Komagataella phaffii Cue5 Piggybacks on Lipid Droplets for Its Vacuolar Degradation during Stationary Phase Lipophagy

Recently, we developed Komagataella phaffii (formerly Pichia pastoris) as a model for lipophagy, the selective autophagy of lipid droplets (LDs). We found that lipophagy pathways induced by acute nitrogen (N) starvation and in stationary (S) phase have different molecular mechanisms. Moreover, both types of lipophagy are independent of Atg11, the scaffold protein that interacts with most autophagic receptors and, therefore, is essential for most types of selective autophagy in yeast. Since yeast aggrephagy, the selective autophagy of ubiquitinated protein aggregates, is also independent of Atg11 and utilizes the ubiquitin-binding receptor, Cue5, we studied the relationship of K. phaffii Cue5 with differentially induced LDs and lipophagy. While there was no relationship of Cue5 with LDs and lipophagy under N-starvation conditions, Cue5 accumulated on LDs in S-phase and degraded together with LDs via S-phase lipophagy. The accumulation of Cue5 on LDs and its degradation by S-phase lipophagy strongly depended on the ubiquitin-binding CUE domain and Prl1, the positive regulator of lipophagy 1. However, unlike Prl1, which is required for S-phase lipophagy, Cue5 was dispensable for it suggesting that Cue5 is rather a new substrate of this pathway. We propose that a similar mechanism (Prl1-dependent accumulation on LDs) might be employed by Prl1 to recruit another ubiquitin-binding protein that is essential for S-phase lipophagy.

Effects of nitrogen and phosphorus on Microcystis aeruginosa growth and microcystin production

In the present study, the effects of nitrogen (N) and phosphorus (P) on the growth of Microcystis aeruginosa and the production of microcystins (MCs) were investigated. The results showed that the growth of M. aeruginosa was not merely determined by N or P, but both nutrients were limiting for the species. Moreover, an excess of N and constant P in the culture medium could stimulate the growth of M. aeruginosa, whereas the growth of the species was inhibited in the culture medium containing excess of P and constant N. The optimal growth of M. aeruginosa was at an N:P ratio of 0.1 with the maximal optical density of 1.197 at 680 nm (OD680), whereas the maximal microcystin-LR (MC-LR) content of 228.2 μg·L−1 observed in the culture medium with an N:P ratio of 5. Interestingly, MC-LR production occurred under conditions of N starvation, thereby suggesting that the growth rate of M. aeruginosa was not related to MC-LR production under conditions of nutrient stress.

TaLBD1, a LOB Transcription Factor Gene In T. Aestivum, Improves Plant N Starvation Adaptation Via Modulating N Acquisition-Associated Processes

Members of transcription factor (TF) families contribute largely to plant N starvation tolerance by regulating downstream stress defensive genes. In this study, we characterized TaLBD1, a Lateral Organ Boundary (LOB) TF gene in T. aestivum, in regulating plant low-N stress adaptation. TaLBD1 harbors the conserved domains specified by plant LOB proteins, targeting onto nucleus after endoplasmic reticulum (ER) assortment. The TaLBD1 transcripts were response sensitively to N starvation (NS) signaling, showing to be gradually upregulated in aerial and root tissues over a 27-h NS condition. The N. tabacum lines overexpressing TaLBD1 improved phenotype, root system architecture (RSA) establishment, biomass, and N contents of plants under NS treatment. The nitrate transporter gene NtNRT2.4 and PIN-FORMED gene NtPIN6 significantly upregulated in expression in NS-challenged lines; knockdown expression of NtNRT2.4 decreased N uptake and that of NtPIN6 alleviated RSA establishment relative to WT. These results validate the function of NRT and PIN genes in regulating plant N uptake and RSA behavior. RNA-seq analyses revealed that a quantity of genes modify expression in N-deprived lines overexpressing TaLBD1, which enriched into functional groups of signal transduction, transcription, protein biosynthesis, primary or secondary metabolism, and stress defensiveness. These findings suggested that the TaLBD1-improved NS adaptation attributes largely to its role in transcriptionally regulating NRT and PIN genes as well as in modulating those functional in various biological processes. TaLBD1 is a crucial regulator in plant N starvation tolerance and valuable target for molecular breeding high N use efficiency (NUE) crop cultivars.

Komagataella phaffii Cue5 Piggybacks on Lipid Droplets for Its Vacuolar Degradation During Stationary Phase Lipophagy

Recently, we developed Komagataella phaffii (formerly Pichia pastoris) as a model for lipophagy, the selective autophagy of lipid droplets (LDs). We found that lipophagy pathways induced by acute nitrogen (N) starvation and in stationary (S) phase have different molecular mechanisms. Moreover, both types of lipophagy are independent of Atg11, the scaffold protein that interacts with most autophagic receptors and, therefore, is essential for most types of selective autophagy in yeast. Since yeast aggrephagy, the selective autophagy of ubiquitinated protein aggregates, is also independent of Atg11 and utilizes the ubiquitin-binding receptor, Cue5, we studied the relationship of K. phaffii Cue5 with differentially induced LDs and lipophagy. While there was no relationship of Cue5 with LDs and lipophagy under N-starvation conditions, Cue5 accumulated on LDs in S-phase and degraded together with LDs via S-phase lipophagy. The accumulation of Cue5 on LDs and its degradation by S-phase lipophagy strongly depended on the ubiquitin-binding CUE domain and Prl1, the positive regulator of lipophagy 1. However, unlike Prl1, which is required for S-phase lipophagy, Cue5 was dispensable for it suggesting that Cue5 is rather a new substrate of this pathway. We propose that a similar mechanism (Prl1-dependent accumulation on LDs) might be employed by Prl1 to recruit another ubiquitin-binding protein that is essential for S-phase lipophagy.

Morphological, physiological, and transcriptional responses to low nitrogen stress in Populus deltoides Marsh. clones with contrasting nitrogen use efficiency

Background Nitrogen (N) is one of the main factors limiting the wood yield in poplar cultivation. Understanding the molecular mechanism of N utilization could play a guiding role in improving the nitrogen use efficiency (NUE) of poplar. Results In this study, three N-efficient genotypes (A1-A3) and three N-inefficient genotypes (C1-C3) of Populus deltoides were cultured under low N stress (5 μM NH4NO3) and normal N supply (750 μM NH4NO3). The dry matter mass, leaf morphology, and chlorophyll content of both genotypes decreased under N starvation. The low nitrogen adaptation coefficients of the leaves and stems biomass of group A were significantly higher than those of group C (p < 0.05). Interestingly, N starvation induced fine root growth in group A, but not in group C. Next, a detailed time-course analysis of enzyme activities and gene expression in leaves identified 2062 specifically differentially expressed genes (DEGs) in group A and 1118 in group C. Moreover, the sensitivity to N starvation of group A was weak, and DEGs related to hormone signal transduction and stimulus response played an important role in the low N response this group. Weighted gene co-expression network analysis identified genes related to membranes, catalytic activity, enzymatic activity, and response to stresses that might be critical for poplar’s adaption to N starvation and these genes participated in the negative regulation of various biological processes. Finally, ten influential hub genes and twelve transcription factors were identified in the response to N starvation. Among them, four hub genes were related to programmed cell death and the defense response, and PodelWRKY18, with high connectivity, was involved in plant signal transduction. The expression of hub genes increased gradually with the extension of low N stress time, and the expression changes in group A were more obvious than those in group C. Conclusions Under N starvation, group A showed stronger adaptability and better NUE than group C in terms of morphology and physiology. The discovery of hub genes and transcription factors might provide new information for the analysis of the molecular mechanism of NUE and its improvement in poplar.

Signaling Responses to N Starvation: Focusing on Wheat and Filling the Putative Gaps With Findings Obtained in Other Plants. A Review

Wheat is one of the most important food crops worldwide. In recent decades, fertilizers, especially nitrogen (N), have been increasingly utilized to maximize wheat productivity. However, a large proportion of N is not used by plants and is in fact lost into the environment and causes serious environmental pollution. Therefore, achieving a low N optimum via efficient physiological and biochemical processes in wheat grown under low-N conditions is highly important for agricultural sustainability. Although N stress-related N capture in wheat has become a heavily researched subject, how this plant adapts and responds to N starvation has not been fully elucidated. This review summarizes the current knowledge on the signaling mechanisms activated in wheat plants in response to N starvation. Furthermore, we filled the putative gaps on this subject with findings obtained in other plants, primarily rice, maize, and Arabidopsis. Phytohormones have been determined to play essential roles in sensing environmental N starvation and transducing this signal into an adjustment of N transporters and phenotypic adaptation. The critical roles played by protein kinases and critical kinases and phosphatases, such as MAPK and PP2C, as well as the multifaceted functions of transcription factors, such as NF-Y, MYB, DOF, and WRKY, in regulating the expression levels of their target genes (proteins) for low-N tolerance are also discussed. Optimization of root system architecture (RSA) via root branching and thinning, improvement of N acquisition and assimilation, and fine-tuned autophagy are pivotal strategies by which plants respond to N starvation. In light of these findings, we attempted to construct regulatory networks for RSA modification and N uptake, transport, assimilation, and remobilization.

Comparative Transcriptome Analysis of Sophora japonica (L.) Roots Reveals Key Pathways and Genes in Response to PEG-Induced Drought Stress under Different Nitrogen Conditions

Sophora japonica is a native leguminous tree species in China. The high stress tolerance contributes to its long lifespan of thousands of years. The lack of genomic resources greatly limits genetic studies on the stress responses of S. japonica. In this study, RNA-seq was conducted for S. japonica roots grown under short-term 20% polyethylene glycol (PEG) 6000-induced drought stress under normal N and N starvation conditions (1 and 0 mM NH4NO3, respectively). In each of the libraries, we generated more than 25 million clean reads, which were then de novo assembled to 46,852 unigenes with an average length of 1310.49 bp. In the differential expression analyses, more differentially expressed genes (DEGs) were found under drought with N starvation than under single stresses. The number of transcripts identified under N starvation and drought in S. japonica was nearly the same, but more upregulated genes were induced by drought, while more downregulated genes were induced by N starvation. Genes involved in “phenylpropanoid biosynthesis” and “biosynthesis of amino acids” pathways were upregulated according to KEGG enrichment analyses, irrespective of the stress treatments. Additionally, upregulated N metabolism genes were enriched upon drought, and downregulated photosynthesis genes were enriched under N starvation. We found 4372 and 5430 drought-responsive DEGs under normal N and N starvation conditions, respectively. N starvation may aggravate drought by downregulating transcripts in the “carbon metabolism”, “ribosome”, “arginine biosynthesis pathway”, “oxidative phosphorylation” and “aminoacyl-tRNA biosynthesis” pathways. We identified 78 genes related to N uptake and assimilation, 38 of which exhibited differential expression under stress. A total of 395 DEGs were categorized as transcription factors, of which AR2/ERF-ERF, WRKY, NAC, MYB, bHLH, C3H and C2C2-Dof families played key roles in drought and N starvation stresses. The transcriptome data obtained, and the genes identified facilitate our understanding of the mechanisms of S. japonica responses to drought and N starvation stresses and provide a molecular foundation for understanding the mechanisms of its long lifespan for breeding resistant varieties for greening.

Comparative Transcriptome Analysis Reveals Different Low-Nitrogen-Responsive Genes in Pepper Cultivars

The molecular mechanisms underlying the variation in N-use efficiency (NUE) in pepper (Capsicum annuum L.) genotypes are poorly understood. In this work, two genotypes (750-1, low-N tolerant; ZCFB, low-N sensitive) with contrasting low-N tolerance were selected from 100 pepper cultivars on the basis of their relative leaf areas, shoot dry weights, root dry weights, and plant dry weights at the seedling stage. Subsequently, using RNA-Seq, the transcriptome of these two pepper genotypes under N starvation for 28 days was analyzed. We detected 2621/2470 and 3936/4218 different expressed genes (DEGs) in the leaves/roots of 750-1 and ZCFB, respectively. The changes in the expression of basic N metabolism genes were similar between 750-1 and ZCFB. However, different DEGs not directly involved in N metabolism were identified between the 750-1 and ZCFB cultivars. In 750-1, 110 unique DEGs were detected in the leaves, of which 103 were down-regulated, including genes associated with protein metabolism, photosynthesis, secondary metabolism, cell wall metabolism, stress response, and disease resistance. In ZCFB, 142 unique DEGs were detected in the roots, of which 117 were up-regulated, resulting in enhancement of processes such as protein degradation, secondary metabolites synthesis, lipid metabolism, endocytosis, the tricarboxylic acid cycle (TCA), transcriptional regulation, stress response, and disease resistance. Our results not only facilitate an understanding of the different regulatory process in low-N-tolerant and low-N-sensitive pepper cultivars, but also provide abundant candidate genes for improving the low-N tolerance of pepper cultivars.

Comparative Analysis Based on Transcriptomics and Metabolomics Data Reveal Differences between Emmer and Durum Wheat in Response to Nitrogen Starvation

Mounting evidence indicates the key role of nitrogen (N) on diverse processes in plant, including development and defense. Using a combined transcriptomics and metabolomics approach, we studied the response of seedlings to N starvation of two different tetraploid wheat genotypes from the two main domesticated subspecies: emmer and durum wheat. We found that durum wheat exhibits broader and stronger response in comparison to emmer as seen from the expression pattern of both genes and metabolites and gene enrichment analysis. They showed major differences in the responses to N starvation for transcription factor families, emmer showed differential reduction in the levels of primary metabolites while durum wheat exhibited increased levels of most of them to N starvation. The correlation-based networks, including the differentially expressed genes and metabolites, revealed tighter regulation of metabolism in durum wheat in comparison to emmer. We also found that glutamate and -aminobutyric acid (GABA) had highest values of centrality in the metabolic correlation network, suggesting their critical role in the genotype-specific response to N starvation of emmer and durum wheat, respectively. Moreover, this finding indicates that there might be contrasting strategies associated to GABA and glutamate signaling modulating shoot vs. root growth in the two different wheat subspecies.

Nitrogen Starvation-Responsive MicroRNAs Are Affected by Transgenerational Stress in Durum Wheat Seedlings

Stress events have transgenerational effects on plant growth and development. In Mediterranean regions, water-deficit and heat (WH) stress is a frequent issue that negatively affects crop yield and quality. Nitrogen (N) is an essential plant macronutrient and often a yield-limiting factor for crops. Here, the response of durum wheat seedlings to N starvation under the transgenerational effects of WH stress was investigated in two genotypes. Both genotypes showed a significant reduction in seedling height, leaf number, shoot and root weight (fresh and dry), primary root length, and chlorophyll content under N starvation stress. However, in the WH stress-tolerant genotype, the percentage reduction of most traits was lower in progeny from the stressed parents than progeny from the control parents. Small RNA sequencing identified 1534 microRNAs in different treatment groups. Differentially expressed microRNAs (DEMs) were characterized subject to N starvation, parental stress and genotype factors, with their target genes identified in silico. GO and KEGG enrichment analyses revealed the biological functions, associated with DEM-target modules in stress adaptation processes, that could contribute to the phenotypic differences observed between the two genotypes. The study provides the first evidence of the transgenerational effects of WH stress on the N starvation response in durum wheat.