Physiological and transcriptomic analyses reveal the molecular networks of responses induced by exogenous trehalose in plant

It is well known that exogenous trehalose can improve resistances of plants to some abiotic and biotic stresses. Nonetheless, information respecting the molecular responses of tobacco leaves to Tre treatment is limited. Here we show that exogenous Tre can rapidly reduce stomatal aperture, up-regulate NADPH oxidase genes and increase O2•-andH2O2 on tobacco leaves at 2 h after treatment. We further demonstrated that imidazole and DPI, inhibitors of NADPH oxidase, can promote recovery of stomatal aperture of tobacco leaves upon trehalose treatment. Exogenous trehalose increased tobacco leaf resistance to tobacco mosaic disease significantly in a concentration-dependent way. To elucidate the molecular mechanisms in response to exogenous trehalose, the transcriptomic responses of tobacco leaves with 10 (low concentration) or 50 (high concentration) mM of trehalose treatment at 2 or 24h were investigated through RNA-seq approach. In total, 1288 differentially expressed genes (DEGs) were found with different conditions of trehalose treatments relative to control. Among them, 1075 (83.5%) were triggered by low concentration of trehalose (10mM), indicating that low concentration of Tre is a better elicitor. Functional annotations with KEGG pathway analysis revealed that the DEGs are involved in metabolic pathway, biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, protein processing in ER, flavonoid synthesis and circadian rhythm and so on. The protein-protein interaction networks generated from the core DEGs regulated by all conditions strikingly revealed that eight proteins, including ClpB1, HSP70, DnaJB1-like protein, universal stress protein (USP) A-like protein, two FTSH6 proteins, GolS1-like protein and chloroplastics HSP, play a core role in responses to exogenous trehalose in tobacco leaves. Our data suggest that trehalose triggers a signal transduction pathway which involves calcium and ROS-mediated signalings. These core components could lead to partial resistance or tolerance to abiotic and biotic stresses. Moreover, 19 DEGs were chosen for analysis of quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR for the 19 candidate genes coincided with the DEGs identified via the RNA-seq analysis, sustaining the reliability of our RNA-seq data.

众所周知，外源海藻糖（trehalose）可提升植物对部分非生物胁迫与生物胁迫的抵抗能力。然而，有关烟草叶片响应海藻糖处理的分子机制相关研究仍较为匮乏。本研究发现，外源海藻糖可在处理后2小时内快速降低烟草叶片的气孔开度，上调NADPH氧化酶（NADPH oxidase）基因的表达，并诱导超氧阴离子自由基（O₂•⁻）与过氧化氢（H₂O₂）的积累。进一步研究表明，NADPH氧化酶抑制剂咪唑与DPI可促进海藻糖处理后烟草叶片气孔开度的恢复。外源海藻糖可通过浓度依赖型方式显著提升烟草对烟草花叶病的抗性。

为阐明烟草叶片响应外源海藻糖的分子机制，本研究通过RNA测序（RNA-seq）技术，分析了分别经10 mM（低浓度）与50 mM（高浓度）海藻糖处理2小时或24小时后的烟草叶片的转录组响应。结果显示，相较于对照组，不同海藻糖处理条件下共鉴定得到1288个差异表达基因（differentially expressed genes, DEGs）。其中，1075个（占比83.5%）差异表达基因由低浓度海藻糖（10 mM）诱导产生，表明低浓度海藻糖是更高效的诱导子。

通过京都基因与基因组百科全书（KEGG）通路分析进行功能注释发现，这些差异表达基因主要参与代谢通路、次生代谢物生物合成、植物激素信号转导、植物-病原体互作、内质网蛋白质加工、黄酮类化合物合成以及昼夜节律等生物学过程。基于所有处理条件下核心差异表达基因构建的蛋白质相互作用网络显示，包括ClpB1、热休克蛋白70（HSP70）、DnaJB1样蛋白、通用应激蛋白（universal stress protein, USP）A样蛋白、2个FTSH6蛋白、GolS1样蛋白以及叶绿体热休克蛋白在内的8种蛋白在烟草叶片响应外源海藻糖的过程中发挥核心调控作用。

本研究数据表明，海藻糖可触发一条依赖于钙信号与活性氧（ROS）介导的信号转导通路，这些核心组分可部分赋予植物对非生物与生物胁迫的抗性或耐受性。此外，本研究选取19个差异表达基因进行实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证，结果显示这19个候选基因的表达趋势与RNA-seq分析鉴定得到的差异表达基因结果一致，证实了本研究RNA-seq数据的可靠性。