites. Besides the well-described accumulation of proline, we observed contrasted accumulations of some "respiratory" amino acids (branched-chain amino acids, lysineand tyrosine) in response to drought under high- and low-N conditions. Drought also induced an increase in sucrose content in sink leaves combined with a decrease in source leaves. N deficiency strongly decreased the levels of major amino acids and subsequently the metabolic response to drought. The drought-rehydration sequence identified proline, phenylalanine, and tryptophan as valuable metabolic indicators of WOSR water status for sink leaves. The results were discussed with respect to the metabolic origin of sucrose and some amino acids in sink leaves and the impact of drought on source-to-sink remobilization processes depending on N nutrition status. Overall, this study identified major metabolic signatures reflecting a similar response of oilseed rape to drought under low- and high-N conditions.

Mapping of Candidate Genes for Nitrogen Uptake and Utilization in Japonica Rice at Seedling Stage.

Nitrogen (N) is one of the essential nutrients for the growth and development of crops. The adequate application of N not only increases the yield of crops but also improves the quality of agricultural products, but the excessive application of N can cause many adverse effects on ecology and the environment. In this study, genome-wide association analysis (GWAS) was performed under low- and high-N conditions based on 788,396 SNPs and phenotypic traits relevant to N uptake and utilization (N content and N accumulation). A total of 75 QTLs were obtained using GWAS, which contained 811 genes. Of 811 genes, 281 genes showed different haplotypes, and 40 genes had significant phenotypic differences among different haplotypes. Of these 40 genes, 5 differentially expressed genes (Os01g0159250, Os02g0618200, Os02g0618400, Os02g0630300, and Os06g0619000) were finally identified as the more valuable candidate genes based on the transcriptome data sequenced from Longjing31 (low-N-tolerant variety) and Songjing 10 (low-N-sensitive variety) under low- and high-N treatments. These new findings enrich the genetic resources for N uptake and utilization in rice, as well as lay a theoretical foundation for improving the efficiency of N uptake and utilization in rice.

Enhancement patterns of potassium on nitrogen transport and functional genes in cotton vary with nitrogen levels

The application of potassium (K) in conjunction with nitrogen (N) has been shown to enhance N use efficiency. However, there is still a need for further understanding of the optimal ratios and molecular regulatory mechanisms, particularly in soil-cotton systems. Here, a field trial was conducted, involving varying rates of N and K, alongside pot and hydroponic experiments. The objective was to assess the impact of N-K interaction on the absorption, transport and distribution of N in cotton. The results showed that K supply at 90 and 240 kg ha−1 had a beneficial impact on N uptake and distribution to both seed and lint, resulting in the highest N use efficiency ranging from 22% to 62% and yield improvements from 20% to 123%. The increase in stem and root diameters, rather than the quantify of xylem vessels and phloem sieve tubes, facilitated the uptake and transport of N due to the provision of K. At the molecular level, K supply upregulated the expression levels of genes encoding GhNRT2.1 transporter and GhSLAH3 channel in cotton roots to promote N uptake and GhNRT1.5/NPF7.3 genes to transport N to shoot under low-N conditions. However, under high-N conditions, K supply induced anion channel genes (GhSLAH4) of roots to promote N uptake and genes encoding GhNRT1.5/NPF7.3 and GhNRT1.8/NPF7.2 transporters to facilitate NO3- unloading from xylem to mesophyll cell in high-N plants. Furthermore, K supply resulted in the upregulation of gene expression for GhGS2 in leaves, while simultaneously downregulating the expression of GhNADH-GOGAT, GhGDH1 and GhGDH3 genes in high-N roots. The enzyme activities of nitrite reductase and glutamine synthetase increased and glutamate dehydrogenase decreased, but the concentration of NO3- and soluble protein exhibited a significant increase and free amino acid decreased in the shoots subsequent to K supply.

Estimating the genetic parameters of yield-related traits under different nitrogen conditions in maize.

Understanding the genetic basis responding to nitrogen (N) fertilization in crop production is a long-standing research topic in plant breeding and genetics. Albeit years of continuous efforts, the genetic architecture parameters, such as heritability, polygenicity, and mode of selection, underlying the N responses in maize remain largely unclear. In this study, about n = 230 maize inbred lines were phenotyped under high N (HN) and low N (LN) conditions for 2 consecutive years to obtain 6 yield-related traits. Heritability analyses suggested that traits highly responsive to N treatments were less heritable. Using publicly available SNP genotypes, the genome-wide association study (GWAS) was conducted to identify n = 237 and n = 130 trait-associated loci under HN and LN conditions, n = 164 for N-responsive (NR) traits, and n = 31 for genotype by N interaction (G × N). Furthermore, genome-wide complex trait Bayesian (GCTB) analysis, a method complementary to GWAS, was performed to estimate genetic parameters, including genetic polygenicity and the mode of selection (S). GCTB results suggested that the NR value of a yield component trait was highly polygenic and that 4 NR traits exhibited negative correlations between SNP effects and their minor allele frequencies (or the S value <0)-a pattern consistent with negative selection to purge deleterious alleles. This study reveals the complex genetic architecture underlying N responses for yield-related traits and provides candidate genetic loci for N resilient maize improvement.

Carbon and nitrogen remobilization during seed filling in Arabidopsis is strongly impaired in the pyrroline-5-carboxylate dehydrogenase mutant.

Proline is an amino acid that is degraded in the mitochondria by the sequential action of proline dehydrogenase (ProDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) to form glutamate. We investigated the phenotypes of Arabidopsis wild-type plants, the knockout prodh1 prodh2 double-mutant, and knockout p5cdh allelic mutants grown at low and high nitrate supplies. Surprisingly, only p5cdh presented lower seed yield and produced lighter seeds. Analyses of elements in above-ground organs revealed lower C concentrations in the p5cdh seeds. Determination of C, N, and dry matter partitioning among the above-ground organs revealed a major defect in stem-to-seed resource allocations in this mutant. Again surprisingly, defects in C, N, and biomass allocation to seeds dramatically increased in high-N conditions. 15N-labelling consistently confirmed the defect in N remobilization from the rosette and stem to seeds in p5cdh. Consequently, the p5cdh mutants produced morphologically abnormal, C-depleted seeds that displayed very low germination rates. The most striking result was the strong amplification of the N-remobilization defects in p5cdh under high nitrate supply, and interestingly this phenotype was not observed in the prodh1 prodh2 double-mutant irrespective of nitrate supply. This study reveals an essential role of P5CDH in carbon and nitrogen remobilization for reserve accumulation during seed development in Arabidopsis.

Alteration of plant immunity in the interaction of roots with the endophytic fungus Phomopsis liquidambaris in response to external nitrogen conditions.

The complex environments of plants force them to prioritize their immune responses to stimuli occurring simultaneously, including colonization by microbes or nutrient availability. Little is known about how the interplay between endophytes and nutrient status affects the immune responses of both plants and fungi. We primarily monitored immune responses in rice following inoculation with the endophytic fungus Phomopsis liquidambaris under different nitrogen (N) conditions. Ph. liquidambaris promoted plant growth under low N (LN) conditions, concomitant with higher root colonization. Plant production of oxidative signals, including hydrogen peroxide and nitric oxide, was activated by Ph. liquidambaris colonization under LN conditions, while salicylic acid (SA) was maintained at high levels and was involved in controlling rice-fungal interactions. High N (HN) conditions enhanced the ability of Ph. liquidambaris in suppressing plant cell death and the ability of roots to degrade Ph. liquidambaris cell walls. Furthermore, under both LN and HN conditions, the activity of plant defence-associated enzymes and fungal antioxidases was not affected in the interactive association. Our data reveal the alteration of plant immunity, including oxidative signalling and plant cell death, by fungal colonization in response to external N conditions and identify SA signalling as a potential controller for rice-Ph. liquidambaris interaction.

Rice transcription factors OsLBD37/38/39 regulate nitrate uptake by repressing OsNRT2.1/2.2/2.3 under high-nitrogen conditions

Nitrate (NO3−) uptake involves a finely regulated and complex multilevel response system. Elucidating the molecular mechanism of nitrate uptake may lead to improving the growth and productivity of plants in the presence of dynamic variation in nitrate concentration. In this study, we identified three lateral organ boundaries domain (LBD) transcription factors, OsLBD37, OsLBD38, and OsLBD39, as regulators of nitrate uptake in response to nitrogen (N) availability. OsLBD37, OsLBD38, and OsLBD39 were induced by ammonium and glutamine in rice roots. Individual or collective knockout of OsLBD37, OsLBD38, and OsLBD39 led to increased concentrations of nitrate and increased expression of OsNRT2.1, OsNRT2.2, and OsNRT2.3 respectively under high-N conditions, whereas overexpression of each of these three LBD genes produced opposite effects where N accumulation was reduced. Dual-luciferase reporter assay further confirmed that OsLBD37, OsLBD38, and OsLBD39 possessed transcription inhibitory activities in rice protoplast cells, downregulating the expression of OsNRT2.1/OsNRT2.2/OsNRT2.3. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that OsLBD37 interacted with OsLBD37, OsLBD38, and OsLBD39 in the nucleus. Together, these results show that OsLBD37, OsLBD38, and OsLBD39 collaborate to inhibit the expression of OsNRT2.1/OsNRT2.2/OsNRT2.3 transporters under N-sufficient conditions, thereby helping rice plants avoid excessive nitrate accumulation that may affect their growth.

Identifying variation for N-use efficiency and associated traits in amphidiploids derived from hybrids of bread wheat and the genera Aegilops, Secale, Thinopyrum and Triticum.

Future genetic progress in wheat grain yield will depend on increasing biomass and this must be achieved without commensurate increases in nitrogen (N) fertilizer inputs to minimize environmental impacts. In recent decades there has been a loss of genetic diversity in wheat through plant breeding. However, new genetic diversity can be created by incorporating genes into bread wheat from wild wheat relatives. Our objectives were to investigate amphidiploids derived from hybrids of bread wheat (Triticum aestivum L.) and related species from the genera Aegilops, Secale, Thinopyrum and Triticum for expression of higher biomass, N-use efficiency (NUE) and leaf photosynthesis rate compared to their bread wheat parents under high and low N conditions. Eighteen amphidiploid lines and their bread wheat parents were examined in high N (HN) and low N (LN) treatments under glasshouse conditions in two years. Averaged across years, grain yield reduced by 38% under LN compared to HN conditions (P = 0.004). Three amphidiploid lines showed positive transgressive segregation compared to their bread wheat parent for biomass per plant under HN conditions. Positive transgressive segregation was also identified for flag-leaf photosynthesis both pre-anthesis and post-anthesis under HN and LN conditions. For N uptake per plant at maturity positive transgressive segregation was identified for one amphidiploid line under LN conditions. Our results indicated that introgressing traits from wild relatives into modern bread wheat germplasm offers scope to raise biomass and N-use effciency in both optimal and low N availability environments.

Does resource exchange in ectomycorrhizal symbiosis vary with competitive context and nitrogen addition?

Ectomycorrhizal symbiosis is essential for the nutrition of most temperate forest trees and helps regulate the movement of carbon (C) and nitrogen (N) through forested ecosystems. The factors governing the exchange of plant C for fungal N, however, remain obscure. Because competition and soil resources may influence ectomycorrhizal resource movement, we performed a 10-month split-root microcosm study using Pinus muricata seedlings with Thelephora terrestris, Suillus pungens, or no ectomycorrhizal fungus, under two N concentrations in artificial soil. Fungi competed directly with roots and indirectly with each other. We used stable isotope enrichment to track plant photosynthate and fungal N. For T. terrestris, plants received N commensurate with the C given to their fungal partners. Thelephora terrestris was a superior mutualist under high-N conditions. For S. pungens, plant C and fungal N exchange were not coupled. However, in low-N conditions, plants preferentially allocated C to S. pungens rather than T. terrestris. Our results suggest that ectomycorrhizal resource transfer depends on competitive and nutritional context. Plants can exchange C for fungal N, but coupling of these resources can depend on the fungal species and soil N. Understanding the diversity of fungal strategies, and how they change with environmental context, reveals mechanisms driving this important symbiosis.

Proteomics characterization nitrogen fertilizer promotes the starch synthesis and metabolism and amino acid biosynthesis in common buckwheat

Nitrogen (N) affects common buckwheat quality by affecting starch and amino acids (AAs) content, but its molecular mechanism is still unclear. We selected two common buckwheat varieties with high and low starch content, and designed two treatments with 180 and 0 kg N/ha. Application of high-N led to significant increases in starch, amylose and amylopectin content. Of 1337 differentially expressed proteins (DEPs) induced by high-N conditions. 472DEPs were significantly upregulated and 176DEPs downregulated for Xinong9976. 239DEPs were significantly upregulated and 126DEPs downregulated for Beizaosheng. The six alpha-glucan phosphorylases, three alpha-amylases, one granule-bound starch synthase 1 and one sucrose synthase exhibited higher expression at the 180 kg N/ha than at the 0 kg N/ha. In addition, high-N application promoted arginine, leucine, isoleucine and valine biosynthesis. This study revealed the effect of N on the starch and AA content of common buckwheat and its mechanism. The crucial proteins identified may develop the quality of common buckwheat.

Tree water uptake enhances nitrogen acquisition in a fertilized boreal forest - but not under nitrogen-poor conditions.

Understanding how plant water uptake interacts with acquisition of soil nitrogen (N) and other nutrients is fundamental for predicting plant responses to a changing environment, but it is an area where models disagree. We present a novel isotopic labelling approach which reveals spatial patterns of water and N uptake, and their interaction, by trees. The stable isotopes 15 N and 2 H were applied to a small area of the forest floor in stands with high and low soil N availability. Uptake by surrounding trees was measured. The sensitivity of N acquisition to water uptake was quantified by statistical modelling. Trees in the high-N stand acquired twice as much 15 N as in the low-N stand and around half of their N uptake was dependent on water uptake (2 H enrichment). By contrast, in the low-N stand there was no positive effect of water uptake on N uptake. We conclude that tree N acquisition was only marginally dependent on water flux toward the root surface under low-N conditions whereas under high-N conditions, the water-associated N uptake was substantial. The results suggest a fundamental shift in N acquisition strategy under high-N conditions.

Nitrogen Uptake Efficiency, Mediated by Fine Root Growth, Early Determines Temporal and Genotypic Variations in Nitrogen Use Efficiency of Winter Oilseed Rape.

Maintaining seed yield under low N inputs is a major issue for breeding, which requires thoroughly exploiting the genetic diversity of processes related to Nitrogen Use Efficiency (NUE). However, dynamic analysis of processes underlying genotypic variations in NUE in response to N availability from sowing to harvest are scarce, particularly at the whole-plant scale. This study aimed to dynamically decipher the contributions of Nitrogen Uptake Efficiency (NUpE) and Nitrogen Utilization Efficiency (NUtE) to NUE and to identify traits underlying NUpE genetic variability throughout the growth cycle of rapeseed. Three experiments were conducted under field-like conditions to evaluate seven genotypes under two N conditions. We developed NUE_DM (ratio of total plant biomass to the amount of N available) as a new proxy of NUE at harvest, valid to discriminate genotypes from the end of inflorescence emergence, and N conditions as early as the beginning of stem elongation. During autumn growth, NUpE explained up to 100% of variations in NUE_DM, validating the major role of NUpE in NUE shaping. During this period, under low N conditions, up to 53% of the plant nitrogen was absorbed and NUpE genetic variability resulted not from differences in Specific N Uptake but in fine-root growth. NUtE mainly contributed to NUE_DM genotypic variation during the reproductive phase under high-N conditions, but NUpE contribution still accounted for 50–75% after flowering. Our study highlights for the first time NUpE and fine-root growth as important processes to optimize NUE, which opens new prospects for breeding.

SLAF-Based Linkage Map Construction and QTL Mapping of Nitrogen Use Efficiency in Rice (Oryza sativa L.)

Rice production relies on large amounts of synthetic nitrogen fertilizer to meet crop nutritional demands. However, the current fertilizer use efficiency is poor, with an average of 30–50% of total applied nitrogen (N) recovered in grains. Consequently, optimizing N availability and N use efficiency (NUE) has become a major target for rice yield improvement, and looking for quantitative trait loci (QTL) associated with NUE can provide useful information for developing nitrogen efficient rice genotypes. The present study aims to identify QTLs associated with NUE and its two components, agronomic NUE (agNUE) and grain nitrogen increase rate (GIR). A recombinant inbred line population (RIL) derived from a cross between Nipponbare and OM052, consisting of 159 F3 individuals was developed and cultivated in low-N (8 kg N/0.67 ha, N8) and high-N (18 kg N/0.67 ha, N18) conditions. Specific length amplified fragment sequencing (SLAF) was used to genotype the RILs and construct a high-density linkage map spanning 1587.70 cM, consisting 2707 SLAF markers with an average interval of 0.59 cM between adjacent marker loci. QTL analysis confirmed four significant QTLs distributed among chromosome 1, 6, and 11, with the proportion of phenotypic variance explained by each QTL ranging from 2.96 to 11.11% and LOD scores from 1.67 to 3.75. Functional annotation of genes located within the QTL intervals revealed NUE putative candidate genes. Overall, the QTLs identified in this study contribute information that could be useful for NUE improvement in rice.

Genetics of extra-early-maturing yellow and orange quality protein maize inbreds and derived hybrids under low soil nitrogen and Striga infestation.

The development and commercialization of extra‐early quality protein maize (QPM)–provitamin A (PVA) hybrids that are tolerant of low soil N (LN) and Striga resistant are essential for addressing the food insecurity and undernourishment challenges currently faced by sub‐Saharan Africa (SSA). This study was designed (a) to determine the genetic effects regulating grain yield (GY) and important secondary traits of extra‐early yellow and orange QPM–PVA inbred lines under LN, Striga‐infested, and high‐N (HN) conditions, (b) to investigate whether maternal genes influenced the inheritance of GY and other secondary traits, (c) to assess the GY and stability of the hybrids across the three management conditions, and (d) to examine the relationship between single nucleotide polymorphism (SNP) marker‐based genetic distances and GY. Twenty‐four inbred lines were used to produce ninety‐six single cross hybrids using the North Carolina Design II. The performance of the hybrids plus four checks was assessed across LN, Striga‐infested, and HN management conditions in Ghana and Nigeria in 2018. Additive genetic variances were preponderant over nonadditive genetic variances for GY and most secondary traits in each and across environments. TZEEQI 358 exhibited significant and positive male and female GCA effects for GY under LN, Striga infestation, HN, and across management conditions indicating that favorable alleles for GY could be donated by TZEEQI 358. Maternal effects regulated the inheritance of plant height under the Striga‐infested conditions. Genetic distances were associated with GY under LN, Striga infestation, and HN conditions. TZEEIORQ 58 × TZEEQI 397 demonstrated high GY and stability of performance; therefore, it should be further tested under multiple environments for commercialization.

Mammalian herbivory in post-fire chaparral impacts herbaceous composition but not N and C cycling

Abstract Aims Classical theory predicts that herbivores impact herb assemblages and soil nitrogen (N) cycling through selective plant consumption and the deposition of N-rich waste, with effects dependent upon ecosystem N availability. Herbivores are predicted to accelerate N cycling when N availability is high and decelerate cycling when availability is low. However, experimental tests of these theories in natural systems are limited and have yielded contradictory results. California’s widespread chaparral shrublands provide a tractable system in which to test these theories. They are prone to periodic crown fire, which temporarily removes living shrub cover, deposits mineral N on soils and allows diverse herbaceous assemblages to dominate the landscape for 3–5 years. Chaparral is also increasingly vulnerable to herbaceous invasion; mammalian herbivory may limit the establishment of non-native herbs in the shrub understory. Methods We implemented a 2-year herbivore-exclosure experiment (Hopland, CA) to assess the impact of mammalian herbivory during early post-fire chaparral succession, both on herbaceous plant assemblages and soil N and C cycling. We predicted that, in high-N post-fire conditions, mammalian herbivory would not demonstrate a strong preference for N-fixing herbs, would accelerate N cycling and would reduce the abundance of non-native herbs. Important Findings Excluding mammalian herbivores increased herb standing biomass by 54%, but changed neither the relative abundance of N-fixing vs. non-N-fixing herbs nor any measure of N or C cycling. Herbivore impacts on nutrient cycling may not be significant over the 2-year time scale of the experiment and physical effects of herbivore activity could have counteracted the influence of plant litter and animal dung/urine inputs. Mammalian herbivores concentrated their feeding on typical non-native herbs, slightly decreasing their relative abundance; however, mammalian herbivory was not sufficient to stem the invasion of chaparral by invasive herbs or alter C and N cycling over the first 2 years after fire.

English

Field studies were conducted at the teaching and research farm of the Osun State University Ejigbo Campus, Osun State, Nigeria, during the 2015 and 2016 cropping seasons to determine the agronomic performance of low N maize varieties under two nitrogen conditions (low and high). The aim of this experiment was to determine the response of low N maize to two levels of nitrogen fertilizer. Ten low N maize varieties were used in this study. Low and high-N conditions of the soil was induced by application of urea fertilizer at the rate of 30 and 90kg ha-1 of nitrogen .The experiments were laid out in a randomized complete block design with three replications. High nitrogen application significantly improved maize vegetative growth, yield components and grain yield. The use of 90 kg ha-1 of N gave the highest maize plant height, and number of leaves per plant, as well as grain yields of 3.50 and 3.58 tha-1 was obtained with the application of 90 kg ha-1 of N in 2015 and 2016 cropping seasons, respectively. The result indicated that application rates of nitrogen (kg ha-1 of N) improved growth attributes of all the varieties with SINT MAR 20CA LARGA gave maximum grain yield that is not significantly different from 72PBPROLC3SYN. Key words: Plant height, agronomic performance, low N maize, nitrogen levels.

Effect of high-nitrogen fertilizer on gliadin and glutenin subproteomes during kernel development in wheat (Triticum aestivum L.)

Nitrogen (N), a macronutrient essential for plant growth and development, is needed for biosynthesis of protein and starch, which affect grain yield and quality. Application of high-N fertilizer increases plant growth, grain yield, and flour quality. In this study, we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE). Application of high-N fertilizer led to significant increases in gluten macropolymer content, total gliadin and glutenin content, and the accumulation of individual storage protein components. Of 126 differentially accumulated proteins (DAPs) induced by high-N conditions, 24 gliadins, 12 high-molecular-weight glutenins, and 27 low-molecular-weight glutenins were significantly upregulated. DAPs during five kernel developmental stages displayed multiple patterns of accumulation. In particular, gliadins and glutenins showed respectively five and six accumulation patterns. The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.

Inoculation with five diazotrophs alters nitrogen metabolism during the initial growth of sugarcane varieties with contrasting responses to added nitrogen

We examined the influence of inoculation with five species/strains of diazotrophic bacteria on the modulation of two enzymes involved in the assimilation of N and on the soluble N fractions in the sugarcane varieties RB867515 (adapted for low fertility soils) and IACSP95-5000 (adapted for medium to high fertility soils) under high- (3 mM) and low (0.3 mM)-N conditions in hydroponic cultivation for 59 days. The sugarcane plants were produced in three steps to obtain the hydroponic cultivation: the supply of 3 mM N for 30 days (first harvest), N depletion for 72 h (second harvest), and cultivation in high- and low-N conditions over 26 days (final harvest). Inoculation was performed by immersion of the minisetts in a diluted solution of five diazotrophic bacteria. After the final harvest, plants were divided into roots and shoots to assess their dry weight and N, P, and K accumulation. The variety played an important role in the interaction with diazotrophs, each showing distinct behavior in the activity of their N-assimilation enzymes. The nitrate reductase activity (NRa—EC 1.7.1.1) was increased in var. RB867515 by 26% in the shoots and by 48% in the roots after 72 h under N depletion, while var. IACSP95-5000 showed a reduced enzymatic activity in the roots (by 62%) but not in the shoots. Under high-N conditions, the inoculated IACSP95-5000 plants showed 31% higher glutamine synthetase activity (GSa—EC 6.31.2) compared with 19% in RB867515. Under low-N conditions, the GSas were 21% and 16% higher in the inoculated RB867515 and IACSP95-5000 plants, respectively, compared with that of the control. The content of nitrogen in the form of nitrate (N-nitrate) confirmed these varietal differences, but the soluble sugar content did not. The varieties utilized N sources differently, and inoculation modified the activity of two N-assimilation enzymes as well as the biomass accumulation, with the highest improvement seen in the low fertility-adapted variety RB867515; it showed a greater response to inoculation compared with that of the high fertility-adapted variety IACSP95-5000, with an increase in biomass and nutrient accumulation (N, P, K), especially when cultivated under low-N conditions. This suggests that the best response to inoculation with diazotrophs will be achieved using low fertility-adapted sugarcane varieties under low-N conditions.

Simultaneous Improvements of Pseudomonas Cell Growth and Polyhydroxyalkanoate Production from a Lignin Derivative for Lignin-Consolidated Bioprocessing.

Cell growth and polyhydroxyalkanoate (PHA) biosynthesis are two key traits in PHA production from lignin or its derivatives. However, the links between them remain poorly understood. Here, the transcription levels of key genes involved in PHA biosynthesis were tracked in Pseudomonas putida strain A514 grown on vanillic acid as the sole carbon source under different levels of nutrient availability. First, enoyl-coenzyme A (CoA) hydratase (encoded by phaJ4) is stress induced and likely to contribute to PHA synthesis under nitrogen starvation conditions. Second, much higher expression levels of 3-hydroxyacyl-acyl carrier protein (ACP) thioesterase (encoded by phaG) and long-chain fatty acid-CoA ligase (encoded by alkK) under both high and low nitrogen (N) led to the hypothesis that they likely not only have a role in PHA biosynthesis but are also essential to cell growth. Third, 40 mg/liter PHA was synthesized by strain AphaJ4C1 (overexpression of phaJ4 and phaC1 in strain A514) under low-N conditions, in contrast to 23 mg/liter PHA synthesized under high-N conditions. Under high-N conditions, strain AalkKphaGC1 (overexpression of phaG, alkK, and phaC1 in A514) produced 90 mg/liter PHA with a cell dry weight of 667 mg/liter, experimentally validating our hypothesis. Finally, further enhancement in cell growth (714 mg/liter) and PHA titer (246 mg/liter) was achieved in strain Axyl_alkKphaGC1 via transcription level optimization, which was regulated by an inducible strong promoter with its regulator, XylR-PxylA, from the xylose catabolic gene cluster of the A514 genome. This study reveals genetic features of genes involved in PHA synthesis from a lignin derivative and provides a novel strategy for rational engineering of these two traits, laying the foundation for lignin-consolidated bioprocessing.IMPORTANCE With the recent advances in processing carbohydrates in lignocellulosics for bioproducts, almost all biological conversion platforms result in the formation of a significant amount of lignin by-products, representing the second most abundant feedstock on earth. However, this resource is greatly underutilized due to its heterogeneity and recalcitrant chemical structure. Thus, exploiting lignin valorization routes would achieve the complete utilization of lignocellulosic biomass and improve cost-effectiveness. The culture conditions that encourage cell growth and polyhydroxyalkanoate (PHA) accumulation are different. Such an inconsistency represents a major hurdle in lignin-to-PHA bioconversion. In this study, we traced and compared transcription levels of key genes involved in PHA biosynthesis pathways in Pseudomonas putida A514 under different nitrogen concentrations to unveil the unusual features of PHA synthesis. Furthermore, an inducible strong promoter was identified. Thus, the molecular features and new genetic tools reveal a strategy to coenhance PHA production and cell growth from a lignin derivative.