Leaf Senescence Regulation Mechanism Based on Comparative Transcriptome Analysis in Foxtail Millet.

Leaf senescence, a pivotal process in plants, directly influences both crop yield and nutritional quality. Foxtail millet (Setaria italica) is a C4 model crop renowned for its exceptional nutritional value and stress tolerance characteristics. However, there is a lack of research on the identification of senescence-associated genes (SAGs) and the underlying molecular regulatory mechanisms governing this process. In this study, a dark-induced senescence (DIS) experimental system was applied to investigate the extensive physiological and transcriptomic changes in two foxtail millet varieties with different degrees of leaf senescence. The physiological and biochemical indices revealed that the light senescence (LS) variety exhibited a delayed senescence phenotype, whereas the severe senescence (SS) variety exhibited an accelerated senescence phenotype. The most evident differences in gene expression profiles between these two varieties during DIS included photosynthesis, chlorophyll, and lipid metabolism. Comparative transcriptome analysis further revealed a significant up-regulation of genes related to polysaccharide and calcium ion binding, nitrogen utilization, defense response, and malate metabolism in LS. In contrast, the expression of genes associated with redox homeostasis, carbohydrate metabolism, lipid homeostasis, and hormone signaling was significantly altered in SS. Through WGCNA and RT-qPCR analyses, we identified three SAGs that exhibit potential negative regulation towards dark-induced leaf senescence in foxtail millet. This study establishes the foundation for a further comprehensive examination of the regulatory network governing leaf senescence and provides potential genetic resources for manipulating senescence in foxtail millet.

Identification and function analysis of yellow-leaf mutant (YX-yl) of broomcorn millet.

BACKGROUND: Broomcorn millet is highly tolerant to drought and barren soil. Changes in chlorophyll content directly affect leaf color, which subsequently leadsleading to poor photosynthetic performance and reduced crop yield. Herein, we isolated a yellow leaf mutant (YX-yl) using a forward genetics approach and evaluated its agronomic traits, photosynthetic pigment content, chloroplast ultrastructure, and chlorophyll precursors. Furthermore, the molecular mechanism of yellowing was explored using transcriptome sequencing. RESULTS: The YX-yl mutant showed significantly decreased plant height and low yield. The leaves exhibited a yellow-green phenotype and poor photosynthetic capacity during the entire growth period. The content of chlorophyll a, chlorophyll b, and carotenoids in YX-yl leaves was lower than that in wild-type leaves. Chlorophyll precursor analysis results showed that chlorophyll biosynthesis in YX-yl was hindered by the conversion of porphobilinogen to protoporphyrin IX. Examination of chloroplast ultrastructure in the leaves revealed that the chloroplasts of YX-yl accumulated on one side of the cell. Moreover, the chloroplast structure of YX-yl was degraded. The inner and outer membranes of the chloroplasts could not be distinguished well. The numbers of grana and grana thylakoids in the chloroplasts were low. The transcriptome of the yellowing mutant YX-yl was sequenced and compared with that of the wild type. Nine chlorophyll-related genes with significantly different expression profiles were identified: PmUROD, PmCPO, PmGSAM, PmPBDG, PmLHCP, PmCAO, PmVDE, PmGluTR, and PmPNPT. The proteins encoded by these genes were located in the chloroplast, chloroplast membrane, chloroplast thylakoid membrane, and chloroplast matrix and were mainly involved in chlorophyll biosynthesis and redox-related enzyme regulation. CONCLUSIONS: YX-yl is an ideal material for studying pigment metabolism mechanisms. Changes in the expression patterns of some genes between YX-yl and the wild type led to differences in chloroplast structures and enzyme activities in the chlorophyll biosynthesis pathway, ultimately resulting in a yellowing phenotype in the YX-yl mutant. Our findings provide an insight to the molecular mechanisms of leaf color formation and chloroplast development in broomcorn millet.

OsATG8c-Mediated Increased Autophagy Regulates the Yield and Nitrogen Use Efficiency in Rice.

Autophagy, a conserved pathway in eukaryotes, degrades and recycles cellular components, thus playing an important role in nitrogen (N) remobilization. N plays an important role in the growth and development of plants, which also affects plant yield and quality. In this research, it was found that the transcriptional level of a core autophagy gene of rice (Oryza sativa), OsATG8c, was increased during N starvation conditions. It was found that the overexpression of OsATG8c significantly enhanced the activity of autophagy and that the number of autophagosomes, dwarfed the plant height and increased the effective tillers' number and yield. The nitrogen uptake efficiency (NUpE) and nitrogen use efficiency (NUE) significantly increased in the transgenic rice under both optimal and suboptimal N conditions. Based on our results, OsATG8c is considered to be a good candidate gene for increasing NUE, especially under suboptimal field conditions.

An Integrated Analysis of the Rice Transcriptome and Metabolome Reveals Root Growth Regulation Mechanisms in Response to Nitrogen Availability.

Nitrogen is an essential nutrient for plant growth and basic metabolic processes. Root systems play an important role in the ability of plants to obtain nutrients from the soil, and are closely related to the growth and development of above-ground plants. Root morphology analysis showed that root growth was induced under low-nitrogen conditions and inhibited under high-nitrogen conditions. To better understand the molecular mechanisms and metabolic basis underlying the rice root response to nitrogen availability, an integrated analysis of the rice root transcriptome and metabolome under three environmental conditions (low-, control, and high-nitrogen conditions) was conducted. A total of 262 and 262 differentially level metabolites were identified under low- and high-nitrogen conditions, respectively. A total of 696 and 808 differentially expressed genes were identified under low- and high-nitrogen conditions, respectively. For both the differentially expressed genes and metabolites, KEGG pathway analysis indicated that amino acid metabolism, carbon and nitrogen metabolism, phenylpropanoid metabolism, and phytohormones' signal transduction were significantly affected by nitrogen availability. Additionally, variable levels of 65 transcription factors (TFs) were identified in rice leaves exposed to high and low nitrogen, covering 22 TF families. These results also indicate that there is a significant difference in the transcriptional regulation mechanisms of rice roots between low and high nitrogen. In summary, our study provides new information for a further understanding of the response of rice roots to low-nitrogen and high-nitrogen conditions.

An Integrated Analysis of the Rice Transcriptome and Metabolome Reveals Differential Regulation of Carbon and Nitrogen Metabolism in Response to Nitrogen Availability.

Nitrogen (N) is an extremely important macronutrient for plant growth and development. It is the main limiting factor in most agricultural production. However, it is well known that the nitrogen use efficiency (NUE) of rice gradually decreases with the increase of the nitrogen application rate. In order to clarify the underlying metabolic and molecular mechanisms of this phenomenon, we performed an integrated analysis of the rice transcriptome and metabolome. Both differentially expressed genes (DEGs) and metabolite Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that carbon and nitrogen metabolism is significantly affected by nitrogen availability. Further analysis of carbon and nitrogen metabolism changes in rice under different nitrogen availability showed that high N inhibits nitrogen assimilation and aromatic metabolism pathways by regulating carbon metabolism pathways such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway (PPP). Under low nitrogen, the TCA cycle is promoted to produce more energy and α-ketoglutarate, thereby enhancing nitrogen transport and assimilation. PPP is also inhibited by low N, which may be consistent with the lower NADPH demand under low nitrogen. Additionally, we performed a co-expression network analysis of genes and metabolites related to carbon and nitrogen metabolism. In total, 15 genes were identified as hub genes. In summary, this study reveals the influence of nitrogen levels on the regulation mechanisms for carbon and nitrogen metabolism in rice and provides new insights into coordinating carbon and nitrogen metabolism and improving nitrogen use efficiency in rice.

Integrated Metabolomic and Transcriptomic Analyses Reveal the Basis for Carotenoid Biosynthesis in Sweet Potato (Ipomoea batatas (L.) Lam.) Storage Roots.

Carotenoids are important compounds of quality and coloration within sweet potato storage roots, but the mechanisms that govern the accumulation of these carotenoids remain poorly understood. In this study, metabolomic and transcriptomic analyses of carotenoids were performed using young storage roots (S2) and old storage roots (S4) from white-fleshed (variety S19) and yellow-fleshed (variety BS) sweet potato types. S19 storage roots exhibited significantly lower total carotenoid levels relative to BS storage roots, and different numbers of carotenoid types were detected in the BS-S2, BS-S4, S19-S2, and S19-S4 samples. ß-cryptoxanthin was identified as a potential key driver of differences in root coloration between the S19 and BS types. Combined transcriptomic and metabolomic analyses revealed significant co-annotation of the carotenoid and abscisic acid (ABA) metabolic pathways, PSY (phytoene synthase), CHYB (ß-carotene 3-hydroxylase), ZEP (zeaxanthin epoxidase), NCED3 (9-cis-epoxycarotenoid dioxygenase 3), ABA2 (xanthoxin dehydrogenase), and CYP707A (abscisic acid 8'-hydroxylase) genes were found to be closely associated with carotenoid and ABA content in these sweet potato storage roots. The expression patterns of the transcription factors OFP and FAR1 were associated with the ABA content in these two sweet potato types. Together, these results provide a valuable foundation for understanding the mechanisms governing carotenoid biosynthesis in storage roots, and offer a theoretical basis for sweet potato breeding and management.

Uncovering the mechanism of anthocyanin accumulation in a purple-leaved variety of foxtail millet (Setaria italica) by transcriptome analysis.

Anthocyanin is a natural pigment that has a functional role in plants to attract pollinating insects and is important in stress response. Foxtail millet (Setaria italica) is known as a nutritional crop with high resistance to drought and barren. However, the molecular mechanism regulating anthocyanin accumulation and the relationship between anthocyanin and the stress resistance of foxtail millet remains obscure. In this study, we screened hundreds of germplasm resources and obtained several varieties with purple plants in foxtail millet. By studying the purple-leaved B100 variety and the control variety, Yugu1 with green leaves, we found that B100 could accumulate a large amount of anthocyanin in the leaf epiderma, and B100 had stronger stress tolerance. Further transcriptome analysis revealed the differences in gene expression patterns between the two varieties. We identified nine genes encoding enzymes related to anthocyanin biosynthesis using quantitative PCR validation that showed significantly higher expression levels in B100 than Yugu1. The results of this study lay the foundation for the analysis of the molecular mechanism of anthocyanin accumulation in foxtail millet, and provided genetic resources for the molecular breeding of crops with high anthocyanin content.

Autophagy mediates grain yield and nitrogen stress resistance by modulating nitrogen remobilization in rice.

Autophagy, a conserved cellular process in eukaryotes, has evolved to a sophisticated process to dispose of intracellular constituents and plays important roles in plant development, metabolism, and efficient nutrients remobilization under suboptimal nutrients conditions. Here, we show that OsATG8b, an AUTOPHAGY-RELATED8 (ATG8) gene in rice, was highly induced by nitrogen (N) starvation. Elevated expression of OsATG8b significantly increased ATG8 lipidation, autophagic flux, and grain yield in rice under both sufficient and deficient N conditions. Overexpressing of OsATG8b could greatly increase the activities of enzymes related to N metabolism. Intriguingly, the 15N-labeling assay further revealed that more N was remobilized to seeds in OsATG8b-overexpressing rice, which significantly increased the N remobilization efficiency (NRE), N harvest index, N utilization efficiency (NUE), and N uptake efficiency (NUpE). Conversely, the osatg8b knock-out mutants had the opposite results on these characters. The substantial transcriptional changes of the overexpressed transgenic lines indicated the presence of complex signaling to developmental, metabolic process, and hormone, etc. Excitingly, the transgenic rice under different backgrounds all similarly be boosted in yield and NUE with OsATG8b overexpression. This work provides an excellent candidate gene for improving N remobilization, utilization, and yield in crops simultaneously.

Adaptation Mechanism of Roots to Low and High Nitrogen Revealed by Proteomic Analysis.

BACKGROUND: Nitrogen-based nutrients are the main factors affecting rice growth and development. Root systems play an important role in helping plants to obtain nutrients from the soil. Root morphology and physiology are often closely related to above-ground plant organs performance. Therefore, it is important to understand the regulatory effects of nitrogen (N) on rice root growth to improve nitrogen use efficiency. RESULTS: In this study, changes in the rice root traits under low N (13.33 ppm), normal N (40 ppm) and high N (120 ppm) conditions were performed through root morphology analysis. These results show that, compared with normal N conditions, root growth is promoted under low N conditions, and inhibited under high N conditions. To understand the molecular mechanism underlying the rice root response to low and high N conditions, comparative proteomics analysis was performed using a tandem mass tag (TMT)-based approach, and differentially abundant proteins (DAPs) were further characterized. Compared with normal N conditions, a total of 291 and 211 DAPs were identified under low and high N conditions, respectively. The abundance of proteins involved in cell differentiation, cell wall modification, phenylpropanoid biosynthesis, and protein synthesis was differentially altered, which was an important reason for changes in root morphology. Furthermore, although both low and high N can cause nitrogen stress, rice roots revealed obvious differences in adaptation to low and high N. CONCLUSIONS: These results provide insights into global changes in the response of rice roots to nitrogen availability and may facilitate the development of rice cultivars with high nitrogen use efficiency through root-based genetic improvements.

Overexpression of rice gene OsATG8b confers tolerance to nitrogen starvation and increases yield and nitrogen use efficiency (NUE) in Arabidopsis.

Nitrogen (N) is an important element required for plant growth and development, which also affects plant yield and quality. Autophagy, a conserved pathway in eukaryotes, degrades and recycles cellular components, thus playing an important role in N remobilization. However, only a few autophagy genes related to N remobilization in rice (Oryza sativa) have been reported. Here, we identified a core autophagy gene in rice, OsATG8b, with increased expression levels under N starvation conditions. It was investigated the function of OsATG8b by generating three independent homozygous 35S-OsATG8b transgenic Arabidopsis thaliana lines. The overexpression of OsATG8b significantly enhanced autophagic flux in the transgenic Arabidopsis plants. It was also showed that over-expressing OsATG8b promoted growth and development of Arabidopsis, in which the rosette leaves were larger than those of the wild type (WT), and the yield increased significantly by 25.25%. In addition, the transgenic lines accumulated more N in seeds than in the rosette leaves. Further examination revealed that overexpression of OsATG8b could effectively alleviate the growth inhibition of transgenic Arabidopsis under nitrogen (N) stress. N partitioning studies revealed that nitrogen-harvest index (NHI) and nitrogen use efficiency (NUE) were significantly increased in the transgenic Arabidopsis, as well as the 15N-tracer experiments revealing that the remobilization of N to seeds in the OsATG8b-overexpressing transgenic Arabidopsis was high and more than WT. Based on our findings, we consider OsATG8b to be a great candidate gene to increase NUE and yield, especially under suboptimal field conditions.

Increased Autophagy of Rice Can Increase Yield and Nitrogen Use Efficiency (NUE).

Autophagy (self-eating), a conserved pathway in eukaryotes, which is designed to handle cytoplasmic material in bulk and plays an important role in the remobilization of nutrient, such as nitrogen (N) under suboptimal nutrient conditions. Here, we identified a core component of an autophagy gene in rice (Oryza sativa), OsATG8a, with increased expression levels under N starvation conditions. Overexpression of OsATG8a significantly enhanced the level of autophagy and the number of effective tillers in the transgenic rice. In addition, the transgenic lines accumulated more N in grains than in the dry remains and the yield was significantly increased under normal N conditions. Further N allocation studies revealed that the nitrogen uptake efficiency (NUpE) and nitrogen use efficiency (NUE) significantly increased. Otherwise, under suboptimal N conditions, overexpression of OsATG8a did not seem to have any effect on yield and NUE, but NUpE was still improved significantly. Based on our findings, we consider OsATG8a to be a great candidate gene to increase NUE and yield.

Differential Uptake and Utilization of Two Forms of Nitrogen in Japonica Rice Cultivars From North-Eastern China.

Japonica rice is widely planted in north-eastern China because of its superior food quality and stable grain yields. Nitrogen (N) is an essential element for rice growth, and development and its availability directly impacts on rice yields. The knowledge of N uptake and its utilization characteristics in japonica are thus important areas of research. Three japonica rice cultivars, SN265, SN1401, and SN9816, which are planted across large areas of north-eastern China, were used here to evaluate the uptake and utilization along the life cycle of both ammonium ( N H 4 + ) and nitrate ( N O 3 - ) in hydroponically grown plants. The plants were grown in one of three different solutions with varying N H 4 + : N O 3 - ratios: 1:0, 0:1, and 1:1 (The total N content was 40 mg L-1 for each treatment). At the tillering stage, when only N O 3 - was provided, lower rates of N uptake and enzyme activities of three rice plants resulted in reduced tiller numbers. During the reproductive stage, the N H 4 + and ( N H 4 + ) uptake rates in SN1401 were consistently maintained at high levels, whereas the rates in SN265 and SN9816 were significantly lower, across all three treatments. At the booting stage, when only N O 3 - was provided, SN1401 plants had significantly higher expression levels of OsNRT2.1 and OsNRT2.2, higher activity of nitrate reductase in the roots, and higher activity levels of glutamine synthetase and glutamate synthase in the leaves, compared with the SN265 and SN9816 plants. The higher enzyme activity was beneficial to the secondary assimilation of N, which ultimately promoted panicle development in SN1401. Consequently, the grain yield per plant of SN1401 was the highest with solutions of both N H 4 + and N O 3 - . These results indicate that selecting a rice cultivar with higher utilization of N O 3 - is beneficial for increasing the number of grains per panicle, grain yield, and N use efficiency.