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青檀扦插苗对不同氮素水平的形态、光合生理响应和转录组分析
郭伟, 韩秀, 张利, 王迎, 杜辉, 燕语, 孙忠奎, 张林, 李国华, 罗磊
南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (5) : 87-96.
PDF(2108 KB)
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青檀扦插苗对不同氮素水平的形态、光合生理响应和转录组分析
Morphological, photosynthetic physiological and transcriptome analyses of Pteroceltis tatarinowii in response to different nitrogen application levels
【目的】 研究不同氮素水平下青檀(Pteroceltis tatarinowii)形态和光合生理变化,筛选氮素响应基因,为后期深入开展氮素响应分子机理研究提供参考。【方法】 选取0、2、50 mmol/L NH4NO3的Hocking’s营养液分别作为缺氮(N0)、中氮(N2)、高氮(N50)处理条件对青檀扦插苗进行盆栽控制试验,28 d时进行形态和光合生理指标测定。对不同氮素水平处理后的青檀混合样本进行3代全长转录组测序,测序结果为2代转录组测序提供参考,通过生物信息学分析筛选不同氮素水平响应的差异表达基因并进行KEGG通路富集分析,利用荧光定量PCR技术对富集基因进行根和叶片中的表达分析。【结果】 氮素处理第28天时青檀的株高、地径、叶片数量、叶面积、比叶面积、叶片生物量、茎生物量,以及根系、叶片与茎氮含量随着氮素处理浓度提高而提高,总根长、总根表面积、总根体积、比根长、根冠比、根系生物量随着氮素浓度提高而降低。高氮素处理明显提高了叶绿素a、叶绿素b、类胡萝卜素含量,增强了净光合速率和气孔导度。不同氮素水平的转录组测序的差异表达基因中76个基因随着氮素水平提高稳定上调表达,32个基因随着氮素水平提高稳定下调表达。对这108个基因进行KEGG通路富集分析发现排在前两位的分别是光合作用和氮代谢通路,光合作用通路富集的基因是OEE2、OEE3、PSBR、PSB27、PSAF和PSAK,氮代谢通路富集的基因是2个NR和1个NiR基因。表达分析结果显示这9个基因在根和叶片中具有相似的表达模式,缺氮处理中表达量最低,高氮处理中表达量最高;光合作用通路的6个基因在每一种氮素浓度处理时在叶中的表达量均高于在根中的。【结论】 随着氮素浓度的提高,光合作用通路基因表达上调,促进了光合作用,硝酸还原酶基因和亚硝酸还原酶基因表达上调,提高了硝酸根的同化效率,最终造成青檀地上部生长得到加强,根系生长受到抑制。在没有参考基因组的情况下,为探讨青檀氮素响应分子机理、发掘关键候选基因提供了有力依据。
【Objective】Our objectives are to study the morphological and photosynthetic physiological changes of Pteroceltis tatarinowii under different nitrogen application levels, and screen nitrogen response related genes in order to provide reference for further research on the molecular mechanisms how nitrogen application trigger changes in P. tatarinowii. 【Method】A pot experiment was conducted with the cuttings of P. tatarinowii fertilized with Hocking’s complete nutrient solution with 0 (limiting, N0), 2 (intermediate, N2) and 50 mmol/L (luxuriant, N50) NH4NO3, respectively. The physiological and photosynthetic morphological parameters were determined at day 28. SMRT-seq, performed with the pooled sample of P. tatarinowii treated with different nitrogen application levels, provided full length transcriptome data as a reference for the RNA-seq. Differentially expressed genes responding to different nitrogen levels were screened using bioinformatics methods. Then, the KEGG pathway enrichment analysis was conducted. The expression of the differentially expressed genes enriched in roots and leaves was quantified by RT-qPCR. 【Result】The physiological and morphological observations, determined at day 28 after being fertilized with different nitrogen application levels, showed that the values of height, stem diameter, number of leaves, leaf area, specific leaf area, biomass of leaves, biomass of stems, N concentration in roots, leaves and stems increased with the increase of nitrogen concentration, while the values of the total root length, total root surface area, total root volume, specific root length, root to shoot ratio, and biomass of roots, all decreased with the increase of nitrogen concentration. The chlorophyll a, chlorophyll b, carotenoid content, net photosynthetic rates and stomatal conductance were significantly promoted by the luxuriant nitrogen level. Of the differentially expressed genes among different nitrogen levels based on the global transcriptomic profiling by RNA-seq, 76 genes were up-regulated following the increase of nitrogen application levels, while 32 genes were opposite. The KEGG pathway enrichment analysis of the 108 genes showed that the top two pathways were photosynthesis and nitrogen metabolism. The photosynthesis pathway was enriched with OEE2, OEE3, PSBR, PSB27, PSAF and PSAK, while the nitrogen metabolism pathway was enriched with two NR genes and one NiR gene. The expression analysis showed that the nine genes were with similar expression patterns in roots and leaves, lowest expression levels in limiting nitrogen level while highest expression levels in luxuriant nitrogen. The expression levels of six genes of photosynthesis pathway in leaves were higher than that in roots under each nitrogen level. 【Conclusion】 Following the increase of nitrogen application levels, photosynthesis pathway genes were up-regulated, which promoted photosynthesis, while, nitrate reductase genes and nitrite reductase genes were up-regulated, which improved the nitrate assimilation efficiency. In the absence of a reference genome sequence, our results provided a basis for exploring the molecular mechanisms and discovering key candidate genes how nitrogen trigger changes in P. tatarinowii.
青檀 / 氮素 / 形态和光合生理响应 / 全长转录组 / 差异表达基因
Pteroceltis tatarinowii / nitrogen / morphological and photosynthetic physiological response / full length transcriptome / differentially expressed gene
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Nitrogen (N) is an essential macronutrient for plants. N levels in soil vary widely, and plants have developed strategies to cope with N deficiency. However, the regulation of these adaptive responses and the coordinating signals that underlie them are still poorly understood. The aim of this study was to characterize N starvation in adult Arabidopsis (Arabidopsis thaliana) plants in a spatiotemporal manner by an integrative, multilevel global approach analyzing growth, metabolites, enzyme activities, and transcript levels. We determined that the remobilization of N and carbon compounds to the growing roots occurred long before the internal N stores became depleted. A global metabolite analysis by gas chromatography-mass spectrometry revealed organ-specific differences in the metabolic adaptation to complete N starvation, for example, for several tricarboxylic acid cycle intermediates, but also for carbohydrates, secondary products, and phosphate. The activities of central N metabolism enzymes and the capacity for nitrate uptake adapted to N starvation by favoring N remobilization and by increasing the high-affinity nitrate uptake capacity after long-term starvation. Changes in the transcriptome confirmed earlier studies and added a new dimension by revealing specific spatiotemporal patterns and several unknown N starvation-regulated genes, including new predicted small RNA genes. No global correlation between metabolites, enzyme activities, and transcripts was evident. However, this multilevel spatiotemporal global study revealed numerous new patterns of adaptation mechanisms to N starvation. In the context of a sustainable agriculture, this work will give new insight for the production of crops with increased N use efficiency.
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Nitrogen (N) starvation and excess have distinct effects on N uptake and metabolism in poplars, but the global transcriptomic changes underlying morphological and physiological acclimation to altered N availability are unknown. We found that N starvation stimulated the fine root length and surface area by 54 and 49%, respectively, decreased the net photosynthetic rate by 15% and reduced the concentrations of NH4+, NO3(-) and total free amino acids in the roots and leaves of Populus simonii Carr. in comparison with normal N supply, whereas N excess had the opposite effect in most cases. Global transcriptome analysis of roots and leaves elucidated the specific molecular responses to N starvation and excess. Under N starvation and excess, gene ontology (GO) terms related to ion transport and response to auxin stimulus were enriched in roots, whereas the GO term for response to abscisic acid stimulus was overrepresented in leaves. Common GO terms for all N treatments in roots and leaves were related to development, N metabolism, response to stress and hormone stimulus. Approximately 30-40% of the differentially expressed genes formed a transcriptomic regulatory network under each condition. These results suggest that global transcriptomic reprogramming plays a key role in the morphological and physiological acclimation of poplar roots and leaves to N starvation and excess.© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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In the process of making full-length cDNA, predicting protein coding regions helps both in the preliminary analysis of genes and in any succeeding process. However, unfinished cDNA contains artifacts including many sequencing errors, which hinder the correct evaluation of coding sequences. Especially, predictions of short sequences are difficult because they provide little information for evaluating coding potential. In this paper, we describe ANGLE, a new program for predicting coding sequences in low quality cDNA. To achieve error-tolerant prediction, ANGLE uses a machine-learning approach, which makes better expression of coding sequence maximizing the use of limited information from input sequences. Our method utilizes not only codon usage, but also protein structure information which is difficult to be used for stochastic model-based algorithms, and optimizes limited information from a short segment when deciding coding potential, with the result that predictive accuracy does not depend on the length of an input sequence. The performance of ANGLE is compared with ESTSCAN on four dataset each of them having a different error rate (one frame-shift error or one substitution error per 200–500 nucleotides) and on one dataset which has no error. ANGLE outperforms ESTSCAN by 9.26% in average Matthews's correlation coefficient on short sequence dataset (< 1000 bases). On long sequence dataset, ANGLE achieves comparable performance.
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Exogenous application of spermidine (Spd) has been reported to modulate physiological processes and alleviate salt-induced damage to growth and productivity of several plants including rice. Employing a proteomic approach, we aimed at identifying rice leaf and grain proteins differentially expressing under salt stress, and in response to Spd prior to NaCl treatment. A total of 9 and 20 differentially expressed protein spots were identified in the leaves of salt-tolerant (Pokkali) and salt-sensitive (KDML105) rice cultivars, respectively. Differential proteins common to both cultivars included a photosynthetic light reaction protein (oxygen-evolving complex protein 1), enzymes of Calvin cycle and glycolysis (fructose-bisphosphate aldolase and triose-phosphate isomerase), malate dehydrogenase, superoxide dismutase and a hypothetical protein (OsI_18213). Most proteins were present at higher intensities in Pokkali leaves. The photosynthetic oxygen-evolving enhancer protein 2 was detected only in Pokkali and was up-regulated by salt-stress and further enhanced by Spd treatment. All three spots identified as superoxide dismutase in KDML105 were up-regulated by NaCl but down-regulated when treated with Spd prior to NaCl, indicating that Spd acted directly as antioxidants. Important differential stress proteins detected in mature grains of both rice cultivars were late embryogenesis abundant proteins with protective roles and an antioxidant protein, 1-Cys-peroxiredoxin. Higher salt tolerance of Pokkali partly resulted from higher intensities and more responsiveness of the proteins relating to photosynthesis light reactions, energy metabolism, antioxidant enzymes in the leaves, and stress proteins with protective roles in the grains. |
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Plants cope with inorganic phosphate (Pi) deficiencies in their environment by adjusting their developmental programs and metabolic activities. For Arabidopsis (Arabidopsis thaliana), the developmental responses include the inhibition of primary root growth and the enhanced formation of lateral roots and root hairs. Pi deficiency also inhibits photosynthesis by suppressing the expression of photosynthetic genes. Early studies showed that photosynthetic gene expression was also suppressed in Pi-deficient roots, a nonphotosynthetic organ; however, the biological relevance of this phenomenon remains unknown. In this work, we characterized an Arabidopsis mutant, hypersensitive to Pi starvation7 (hps7), that is hypersensitive to Pi deficiency; the hypersensitivity includes an increased inhibition of root growth. HPS7 encodes a tyrosylprotein sulfotransferase. Accumulation of HPS7 proteins in root tips is enhanced by Pi deficiency. Comparative RNA sequencing analyses indicated that the expression of many photosynthetic genes is activated in roots of hps7. Under Pi deficiency, the expression of photosynthetic genes in hps7 is further increased, which leads to enhanced accumulation of chlorophyll, starch, and sucrose. Pi-deficient hps7 roots also produce a high level of reactive oxygen species. Previous research showed that the overexpression of GOLDEN-like (GLK) transcription factors in transgenic Arabidopsis activates photosynthesis in roots. The GLK overexpressing (GLK OX) lines also exhibit increased inhibition of root growth under Pi deficiency. The increased inhibition of root growth in hps7 and GLK OX lines by Pi deficiency was completely reversed by growing the plants in the dark. Based on these results, we propose that suppression of photosynthetic gene expression is required for sustained root growth under Pi deficiency.© 2014 American Society of Plant Biologists. All Rights Reserved.
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Nitrogen is a major limiting factor for crop productivity. The relationship between photosynthesis and nitrogen nutrition has been widely studied. However, the molecular response of leaf photosynthesis to low nitrogen supply in crops is less clear. In this study, RNA sequencing technology (RNA-Seq) was used to investigate the gene expressions related to photosynthesis in maize in response to low nitrogen supply. It was found that low nitrogen supply down-regulated the expression of genes involved in photosystem I (PSI) and photosystem II (PSII). Thus, low nitrogen supply down-regulated the expression of genes related to the antenna system, reduced light absorption, light transport, and electron transport. Correspondingly, the parameters related to chlorophyll fluorescence were very sensitive to nitrogen deficiency. Under low nitrogen supply, leaf chlorophyll content, actual quantum yield of PSII photochemistry, photochemical quenching, and electron transport rate, were reduced. However, the thermal diffusion and chlorophyll fluorescence were increased. RNA-Seq was used to analyze the genes involved in the response of leaf photosynthesis to low nitrogen supply in maize. These results highlight the possibility of utilizing chlorophyll fluorescence parameters, and the related genes, as indicators for plant nitrogen nutrition. This could lead to the development of new tools to make precise nitrogen fertilizer recommendations and select nitrogen-efficient genotypes.
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Background: Nitrogen (N) is a key macronutrient required for plant growth and development. In this study, watermelon plants were grown under hydroponic conditions at 0.2 mM N, 4.5 mM N, and 9 mM N for 14 days.Results: Dry weight and photosynthetic assimilation at low N (0.2 mM) was reduced by 29 and 74% compared with high N (9 mM). The photochemical activity (Fv/Fm) was also reduced from 0.78 at high N to 0.71 at low N. The N concentration in the leaf, stem, and root of watermelon under low N conditions was reduced by 68, 104, and 108%, respectively compared with 9 mM N treatment after 14 days of N treatment. In the leaf tissues of watermelon grown under low N conditions, 9598 genes were differentially expressed, out of which 4533 genes (47.22%) were up-regulated whereas, 5065 genes (52.78%) were down-regulated compared with high N. Similarly in the root tissues, 3956 genes were differentially expressed, out of which 1605 genes were up-regulated (40.57%) and 2351 genes were down-regulated (59.43%), compared with high N. Our results suggest that leaf tissues are more sensitive to N deficiency compared with root tissues. The gene ontology (GO) analysis showed that the availability of N significantly affected 19 biological processes, 8 cell component metabolic pathways, and 3 molecular functions in the leaves; and 13 biological processes, 12 molecular functions, and 5 cell component metabolic pathways in the roots of watermelon. The low affinity nitrate transporters, high affinity nitrate transporters, ammonium transporters, genes related with nitrogen assimilation, and chlorophyll and photosynthesis were expressed differentially in response to low N. Three nitrate transporters (Cla010066, Cla009721, Cla012765) substantially responded to low nitrate supply in the root and leaf tissues. Additionally, a large number of transcription factors (1365) were involved in adaptation to low N availability. The major transcription factor families identified in this study includes MYB, AP2-EREBP, bHLH, C2H2 and NAC.Conclusion: Candidate genes identified in this study for nitrate uptake and transport can be targeted and utilized for further studies in watermelon breeding and improvement programs to improve N uptake and utilization efficiency.
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