Nitrogen-dependent binding of the transcription factor PBF1 contributes to the balance of protein and carbohydrate storage in maize endosperm.

Fluctuations in nitrogen (N) availability influence protein and starch levels in maize (Zea mays) seeds, yet the underlying mechanism is not well understood. Here, we report that N limitation impacted the expression of many key genes in N and carbon (C) metabolism in the developing endosperm of maize. Notably, the promoter regions of those genes were enriched for P-box sequences, the binding motif of the transcription factor prolamin-box binding factor 1 (PBF1). Loss of PBF1 altered accumulation of starch and proteins in endosperm. Under different N conditions, PBF1 protein levels remained stable but PBF1 bound different sets of target genes, especially genes related to the biosynthesis and accumulation of N and C storage products. Upon N-starvation, the absence of PBF1 from the promoters of some zein genes coincided with their reduced expression, suggesting that PBF1 promotes zein accumulation in the endosperm. In addition, PBF1 repressed the expression of sugary1 (Su1) and starch branching enzyme 2b (Sbe2b) under normal N supply, suggesting that, under N-deficiency, PBF1 redirects the flow of C skeletons for zein toward the formation of C compounds. Overall, our study demonstrates that PBF1 modulates C and N metabolism during endosperm development in an N-dependent manner.

Transcription factor ZmEREB97 regulates nitrate uptake in maize (Zea mays) roots.

Maize (Zea mays L.) has very strong requirements for nitrogen. However, the molecular mechanisms underlying the regulations of nitrogen uptake and translocation in this species are not fully understood. Here, we report that an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ZmEREB97 functions as an important regulator in the N signaling network in maize. Predominantly expressed and accumulated in main root and lateral root primordia, ZmEREB97 rapidly responded to nitrate treatment. By overlapping the analyses of differentially expressed genes and conducting a DAP-seq assay, we identified 1,446 potential target genes of ZmEREB97. Among these, 764 genes were coregulated in 2 lines of zmereb97 mutants. Loss of function of ZmEREB97 substantially weakened plant growth under both hydroponic and soil conditions. Physiological characterization of zmereb97 mutant plants demonstrated that reduced biomass and grain yield were both associated with reduced nitrate influx, decreased nitrate content, and less N accumulation. We further demonstrated that ZmEREB97 directly targets and regulates the expression of 6 ZmNRT genes by binding to the GCC-box-related sequences in gene promoters. Collectively, these data suggest that ZmEREB97 is a major positive regulator of the nitrate response and that it plays an important role in optimizing nitrate uptake, offering a target for improvement of nitrogen use efficiency in crops.

Expansion and improvement of ChinaMu by MuT-seq and chromosome-level assembly of the Mu-starter genome.

ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F1 families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.

The NIN-like protein 5 (ZmNLP5) transcription factor is involved in modulating the nitrogen response in maize.

Maize exhibits marked growth and yield response to supplemental nitrogen (N). Here, we report the functional characterization of a maize NIN-like protein ZmNLP5 as a central hub in a molecular network associated with N metabolism. Predominantly expressed and accumulated in roots and vascular tissues, ZmNLP5 was shown to rapidly respond to nitrate treatment. Under limited N supply, compared with that of wild-type (WT) seedlings, the zmnlp5 mutant seedlings accumulated less nitrate and nitrite in the root tissues and ammonium in the shoot tissues. The zmnlp5 mutant plants accumulated less nitrogen than the WT plants in the ear leaves and seed kernels. Furthermore, the mutants carrying the transgenic ZmNLP5 cDNA fragment significantly increased the nitrate content in the root tissues compared with that of the zmnlp5 mutants. In the zmnlp5 mutant plants, loss of the ZmNLP5 function led to changes in expression for a significant number of genes involved in N signalling and metabolism. We further show that ZmNLP5 directly regulates the expression of nitrite reductase 1.1 (ZmNIR1.1) by binding to the nitrate-responsive cis-element at the 5' UTR of the gene. Interestingly, a natural loss-of-function allele of ZmNLP5 in Mo17 conferred less N accumulation in the ear leaves and seed kernels resembling that of the zmnlp5 mutant plants. Our findings show that ZmNLP5 is involved in mediating the plant response to N in maize.

High-resolution profile of transcriptomes reveals a role of alternative splicing for modulating response to nitrogen in maize.

BACKGROUND: The fluctuation of nitrogen (N) contents profoundly affects the root growth and architecture in maize by altering the expression of thousands of genes. The differentially expressed genes (DEGs) in response to N have been extensively reported. However, information about the effects of N variation on the alternative splicing in genes is limited. RESULTS: To reveal the effects of N on the transcriptome comprehensively, we studied the N-starved roots of B73 in response to nitrate treatment, using a combination of short-read sequencing (RNA-seq) and long-read sequencing (PacBio-sequencing) techniques. Samples were collected before and 30 min after nitrate supply. RNA-seq analysis revealed that the DEGs in response to N treatment were mainly associated with N metabolism and signal transduction. In addition, we developed a workflow that utilizes the RNA-seq data to improve the quality of long reads, increasing the number of high-quality long reads to about 2.5 times. Using this workflow, we identified thousands of novel isoforms; most of them encoded the known functional domains and were supported by the RNA-seq data. Moreover, we found more than 1000 genes that experienced AS events specifically in the N-treated samples, most of them were not differentially expressed after nitrate supply-these genes mainly related to immunity, molecular modification, and transportation. Notably, we found a transcription factor ZmNLP6, a homolog of AtNLP7-a well-known regulator for N-response and root growth-generates several isoforms varied in capacities of activating downstream targets specifically after nitrate supply. We found that one of its isoforms has an increased ability to activate downstream genes. Overlaying DEGs and DAP-seq results revealed that many putative targets of ZmNLP6 are involved in regulating N metabolism, suggesting the involvement of ZmNLP6 in the N-response. CONCLUSIONS: Our study shows that many genes, including the transcription factor ZmNLP6, are involved in modulating early N-responses in maize through the mechanism of AS rather than altering the transcriptional abundance. Thus, AS plays an important role in maize to adapt N fluctuation.

WIT120 data mining technology based on internet of things.

In recent years, with the increasing aging of society, the number of patients with chronic heart disease, hypertension and diabetes has increased dramatically. It has guiding significance for the prevention and treatment by long-term monitoring of the physiological signs of patients with chronic diseases, scoring statistical data, and predicting the development trend of users' health. The work used the data collected by WIT120 system to analyze the pre-processed thick data based on adaptive k-means clustering method under the MapReduce framework, and the GM (1,1) grey model was used to predict the future health status of users. The simulation results have verified the effectiveness of the proposed algorithm.

Cross-Species Root Transcriptional Network Analysis Highlights Conserved Modules in Response to Nitrate between Maize and Sorghum.

Plants have evolved complex mechanisms to respond to the fluctuation of available nitrogen (N) in soil, but the genetic mechanisms underlying the N response in crops are not well-documented. In this study, we generated a time series of NO3--mediated transcriptional profiles in roots of maize and sorghum, respectively. Using weighted gene co-expression network analysis, we identified modules of co-expressed genes that related to NO3- treatments. A cross-species comparison revealed 22 conserved modules, of which four were related to hormone signaling, suggesting that hormones participate in the early nitrate response. Three other modules are composed of genes that are mainly upregulated by NO3- and involved in nitrogen and carbohydrate metabolism, including NRT, NIR, NIA, FNR, and G6PD2. Two G2-like transcription factors (ZmNIGT1 and SbNIGT1), induced by NO3- stimulation, were identified as hub transcription factors (TFs) in the modules. Transient assays demonstrated that ZmNIGT1 and SbNIGT1 are transcriptional repressors. We identified the target genes of ZmNIGT1 by DNA affinity-purification sequencing (DAP-Seq) and found that they were significantly enriched in catalytic activity, including carbon, nitrogen, and other nutrient metabolism. A set of ZmNIGT1 targets encode transcription factors (ERF, ARF, and AGL) that are involved in hormone signaling and root development. We propose that ZmNIGT1 and SbNIGT1 are negative regulators of nitrate responses that play an important role in optimizing nutrition metabolism and root morphogenesis. Together with conserved N responsive modules, our study indicated that, to encounter N variation in soil, maize and sorghum have evolved an NO3--regulatory network containing a set of conserved modules and transcription factors.

Temperature-dependent autoimmunity mediated by chs1 requires its neighboring TNL gene SOC3.

Toll/interleukin receptor (TIR)-nucleotide binding site (NB)-type (TN) proteins are encoded by a family of 21 genes in the Arabidopsis genome. Previous studies have shown that a mutation in the TN gene CHS1 activates the activation of defense responses at low temperatures. However, the underlying molecular mechanism remains unknown. To genetically dissect chs1-mediated signaling, we isolated genetic suppressors of chs1-2 (soc). Several independent soc mutants carried mutations in the same TIR-NB-leucine-rich repeat (LRR) (TNL)-encoding gene SOC3, which is adjacent to CHS1 on chromosome 1. Expression of SOC3 was upregulated in the chs1-2 mutant. Mutations in six soc3 alleles and downregulation of SOC3 by an artificial microRNA construct fully rescued the chilling sensitivity and defense defects of chs1-2. Biochemical studies showed that CHS1 interacted with the NB and LRR domains of SOC3; however, mutated chs1 interacted with the TIR, NB and LRR domains of SOC3 in vitro and in vivo. This study reveals that the TN protein CHS1 interacts with the TNL protein SOC3 to modulate temperature-dependent autoimmunity.

Enhanced lipid accumulation of photoautotrophic microalgae by high-dose CO2 mimics a heterotrophic characterization.

Microalgae possess higher photosynthetic efficiency and accumulate more neutral lipids when supplied with high-dose CO2. However, the nature of lipid accumulation under conditions of elevated CO2 has not been fully elucidated so far. We now revealed that the enhanced lipid accumulation of Chlorella in high-dose CO2 was as efficient as under heterotrophic conditions and this may be attributed to the driving of enlarged carbon source. Both photoautotrophic and heterotrophic cultures were established by using Chlorella sorokiniana CS-1. A series of changes in the carbon fixation, lipid accumulation, energy conversion, and carbon-lipid conversion under high-dose CO2 (1-10%) treatment were characterized subsequently. The daily carbon fixation rate of C. sorokiniana LS-2 in 10% CO2 aeration was significantly increased compared with air CO2. Correspondingly, double oil content (28%) was observed in 10% CO2 aeration, close to 32.3% produced under heterotrophic conditions. In addition, with 10% CO2 aeration, the overall energy yield (Ψ) in Chlorella reached 12.4 from 7.3% (with air aeration) because of the enhanced daily carbon fixation rates. This treatment also improved the energetic lipid yield (Ylipid/Es) with 4.7-fold, tending to the heterotrophic parameters. More significantly, 2.2 times of carbon-lipid conversion efficiency (ηClipid/Ctotal, 42.4%) was observed in 10% CO2 aeration, towards to 53.7% in heterotrophic cultures, suggesting that more fixed carbon might flow into lipid synthesis under both 10% CO2 aeration and heterotrophic conditions. Taken together, all our evidence showed that 10% CO2 may push photoautotrophic Chlorella to display heterotrophic-like efficiency at least in lipid production. It might bring us an efficient model of lipid production based on microalgal cells with high-dose CO2, which is essential to sustain biodiesel production at large scales.

A missense mutation in CHS1, a TIR-NB protein, induces chilling sensitivity in Arabidopsis.

Low temperature is an environmental factor that affects plant growth and development and plant-pathogen interactions. How temperature regulates plant defense responses is not well understood. In this study, we characterized chilling-sensitive mutant 1 (chs1), and functionally analyzed the role of the CHS1 gene in plant responses to chilling stress. The chs1 mutant displayed a chilling-sensitive phenotype, and also displayed defense-associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes: these phenotypes indicated that the mutation in chs1 activates the defense responses under chilling stress. A map-based cloning analysis revealed that CHS1 encodes a TIR-NB-type protein. The chilling sensitivity of chs1 was fully rescued by pad4 and eds1, but not by ndr1. The overexpression of the TIR and NB domains can suppress the chs1-conferred phenotypes. Interestingly, the stability of the CHS1 protein was positively regulated by low temperatures independently of the 26S proteasome pathway. This study revealed the role of a TIR-NB-type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR-NB protein in Arabidopsis.

A chromosome-level genome assembly and annotation of the maize elite breeding line Dan340.

Background: Maize is an important model organism for genetics and genomics research. Though reference genomes of maize are available, some genomes of important genetic germplasms for maize breeding are still lacking, for instance, the cultivar Dan340, which is a backbone inbred line of the LvDa Red Cob Group with several desirable characteristics. In this study, we constructed a high-quality chromosome-level reference genome for Dan340 by using long HiFi reads, short reads, and Hi-C. The final assembly of the Dan340 genome was 2348.72 Mb, which was anchored to 10 chromosomes. Repeat sequences accounted for 73.40% of the genome and 39,733 protein-coding genes were annotated. Comparative genomic analysis between Dan340 and other maize lines identified that 1806 genes from 359 gene families were specific to Dan340. Conclusions: Our genome assembly and annotation provide a valuable resource for improving maize breeding and further understanding the intraspecific genome diversity in maize.

Nicotiana tabacum TTG1 contributes to ParA1-induced signalling and cell death in leaf trichomes.

Leaf trichomes serve as a physical barrier and can also secrete antimicrobial compounds to protect plants from attacks by insects and pathogens. Besides the use of the physical and chemical mechanisms, leaf trichomes might also support plant responses by communicating the extrinsic cues to plant intrinsic signalling pathways. Here we report a role of leaf trichomes in tobacco (Nicotiana tabacum) hypersensitive cell death (HCD) induced by ParA1, an elicitin protein from a plant-pathogenic oomycete. After localized treatment with ParA1, reactive oxygen species were produced first in the leaf trichomes and then in mesophylls. Reactive oxygen species are a group of intracellular signals that are crucial for HCD to develop and for cells to undergo cell death subsequent to chromatin condensation, a hallmark of HCD. These events were impaired when the production of hydrogen peroxide (H(2)O(2)) was inhibited by catalase or a NADPH-oxidase inhibitor applied to trichomes, suggesting the importance of H(2)O(2) in the pathway of HCD signal transduction from the trichomes to mesophylls. This pathway was no longer activated when leaf trichomes were treated with C51S, a ParA1 mutant protein defective in its interaction with N. tabacum TTG1 (NtTTG1), which is a trichome protein that binds ParA1, rather than C51S, in vitro and in trichome cells. The ParA1-NtTTG1 interaction and the HCD pathway were also abrogated when NtTTG1 was silenced in the trichomes. These observations suggest that NtTTG1 plays an essential role in HCD signal transduction from leaf trichomes to mesophylls.