Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress.

Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

Evidence, causes, and consequences of declining nitrogen availability in terrestrial ecosystems.

The productivity of ecosystems and their capacity to support life depends on access to reactive nitrogen (N). Over the past century, humans have more than doubled the global supply of reactive N through industrial and agricultural activities. However, long-term records demonstrate that N availability is declining in many regions of the world. Reactive N inputs are not evenly distributed, and global changes-including elevated atmospheric carbon dioxide (CO2) levels and rising temperatures-are affecting ecosystem N supply relative to demand. Declining N availability is constraining primary productivity, contributing to lower leaf N concentrations, and reducing the quality of herbivore diets in many ecosystems. We outline the current state of knowledge about declining N availability and propose actions aimed at characterizing and responding to this emerging challenge.

Drought, Low Nitrogen Stress, and Ultraviolet-B Radiation Effects on Growth, Development, and Physiology of Sweetpotato Cultivars during Early Season.

Drought, ultraviolet-B (UV-B), and nitrogen stress are significant constraints for sweetpotato productivity. Their impact on plant growth and development can be acute, resulting in low productivity. Identifying phenotypes that govern stress tolerance in sweetpotatoes is highly desirable to develop elite cultivars with better yield. Ten sweetpotato cultivars were grown under nonstress (100% replacement of evapotranspiration (ET)), drought-stress (50% replacement of ET), UV-B (10 kJ), and low-nitrogen (20% LN) conditions. Various shoot and root morphological, physiological, and gas-exchange traits were measured at the early stage of the crop growth to assess its performance and association with the storage root number. All three stress factors caused significant changes in the physiological and root- and shoot-related traits. Drought stress reduced most shoot developmental traits (29%) to maintain root growth. UV-B stress increased the accumulation of plant pigments and decreased the photosynthetic rate. Low-nitrogen treatment decreased shoot growth (11%) and increased the root traits (18%). The highly stable and productive cultivars under all four treatments were identified using multitrait stability index analysis and weighted average of absolute scores (WAASB) analyses. Further, based on the total stress response indices, 'Evangeline', 'O'Henry', and 'Beauregard B-14' were identified as vigorous under drought; 'Evangeline', 'Orleans', and 'Covington' under UV-B; and 'Bonita', 'Orleans', and 'Beauregard B-14' cultivars showed greater tolerance to low nitrogen. The cultivars 'Vardaman' and 'NC05-198' recorded a low tolerance index across stress treatments. This information could help determine which plant phenotypes are desirable under stress treatment for better productivity. The cultivars identified as tolerant, sensitive, and well-adapted within and across stress treatments can be used as source materials for abiotic stress tolerance breeding programs.

Physiological Characteristics and Transcriptomic Dissection in Two Root Segments with Contrasting Net Fluxes of Ammonium and Nitrate of Poplar Under Low Nitrogen Availability.

To investigate physiological and transcriptomic regulation mechanisms underlying the distinct net fluxes of NH4+ and NO3- in different root segments of Populus species under low nitrogen (N) conditions, we used saplings of Populus × canescens supplied with either 500 (normal N) or 50 (low N) µM NH4NO3. The net fluxes of NH4+ and NO3-, the concentrations of NH4+, amino acids and organic acids and the enzymatic activities of nitrite reductase (NiR) and glutamine synthetase (GS) in root segment II (SII, 35-70 mm to the apex) were lower than those in root segment I (SI, 0-35 mm to the apex). The net NH4+ influxes and the concentrations of organic acids were elevated, whereas the concentrations of NH4+ and NO3- and the activities of NiR and GS were reduced in SI and SII in response to low N. A number of genes were significantly differentially expressed in SII vs SI and in both segments grown under low vs normal N conditions, and these genes were mainly involved in the transport of NH4+ and NO3-, N metabolism and adenosine triphosphate synthesis. Moreover, the hub gene coexpression networks were dissected and correlated with N physiological processes in SI and SII under normal and low N conditions. These results suggest that the hub gene coexpression networks play pivotal roles in regulating N uptake and assimilation, amino acid metabolism and the levels of organic acids from the tricarboxylic acid cycle in the two root segments of poplars in acclimation to low N availability.

The association between Hfq and RNase E in long-term nitrogen-starved Escherichia coli.

Under conditions of nutrient adversity, bacteria adjust metabolism to minimize cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualized together in live bacteria. We now show that in long-term nitrogen starved E. coli, both RNase E and Hfq co-localize in a single, large focus. This subcellular assembly, which we refer to as the H-body, forms by a liquid-liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of a hitherto unreported subcellular compartmentalization of a process(s) associated with RNA management in stressed bacteria.

Nitrogen deficiency results in changes to cell wall composition of sorghum seedlings.

Sorghum [Sorghum bicolor (L.) Moench] has been gaining attention as a feedstock for biomass energy production. While it is obvious that nitrogen (N) supply significantly affects sorghum growth and biomass accumulation, our knowledge is still limited regarding the effect of N on the biomass quality of sorghum, such as the contents and structures of lignin and other cell wall components. Therefore, in this study, we investigated the effects of N supply on the structure and composition of sorghum cell walls. The cell walls of hydroponically cultured sorghum seedlings grown under sufficient or deficient N conditions were analyzed using chemical, two-dimensional nuclear magnetic resonance, gene expression, and immunohistochemical methods. We found that the level of N supply considerably affected the cell wall structure and composition of sorghum seedlings. Limitation of N led to a decrease in the syringyl/guaiacyl lignin unit ratio and an increase in the amount and alteration of tissue distribution of several hemicelluloses, including mixed linkage (1 → 3), (1 → 4)-ß-D-glucan, and arabinoxylan. At least some of these cell wall alterations could be associated with changes in gene expression. Nitrogen status is thus one of the factors affecting the cell wall properties of sorghum seedlings.

The Overexpression of NUC Promotes Development and Increases Resistance to Nitrogen Deficiency in Arabidopsis thaliana.

NUTCRACKER (NUC) is a transcription factor expressed in multiple tissues, but little is known about its physiological roles. In this study, we explored the physiological function of NUC with the Arabidopsis knockout, rescue, and overexpression lines. We found that NUC overexpression promoted development at the germination, seedling, and juvenile stages. NUC overexpression increased resistance to nitrogen (N) deficiency stress by increasing the chlorophyll content, suppressing anthocyanin accumulation, and increasing the biomass under N deficiency. In contrast, the absence of NUC did not affect such characteristics. N deficiency significantly increased the expression of NUC in leaves but did not affect the expression of NUC in roots. The overexpression of NUC promoted primary root length under both normal and N deficiency conditions. Furthermore, we found that the N-responsive and lateral-root-related genes TGA1 and NRT2.4 had NUC-binding sites in their promoter regions and that their expression was upregulated by NUC under N deficiency. The overexpression of the NUC increased the number and length of the lateral roots under N deficiency through inducible promotion. Multiple lines of investigation suggest that the regulatory function of the NUC could be bypassed through its redundant MAGPIE (MGP) when the NUC is absent. Our findings provide novel insight into NUC's functions and will assist efforts to improve plants' development and resistance to nutrient stresses.

Dynamic transcriptome and co-expression analysis suggest the potential roles of small secreted peptides from Tartary buckwheat (Fagopyrum tataricum) in low nitrogen stress response.

Small secreted peptides (SSPs) regulate nitrogen (N) response and signaling in plants. Although much progress has been made in understanding the functions of SSPs in N response, very little information is available regarding non-model plants. Tartary buckwheat (Fagopyrum tataricum), a dicotyledonous crop, has a good adaptability to low N (LN) stress; however, little is known regarding the associated mechanisms underlying this adaptation. In this study, 932 putative SSPs were genome-wide characterized in TB genome. Of these SSPs, 233 SSPs were annotated as established SSPs, such as CLE, RALF, PSK, and CEP peptides. The gene expression of 675 putative SSPs was detected in five tissues and 258 SSPs were tissue-specific expressed genes. To analyze the responses of TB SSPs to LN, the dynamic expression analysis of TB roots under LN stress was conducted by RNA-seq. The expression of 378 putative TB SSP genes was detected with diverse expression patterns under LN stress, and some important LN-responsive SSPs were identified. Co-expression analysis suggested SSPs may regulate the adaptability of TB under LN conditions by modulating the expression of the genes involved in N transport and assimilation and IAA signaling. Furthermore, 53 LN stress-responsive RLKs encoding genes were identified and they were predicted as potential SSP receptors. This study expands the repertoire of SSPs in plants and provides useful information for further investigation of the functions of Tartary buckwheat SSPs in LN stress responses.

The GC-TOF/MS-based Metabolomic analysis reveals altered metabolic profiles in nitrogen-deficient leaves and roots of tea plants (Camellia sinensis).

BACKGROUND: Nitrogen (N) fertilizer is commonly considered as one of the most important limiting factors in the agricultural production. As a result, a large amount of N fertilizer is used to improve the yield in modern tea production. Unfortunately, the large amount of N fertilizer input has led to increased plant nitrogen-tolerance and decreased amplitude of yield improvement, which results in significant N loss, energy waste and environment pollution. However, the effects of N-deficiency on the metabolic profiles of tea leaves and roots are not well understood. RESULTS: In this study, seedlings of Camellia sinensis (L.) O. Kuntze Chunlv 2 were treated with 3 mM NH4NO3 (Control) or without NH4NO3 (N-deficiency) for 4 months by sandy culture. The results suggested that N-deficiency induced tea leaf chlorosis, impaired biomass accumulation, decreased the leaf chlorophyll content and N absorption when they were compared to the Control samples. The untargeted metabolomics based on GC-TOF/MS approach revealed a discrimination of the metabolic profiles between N-deficient tea leaves and roots. The identification and classification of the altered metabolites indicated that N deficiency upregulated the relative abundances of most phenylpropanoids and organic acids, while downregulated the relative abundances of most amino acids in tea leaves. Differentially, N-deficiency induced the accumulation of most carbohydrates, organic acids and amino acids in tea roots. The potential biomarkers screened in N-deficient leaves compared to Control implied that N deficiency might reduce the tea quality. Unlike the N-deficient leaves, the potential biomarkers in N-deficient roots indicated an improved stress response might occur in tea roots. CONCLUSIONS: The results demonstrated N deficiency had different effects on the primary and secondary metabolism in tea leaves and roots. The findings of this study will facilitate a comprehensive understanding of the N-deficient tea plants and provide a valuable reference for the optimized N nutrient management and the sustainable development in the tea plantations.

Integration of the metabolome and transcriptome reveals the mechanism of resistance to low nitrogen supply in wild bermudagrass (Cynodon dactylon (L.) Pers.) roots.

BACKGROUND: Nitrogen (N) is an essential macronutrient that significantly affects turf quality. Commercial cultivars of bermudagrass (Cynodon dactylon (L.) Pers.) require large amounts of nitrogenous fertilizer. Wild bermudagrass germplasm from natural habitats with poor nutrition and diverse N distributions is an important source for low-N-tolerant cultivated bermudagrass breeding. However, the mechanisms underlying the differences in N utilization among wild germplasm resources of bermudagrass are not clear. RESULTS: To clarify the low N tolerance mechanism in wild bermudagrass germplasm, the growth, physiology, metabolome and transcriptome of two wild accessions, C291 (low-N-tolerant) and C716 (low-N-sensitive), were investigated. The results showed that root growth was less inhibited in low-N-tolerant C291 than in low-N-sensitive C716 under low N conditions; the root dry weight, soluble protein content and free amino acid content of C291 did not differ from those of the control, while those of C716 were significantly decreased. Down-regulation of N acquisition, primary N assimilation and amino acid biosynthesis was less pronounced in C291 than in C716 under low N conditions; glycolysis and the tricarboxylic acid (TCA) cycle pathway were also down-regulated, accompanied by a decrease in the biosynthesis of amino acids; strikingly, processes such as translation, biosynthesis of the structural constituent of ribosome, and the expression of individual aminoacyl-tRNA synthetase genes, most of genes associated with ribosomes related to protein synthesis were all up-regulated in C291, but down-regulated in C716. CONCLUSIONS: Overall, low-N-tolerant wild bermudagrass tolerated low N nutrition by reducing N primary assimilation and amino acid biosynthesis, while promoting the root protein synthesis process and thereby maintaining root N status and normal growth.

Local auxin biosynthesis acts downstream of brassinosteroids to trigger root foraging for nitrogen.

Lateral roots (LRs) dominate the overall root surface of adult plants and are crucial for soil exploration and nutrient acquisition. When grown under mild nitrogen (N) deficiency, flowering plants develop longer LRs to enhance nutrient acquisition. This response is partly mediated by brassinosteroids (BR) and yet unknown mechanisms. Here, we show that local auxin biosynthesis modulates LR elongation while allelic coding variants of YUCCA8 determine the extent of elongation under N deficiency. By up-regulating the expression of YUCCA8/3/5/7 and of Tryptophan Aminotransferase of Arabidopsis 1 (TAA1) under mild N deficiency auxin accumulation increases in LR tips. We further demonstrate that N-dependent auxin biosynthesis in LRs acts epistatic to and downstream of a canonical BR signaling cascade. The uncovered BR-auxin hormonal module and its allelic variants emphasize the importance of fine-tuning hormonal crosstalk to boost adaptive root responses to N availability and offer a path to improve soil exploration by expanded root systems in plants.

RNA-Seq, physiological, and biochemical analysis of burley tobacco response to nitrogen deficiency.

To explore the effects of nitrogen deficiency in burley tobacco, two varieties were cultivated and subjected to conditions of sufficient and deficient nitrogen. The natural characteristics of varieties TN90 and TN86 during tobacco cultivation were similar for nitrogen metabolism. Both carbon and nitrogen metabolism were significantly affected by reducing amounts of applied nitrogen. Under nitrogen-deficient conditions, average leaf biomass, root weight, photosynthetic rate (Pn), pigment levels, total nitrogen, and nitrate content of TN86 and TN90 were significantly decreased by 52.88%, 69.19%, 22.65%, 46.80%, 37.42%, and 79.15%, respectively (p < 0.01). Nicotine and soluble reducing sugar contents were significantly decreased by 96.67% and 95.12%, respectively, in TN86 roots (p < 0.01), which was consistent with the reductions in root surf area, average diameter, and root volume. Nitrogen deficiency induced 6318 differentially expressed genes in both TN90 and TN86, which were highly expressed. In total, 428 upregulated genes were analysed and found to be mainly enriched in the MAPK signalling pathway, sesquiterpenoid and triterpenoid biosynthesis, and arginine and proline metabolism. Meanwhile, 213 downregulated genes were analysed and found to be mainly enriched in photosynthesis, nitrogen metabolism, and amino acid biosynthesis. Reduced pigment content and Pn may result in low carbohydrate formation and decreased leaf biomass in burley tobacco under nitrogen-deficient conditions.

Mesophyll conductance, photoprotective process and optimal N partitioning are essential to the maintenance of photosynthesis at N deficient condition in a wheat yellow-green mutant (Triticum aestivum L.).

The major effect of nitrogen (N) deficiency is the inhibition on CO2 assimilation regulated by light energy absorption, transport and conversion, as well as N allocation. In this study, a yellow-green wheat mutant (Jimai5265yg) and its wild type (Jimai5265, WT) were compared between 0 mM N (N0) and 14 mM N (N14) treatments using hydroponic experiments. The mutant exhibited higher photosynthetic efficiency (An) than WT despite low chlorophyll (Chl) content in non-stressed conditions. The photosynthetic advantages of the mutant were maintained under N deficient condition. The quantitative analysis of limitations to photosynthesis revealed that CO2 diffusion associated with mesophyll conductance (gm) was the dominant limitation. Relative easiness to gain CO2 in the chloroplast contributed to the higher An of Jimai5265yg. N deficiency induced the photoinhibition of PSII, but the cyclic electron transport and photochemical activity of PSI was higher in Jimai5265yg compared to Jimai5265, which was a protective mechanism to avoid photodamage. Because of the sharp drop of An, N deficient seedlings had much lower photosynthetic N use efficiency (PNUE). However, N deficiency increased the relative content of photosynthetic N (Npsn) and decreased the relative content of storage N (Nstore). The range of change in N partitioning induced by N deficiency was smaller for Jimai5265yg compared to WT. The less insensitive to N deficiency for the mutant in terms of photosynthetic property and N partitioning suggested that gm, cyclic electron transport around PSI and more optimal N partitioning pattern is necessary to sustain photosynthesis under N deficient condition.

Integrative Transcriptomic and Proteomic Analysis Reveals an Alternative Molecular Network of Glutamine Synthetase 2 Corresponding to Nitrogen Deficiency in Rice (Oryza sativa L.).

Nitrogen (N) is an essential nutrient for plant growth and development. The root system architecture is a highly regulated morphological system, which is sensitive to the availability of nutrients, such as N. Phenotypic characterization of roots from LY9348 (a rice variety with high nitrogen use efficiency (NUE)) treated with 0.725 mM NH4NO3 (1/4N) was remarkable, especially primary root (PR) elongation, which was the highest. A comprehensive analysis was performed for transcriptome and proteome profiling of LY9348 roots between 1/4N and 2.9 mM NH4NO3 (1N) treatments. The results indicated 3908 differential expression genes (DEGs; 2569 upregulated and 1339 downregulated) and 411 differential abundance proteins (DAPs; 192 upregulated and 219 downregulated). Among all DAPs in the proteome, glutamine synthetase (GS2), a chloroplastic ammonium assimilation protein, was the most upregulated protein identified. The unexpected concentration of GS2 from the shoot to the root in the 1/4N treatment indicated that the presence of an alternative pathway of N assimilation regulated by GS2 in LY9348 corresponded to the low N signal, which was supported by GS enzyme activity and glutamine/glutamate (Gln/Glu) contents analysis. In addition, N transporters (NRT2.1, NRT2.2, NRT2.3, NRT2.4, NAR2.1, AMT1.3, AMT1.2, and putative AMT3.3) and N assimilators (NR2, GS1;1, GS1;2, GS1;3, NADH-GOGAT2, and AS2) were significantly induced during the long-term N-deficiency response at the transcription level (14 days). Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that phenylpropanoid biosynthesis and glutathione metabolism were significantly modulated by N deficiency. Notably, many transcription factors and plant hormones were found to participate in root morphological adaptation. In conclusion, our study provides valuable information to further understand the response of rice roots to N-deficiency stress.

The Expression Characteristics of NPF Genes and Their Response to Vernalization and Nitrogen Deficiency in Rapeseed.

The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY (NPF) genes, initially characterized as nitrate or peptide transporters in plants, are involved in the transport of a large variety of substrates, including amino acids, nitrate, auxin (IAA), jasmonates (JAs), abscisic acid (ABA) and gibberellins (GAs) and glucosinolates. A total of 169 potential functional NPF genes were excavated in Brassica napus, and they showed diversified expression patterns in 90 different organs or tissues based on transcriptome profile data. The complex time-serial expression changes were found for most functional NPF genes in the development process of leaves, silique walls and seeds, which indicated that the expression of Brassica napus NPF (BnaNPF) genes may respond to altered phytohormone and secondary metabolite content through combining with promoter element enrichment analysis. Furthermore, many BnaNPF genes were detected to respond to vernalization with two different patterns, and 20 BnaNPF genes responded to nitrate deficiency. These results will provide useful information for further investigation of the biological function of BnaNPF genes for growth and development in rapeseed.

Overexpression of differentially expressed AhCytb6 gene during plant-microbe interaction improves tolerance to N2 deficit and salt stress in transgenic tobacco.

Stenotrophomonas maltophilia has plant growth-promoting potential, and interaction with Arachis hypogaea changes host-plant physiology, biochemistry, and metabolomics, which provides tolerance under the N2 starvation conditions. About 226 suppression subtractive hybridization clones were obtained from plant-microbe interaction, of which, about 62% of gene sequences were uncharacterized, whereas 23% of sequences were involved in photosynthesis. An uncharacterized SSH clone, SM409 (full-length sequence showed resemblance with Cytb6), showed about 4-fold upregulation during the interaction was transformed to tobacco for functional validation. Overexpression of the AhCytb6 gene enhanced the seed germination efficiency and plant growth under N2 deficit and salt stress conditions compared to wild-type and vector control plants. Results confirmed that transgenic lines maintained high photosynthesis and protected plants from reactive oxygen species buildup during stress conditions. Microarray-based whole-transcript expression of host plants showed that out of 272,410 genes, 8704 and 24,409 genes were significantly (p < 0.05) differentially expressed (> 2 up or down-regulated) under N2 starvation and salt stress conditions, respectively. The differentially expressed genes belonged to different regulatory pathways. Overall, results suggested that overexpression of AhCytb6 regulates the expression of various genes to enhance plant growth under N2 deficit and abiotic stress conditions by modulating plant physiology.

Mitochondrial dynamics and degradation in the oleaginous yeast Lipomyces starkeyi.

Emerging evidence implicates the vital role of mitochondria in lipid consumption and storage, highlighting the intimate link between energy production and saving. Although formation of giant lipid droplets, which is the key hallmark of the oleaginous yeast Lipomyces starkeyi, appears to be regulated in response to changes in mitochondrial shape and metabolism, technical limitations of genetic manipulation have become an obstacle to uncover the mitochondrial behavior in this nonconventional yeast. Here, we established an L. starkeyi strain stably expressing a fluorescent marker for monitoring mitochondrial morphology and degradation and found that mitochondria are mostly fragmented in L. starkeyi cells under fermentable, nonfermentable, and nitrogen depletion conditions. Notably, a fraction of mitochondria-specific fluorescent signals was localized to the vacuole, a lytic organelle in yeast, indicating degradation of mitochondria in those cells. This possible catabolic event was more predominant in cells under nutrient-poor conditions than that in cells under nutrient-rich conditions, concomitantly with lipid droplet formation. Collectively, our studies provide a new tool to investigate mitochondrial dynamics in L. starkeyi and decipher the potential role of mitochondrial degradation in lipid metabolism.

Role of bundle sheath conductance in sustaining photosynthesis competence in sugarcane plants under nitrogen deficiency.

The role of bundle sheath conductance (gbs) in sustaining sugarcane photosynthesis under nitrogen deficiency was investigated. Sugarcane was grown under different levels of nitrogen supply and gbs was estimated using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence at 21% or 2% [O2] and varying air [CO2] and light intensity. Maximum rates of PEPC carboxylation, Rubisco carboxylation, and ATP production increased with an increase in leaf nitrogen concentration (LNC) from 1 to 3 g m-2. Low nitrogen supply reduced Rubisco and PEPC abundancies, the quantum efficiency of CO2 assimilation and gbs. Because of reduced gbs, low photosynthetic rates were not associated with increased leakiness under nitrogen deficiency. In fact, low nitrogen supply increased bundle sheath cell wall thickness, probably accounting for low gbs and increased estimates of [CO2] at Rubisco sites. Effects of nitrogen on expression of ShPIP2;1 and ShPIP1;2 aquaporins did not explain changes in gbs. Our data revealed that reduced Rubisco carboxylation was the main factor causing low sugarcane photosynthesis at low nitrogen supply, in contrast to the previous report on the importance of an impaired CO2 concentration mechanism under N deficiency. Our findings suggest higher investment of nitrogen into Rubisco protein would favour photosynthesis and plant performance under low nitrogen availability.

Xanthine-derived metabolites enhance chlorophyll degradation in cotyledons and seedling growth during nitrogen deficient condition in Brassica rapa.

Nitrogen (N) deficiency is a main environmental factor that induces early senescence. Cotyledons provide an important N source during germination and early seedling development. In this study, we observed that N deficient condition enhanced gene expression involved in purine catabolism in cotyledons of Chinese cabbage (Brassica rapa ssp. Pekinensis). Seedlings grown with added allopurinol, an inhibitor of xanthine dehydrogenase, in the growth medium showed reduced chlorophyll degradation in cotyledons and lower fresh weight, compared with seedlings grown on normal medium. On the basis of these results, we speculated that xanthine-derived metabolites might affect both seedling growth and early senescence in cotyledons. To confirm this, seedlings were grown with exogenous xanthine to analyze the role of xanthine-derived metabolites under N deficient condition. Seedlings with xanthine as the sole N-source grew faster, and more cotyledon chlorophyll was broken down, compared with seedlings grown without xanthine. The expression levels of senescence- and purine metabolism-related genes in cotyledons were higher than those in seedlings grown without xanthine. These results indicate the possibility that xanthine plays a role as an activator in both purine catabolism and chlorophyll degradation in cotyledons under N deficient condition.

Expression of potential reference genes in response to macronutrient stress in rice and soybean.

Given the complexity of nutrient stress responses and the availability of a few validated reference genes, we aimed to identify robust and stable reference genes for macronutrient stress in rice and soybean. Ten potential reference genes were evaluated using geNorm, NormFinder, BestKeeper, Comparative ΔCt method, and RefFinder algorithms under low and completely starved conditions of nitrogen (N), phosphorus (P), potassium (K), and sulphur (S). Results revealed distinct sets of reference gene pairs, showing stable expression under different experimental conditions. The gene pairs TIP41/UBC(9/10/18) and F-box/UBC10 were most stable in rice and soybean, respectively under N stress. Under P stress, UBC9/UBC10 in rice and F-Box/UBC10 in soybean were most stable. Similarly, TIP41/UBC10 in rice and RING FINGER/UBC9 in soybean were the best gene pairs under K stress while F-Box/TIP41 in rice and UBC9/UBC10 in soybean were the most stable gene pairs under S stress. These reference gene pairs were validated by quantifying the expression levels of high-affinity transporters like NRT2.1/NRT2.5, PT1, AKT1, and SULTR1 for N, P, K, and S stress, respectively. This study reiterates the importance of choosing reference genes based on crop species and the experimental conditions, in order to obtain concrete answers to missing links of gene regulation in response to macronutrient deficiencies.