Integration of nitrate and abscisic acid signaling in plants.

To meet the demands of the new Green Revolution and sustainable agriculture, it is important to develop crop varieties with improved yield, nitrogen use efficiency, and stress resistance. Nitrate is the major form of inorganic nitrogen available for plant growth in many well-aerated agricultural soils, and acts as a signaling molecule regulating plant development, growth, and stress responses. Abscisic acid (ABA), an important phytohormone, plays vital roles in integrating extrinsic and intrinsic responses and mediating plant growth and development in response to biotic and abiotic stresses. Therefore, elucidating the interplay between nitrate and ABA can contribute to crop breeding and sustainable agriculture. Here, we review studies that have investigated the interplay between nitrate and ABA in root growth modulation, nitrate and ABA transport processes, seed germination regulation, and drought responses. We also focus on nitrate and ABA interplay in several reported omics analyses with some important nodes in the crosstalk between nitrate and ABA. Through these insights, we proposed some research perspectives that could help to develop crop varieties adapted to a changing environment and to improve crop yield with high nitrogen use efficiency and strong stress resistance.

NRG2 family members of Arabidopsis and maize regulate nitrate signalling and promote nitrogen use efficiency.

Nitrogen (N) is an essential nutrient for plant growth, and most plants absorb it as nitrate. AtNRG2 has been reported to play an important role in nitrate regulation. In this study, we investigated the functions of AtNRG2 family members of Arabidopsis thaliana and maize in nitrate signalling and metabolism. Our results showed that both AtNRG2.10 and AtNRG2.15 regulated nitrate signalling and metabolism. Overexpression of AtNRG2.11 (AtNRG2) could promote plant growth and improve nitrogen use efficiency (NUE). In addition, the maize genome harbors 23 ZmNRG2 members. We detected the expression of these genes treated with nitrate and the expression of four genes was strongly induced with ZmNRG2.7 having the highest levels. Overexpression of ZmNRG2.7 in the atnrg2 mutant could restore the defects of atnrg2, suggesting that ZmNRG2.7 is involved in nitrate signalling and metabolism. Moreover, the overexpression lines of ZmNRG2.7 showed increased biomass and NUE. These findings demonstrate that at least a part of NRG2 family genes in Arabidopsis and maize regulate nitrate signalling and provide a molecular basis for improving the NUE of crops.

The Arabidopsis eIF4E1 regulates NRT1.1-mediated nitrate signaling at both translational and transcriptional levels.

Identifying new nitrate regulatory genes and illustrating their mechanisms in modulating nitrate signaling are of great significance for achieving the high yield and nitrogen use efficiency (NUE) of crops. Here, we screened a mutant with defects in nitrate response and mapped the mutation to the gene eIF4E1 in Arabidopsis. Our results showed that eIF4E1 regulated nitrate signaling and metabolism. Ribo-seq and polysome profiling analysis revealed that eIF4E1 modulated the amount of some nitrogen (N)-related mRNAs being translated, especially the mRNA of NRT1.1 was reduced in the eif4e1 mutant. RNA-Seq results enriched some N-related genes, supporting that eIF4E1 is involved in nitrate regulation. The genetic analysis indicated that eIF4E1 worked upstream of NRT1.1 in nitrate signaling. In addition, an eIF4E1-interacting protein GEMIN2 was identified and found to be involved in nitrate signaling. Further investigation showed that overexpression of eIF4E1 promoted plant growth and enhanced yield and NUE. These results demonstrate that eIF4E1 regulates nitrate signaling by modulating NRT1.1 at both translational and transcriptional levels, laying the foundation for future research on the regulation of mineral nutrition at the translational level.

Nitrate signaling and use efficiency in crops.

Nitrate (NO3-) is not only an essential nutrient but also an important signaling molecule for plant growth. Low nitrogen use efficiency (NUE) of crops is causing increasingly serious environmental and ecological problems. Understanding the molecular mechanisms of NO3- regulation in crops is crucial for NUE improvement in agriculture. During the last several years, significant progress has been made in understanding the regulation of NO3- signaling in crops, and some key NO3- signaling factors have been shown to play important roles in NO3- utilization. However, no detailed reviews have yet summarized these advances. Here, we focus mainly on recent advances in crop NO3- signaling, including short-term signaling, long-term signaling, and the impact of environmental factors. We also review the regulation of crop NUE by crucial genes involved in NO3- signaling. This review provides useful information for further research on NO3- signaling in crops and a theoretical basis for breeding new crop varieties with high NUE, which has great significance for sustainable agriculture.

Barley transcription factor HvNLP2 mediates nitrate signaling and affects nitrogen use efficiency.

Plants have evolved complex mechanisms to adapt to the changing nitrogen levels in the environment. In Arabidopsis, more than a dozen nitrate signaling regulatory genes have been characterized, including the NODULE INCEPTION-LIKE PROTEIN (AtNLP) genes, which play essential roles in nitrate signaling. However, whether NLP genes in the Triticeae crops are involved in nitrate regulation and nitrogen use efficiency (NUE) remains unknown. Here, we isolated a barley (Hordeum vulgare L.) mutant, hvnlp2-1, from a TILLING (Targeting Local Lesions IN Genomes) population and constructed two RNAi lines, hvnlp2-2 and hvnlp2-3, to study the function of HvNLP2. The expression of the nitrate-responsive genes was substantially inhibited after nitrate treatment in the hvnlp2 mutants, indicating that HvNLP2 controls nitrate signaling. Nitrate content was significantly higher in the hvnlp2 mutants, which may result from the decreased assimilation of nitrogen caused by reduced nitrate reductase activity and expression of nitrate assimilatory genes. HvNLP2 is localized to the nucleus in the presence of nitrate. Further investigation showed that HvNLP2 binds to and activates the nitrate-responsive cis-elements. Moreover, hvnlp2 exhibited reduced biomass, seed yield, and NUE. Therefore, HvNLP2 controls nitrate signaling and plays an important role in NUE.

Novel Aspects of Nitrate Regulation in Arabidopsis.

Nitrogen (N) is one of the most essential macronutrients for plant growth and development. Nitrate (NO3 -), the major form of N that plants uptake from the soil, acts as an important signaling molecule in addition to its nutritional function. Over the past decade, significant progress has been made in identifying new components involved in NO3 - regulation and starting to unravel the NO3 - regulatory network. Great reviews have been made recently by scientists on the key regulators in NO3 - signaling, NO3 - effects on plant development, and its crosstalk with phosphorus (P), potassium (K), hormones, and calcium signaling. However, several novel aspects of NO3 - regulation have not been previously reviewed in detail. Here, we mainly focused on the recent advances of post-transcriptional regulation and non-coding RNA (ncRNAs) in NO3 - signaling, and NO3 - regulation on leaf senescence and the circadian clock. It will help us to extend the general picture of NO3 - regulation and provide a basis for further exploration of NO3 - regulatory network.

The long noncoding RNA T5120 regulates nitrate response and assimilation in Arabidopsis.

Long noncoding RNAs (lncRNAs) are crucial regulators in many plant biological processes. However, it remains unknown whether lncRNAs can respond to nitrate or function in nitrate regulation. We detected 695 lncRNAs, 480 known and 215 novel, in Arabidopsis seedling roots; six showed altered expression in response to nitrate treatment, among which T5120 showed the highest induction. Overexpression of T5120 in Arabidopsis promoted the response to nitrate, enhanced nitrate assimilation and improved biomass and root development. Biochemical and molecular analyses revealed that NLP7, a master nitrate regulatory transcription factor, directly bound to the nitrate-responsive cis-element (NRE)-like motif of the T5120 promoter and activated T5120 transcription. In addition, T5120 partially restored the nitrate signalling and assimilation phenotypes of nlp7 mutant, suggesting that T5120 is involved in NLP7-mediated nitrate regulation. Interestingly, the expression of T5120 was regulated by the nitrate sensor NRT1.1. Therefore, T5120 is modulated by NLP7 and NRT1.1 to regulate nitrate signalling. Our work reveals a new regulatory mechanism in which lncRNA T5120 functions in nitrate regulation, providing new insights into the nitrate signalling network. Importantly, lncRNA T5120 can promote nitrate assimilation and plant growth to improve nitrogen use efficiency.

The study on pyrolysis of oil-based drilling cuttings by microwave and electric heating.

In this paper, the following questions were investigated: the proportion of mass loss, the mass fraction of oil, the structure, composition and ultimate analysis of solid residues and gas products. By comparing the treatment effect of using both microwave and electric as the source of heat to dispose the oil-based drilling cuttings (OBDC), the advantages of microwave heating treatment were demonstrated. Meanwhile, the composition of liquid products by microwave pyrolysis was analyzed. The results show that the microwave heating is better than electric heating and the former can promote the pyrolysis of petroleum hydrocarbons. The results of component analysis of the liquid products from OBDC by microwave pyrolysis show that C12∼C20 components pyrolyze at 500 °C. At the same time, a mass of C21∼C24 components volatilize. At the temperature above 500 °C, the thermal cracking reactions of >C25 components occur and a maximum content of paraffin in liquid products is obtained. As the temperature increases, the components obtained by pyrolysis become more and more complex.

The Arabidopsis NLP7 gene regulates nitrate signaling via NRT1.1-dependent pathway in the presence of ammonium.

Nitrate is not only an important nutrient but also a signaling molecule for plants. A few of key molecular components involved in primary nitrate responses have been identified mainly by forward and reverse genetics as well as systems biology, however, many underlining mechanisms of nitrate regulation remain unclear. In this study, we show that the expression of NRT1.1, which encodes a nitrate sensor and transporter (also known as CHL1 and NPF6.3), is modulated by NIN-like protein 7 (NLP7). Genetic and molecular analyses indicate that NLP7 works upstream of NRT1.1 in nitrate regulation when NH4+ is present, while in absence of NH4+, it functions in nitrate signaling independently of NRT1.1. Ectopic expression of NRT1.1 in nlp7 resulted in partial or complete restoration of nitrate signaling (expression from nitrate-regulated promoter NRP), nitrate content and nitrate reductase activity in the transgenic lines. Transcriptome analysis revealed that four nitrogen-related clusters including amino acid synthesis-related genes and members of NRT1/PTR family were modulated by both NLP7 and NRT1.1. In addition, ChIP and EMSA assays results indicated that NLP7 may bind to specific regions of the NRT1.1 promoter. Thus, NLP7 acts as an important factor in nitrate signaling via regulating NRT1.1 under NH4+ conditions.

Overexpression of the Maize ZmNLP6 and ZmNLP8 Can Complement the Arabidopsis Nitrate Regulatory Mutant nlp7 by Restoring Nitrate Signaling and Assimilation.

Nitrate is a key nutrient that affects maize growth and yield, and much has yet to be learned about nitrate regulatory genes and mechanisms in maize. Here, we identified nine ZmNLP genes in maize and analyzed the functions of two ZmNLP members in nitrate signaling. qPCR results revealed a broad pattern of expression for ZmNLP genes in different stages and organs with the highest levels of transcript expression of ZmNLP6 and ZmNLP8. When ZmNLP6 and ZmNLP8 were overexpressed in the Arabidopsis nitrate regulatory gene mutant nlp7-4, nitrate assimilation and induction of nitrate-responsive genes in the transgenic plants were recovered to WT levels, indicating that ZmNLP6 and ZmNLP8 can replace the essential roles of the master nitrate regulatory gene AtNLP7 in nitrate signaling and metabolism. ZmNLP6 and ZmNLP8 are localized in the nucleus and can bind candidate nitrate-responsive cis-elements in vitro. The biomass and yield of transgenic Arabidopsis lines overexpressing ZmNLP6 and ZmNLP8 showed significant increase compared with WT and nlp7-4 mutant line in low nitrate conditions. Thus, ZmNLP6 and ZmNLP8 regulate nitrate signaling in transgenic Arabidopsis plants and may be potential candidates for improving nitrogen use efficiency of maize.

The Arabidopsis CPSF30-L gene plays an essential role in nitrate signaling and regulates the nitrate transceptor gene NRT1.1.

Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitrogen availability. However, more underlying genes regulating the response to nitrate have yet to be characterized. We report here the identification of a nitrate regulatory mutant whose mutation mapped to the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant, induction of nitrate-responsive genes was inhibited independent of the ammonium conditions and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L. Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and independently of NLP7 in response to nitrate. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis revealed that four nitrogen-related clusters were enriched in the differentially expressed genes of the cpsf30 mutant. Nitrate uptake was decreased in the mutant along with reduced expression of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid content were enhanced in roots along with increased expression of several nitrate assimilatory genes. These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate signaling in part by regulating NRT1.1 expression, thus adding an important component to the nitrate signaling network.

Delayed germination of Arabidopsis seeds under chilling stress by overexpressing an abiotic stress inducible GhTPS11.

Trehalose-6-phosphate synthase (TPS) plays an important role in metabolic regulation and stress responses in a variety of organisms. However information about cotton TPS is poor. Here a cotton TPS gene GhTPS11 was isolated and characterized. Expression analysis revealed that GhTPS11 was induced in 20-day old cotton seedlings by heat drought and high salt stresses as well as GA and ABA. Moreover GhTPS11 was induced by chilling stress and mannitol while was depressed by sucrose. Tissue expression analysis indicated that GhTPS11 expressed higher in leaves than in stems and roots of 20-day old cotton seedlings. The GhTPS11 overexpressing Arabidopsis seeds germinated slower than the wild-type (WT) under chilling stress. Trehalose-6-phosphate (T6P) and trehalose contents were evidently higher in GhTPS11 overexpressing lines 3, 5, and 22 than in WT under normal germination condition as well as chilling stress. Further analysis demonstrated that the expression of ICE1 CBF3 and RCI2A was induced lower whereas that of CBF1 and CBF2 was induced higher under chilling stress in the GhTPS11 overexpressing seeds than WT respectively. These results suggested that GhTPS11 encoded a stress-responsive TPS protein and functioned in chilling stress during seed germination. Perhaps the chilling stress sensitivity of transgenic Arabidopsis seeds was caused by the expression changes of at least some chilling-related genes such as ICE1 CBFs and RCI2A other than HOS1. So this article provided the useful information for GhTPS11 usage for crop molecular breeding.

Overexpression of Late Embryogenesis Abundant 14 enhances Arabidopsis salt stress tolerance.

Late embryogenesis abundant (LEA) proteins are implicated in various abiotic stresses in higher plants. In this study, we identified a LEA protein from Arabidopsis thaliana, AtLEA14, which was ubiquitously expressed in different tissues and remarkably induced with increased duration of salt treatment. Subcellular distribution analysis demonstrated that AtLEA14 was mainly localized in the cytoplasm. Transgenic Arabidopsis and yeast overexpressing AtLEA14 all exhibited enhanced tolerance to high salinity. The transcripts of salt stress-responsive marker genes (COR15a, KIN1, RD29B and ERD10) were overactivated in AtLEA14 overexpressing lines compared with those in wild type plants under normal or salt stress conditions. In vivo and in vitro analysis showed that AtLEA14 could effectively stabilize AtPP2-B11, an important E3 ligase. These results suggested that AtLEA14 had important protective functions under salt stress conditions in Arabidopsis.

Identification and characterization of fructose 1,6-bisphosphate aldolase genes in Arabidopsis reveal a gene family with diverse responses to abiotic stresses.

Fructose 1,6-biphosphate aldolase (FBA) is a key enzyme in plants, which is involved not only in glycolysis and gluconeogenesis in the cytoplasm, but also in the Calvin cycle in plastids. Research on FBAs in various organisms has been reported, but there is none on FBAs in Arabidopsis at the molecular level. In the current study, eight FBA family genes (AtFBA1-8) were identified and analyzed in Arabidopsis thaliana. These genes have a highly conserved aldolase-type TIM barrel domain and a C-terminal peptide, but variable N-terminal peptides. Based on the phylogenetic analysis of FBA protein sequences from Arabidopsis and other plant species, AtFBA family was classified into two subfamilies, including three members (AtFBA1-3) with high similarities to FBAs occurring at plastid, and five (AtFBA4-8) with high similarities to FBAs localized in the cytoplasm. By confocal microscopy analysis with GFP fusion protein, AtFBA3 and AtFBA4 as well as AtFBA6 were observed to be localized in the plastid and cytoplasm, respectively. At least two duplicated gene pairs of AtFBA1 and AtFBA2, as well as AtFBA4 and AtFBA8 were found. Transcript level analysis of AtFBA genes in various tissues revealed the unique and overlapping expression patterns of plastid and cytosol AtFBA genes, suggesting that these genes may function at different stages of plant growth and development. Interestingly, AtFBA1, AtFBA2, AtFBA5 and AtFBA7 showed undetectable expression in roots. The expression patterns of AtFBA genes under different stress conditions suggested that all the members showed different expression patterns in response to stresses, including ABA, NaCl, Cd, abnormal temperature and drought, and, except for AtFBA3, most of the AtFBA genes were significantly responsive to drought stress in roots. Moreover, AtFBA1, AtFBA2, AtFBA5, AtFBA7 and AtFBA8 were induced by at least one of three sugars (sucrose, glucose and fructose) after 24h of treatment. Further functional analyses indicated important clues of AtFBA2, AtFBA6 and AtFBA8 in plant growth, stress responses and development, respectively. Thus these results provide additional knowledge on AtFBA families and their roles.

A DExD/H box RNA helicase is important for K+ deprivation responses and tolerance in Arabidopsis thaliana.

The molecular mechanism for sensing and transducing the stress signals initiated by K(+) deprivation in plants remains unknown. Here, we found that the expression of AtHELPS, an Arabidopsis DExD/H box RNA helicase gene, was induced by low-K(+), zeatin and cold treatments, and downregulated by high-K(+) stress. To further investigate the expression pattern of AtHELPS, pAtHELPS::GUS transgenic plants were generated. Histochemical staining indicated that AtHELPS is mainly expressed in the young seedlings and vascular tissues of leaves and roots. Using both helps mutants and overexpression lines, we observed that, in the low-K(+) condition, AtHELPS affected Arabidopsis seed germination and plant weight. Interestingly, the mRNA levels of AKT1, CBL1/9 and CIPK23 in the helps mutants were much higher than in the overexpression lines under low-K(+) stress. Moreover, under low-K(+) stress, the helps mutants displayed increased K(+) influx, whereas the overexpression line of AtHELPS had a lower flux rate in the roots by the noninvasive micro-test technique. Taken together, these results provide information for the functional analysis of plant DEVH box RNA helicases, and suggest that AtHELPS, as an important negative regulator, plays a role in K(+) deprivation stress.

NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco's defense response.

A cDNA library from tobacco inoculated with Rhizoctonia solani was constructed, and several cDNA fragments were identified by differential hybridization screening. One cDNA clone that was dramatically repressed, NtKTI1, was confirmed as a member of the Kunitz plant proteinase inhibitor family. RT-PCR analysis revealed that NtKTI1 was constitutively expressed throughout the whole plant and preferentially expressed in the roots and stems. Furthermore, RT-PCR analysis showed that NtKTI1 expression was repressed after R. solani inoculation, mechanical wounding and salicylic acid treatment, but was unaffected by methyl jasmonate, abscisic acid and NaCl treatment. In vitro assays showed that NtKTI1 exerted prominent antifungal activity towards R. solani and moderate antifungal activity against Rhizopus nigricans and Phytophthora parasitica var. nicotianae. Bioassays of transgenic tobacco demonstrated that overexpression of NtKTI1 enhanced significantly the resistance of tobacco against R. solani, and the antisense lines exhibited higher susceptibility than control lines towards the phytopathogen. Taken together, these studies suggest that NtKTI1 may be a functional Kunitz trypsin inhibitor with antifungal activity against several important phytopathogens in the tobacco defense response.