Physiological and transcriptome analysis reveals the differences in nitrate content between lamina and midrib of flue-cured tobacco

Background: Nitrate is an important precursor of tobacco-specific nitrosamines (TSNAs) and remarkable difference in nitrate accumulation between lamina and midrib of flue-cured tobacco has long been observed. However, the physiological and molecular mechanisms underpinning this difference remain poorly understood. In this study, physiological and genetic factors impacting nitrate accumulation were identified in pot experiments using flue-cured tobacco K326 with contrasting nitrate content between lamina and midrib.Results: The results showed that three times higher of NO3-N content was observed in midrib than that in lamina, along with lower pigment, NH4-N content, NRA, SSA and GSA in midrib. Transcriptome analysis revealed that expression of genes involved in porphyrin and chlorophyll metabolism, carotenoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, starch and sucrose metabolism, nitrogen metabolism and biosynthesis of amino acids were significantly lower in midrib than in lamina. qRT-PCR results showed that the expression level of nitrate transporter genes LOC107782967, LOC107806749, LOC107775674, LOC107829632, LOC107799198, LOC107768465 decreased by 2.74, 1.81, 49.5, 3.5, 2.64 and 2.96 folds while LOC107789301 increased by 8.23 folds in midrib but not in lamina. Conclusions: Reduced chlorophyll content might result in low carbohydrate formation which is the source of energy and carbon skeleton supply, then the low capacity of nitrogen reduction, assimilation and transportation, and the poor ability of nitrate reallocation but high capacity of accumulation might lead to nitrate accumulation in midrib. The results laid the foundation for reducing nitrate content and TSNA formation in tobacco midribs and their products.

研究背景：硝酸盐是烟草特异性亚硝胺（tobacco-specific nitrosamines, TSNAs）的重要前体物质，长期以来烤烟叶片与主脉间硝酸盐积累的显著差异已被广泛观测到。然而，支撑这一差异的生理与分子机制仍不甚明晰。本研究以叶片与主脉硝酸盐含量差异显著的烤烟K326为试验材料，通过盆栽试验鉴定出影响硝酸盐积累的生理与遗传调控因素。
结果：结果显示，主脉中的硝态氮（nitrate nitrogen, NO3-N）含量为叶片的3倍，且主脉的色素含量、铵态氮（ammonium nitrogen, NH4-N）含量、硝酸还原酶活性（nitrate reductase activity, NRA）、可溶性固形物含量（soluble solid content, SSA）及谷氨酰胺合成酶活性（glutamine synthetase activity, GSA）均低于叶片。转录组分析结果表明，主脉中参与卟啉与叶绿素代谢、类胡萝卜素生物合成、光系统天线蛋白、光合作用、光合生物碳固定、淀粉与蔗糖代谢、氮代谢以及氨基酸生物合成的基因表达水平均显著低于叶片。实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）结果显示，主脉中硝酸盐转运蛋白基因LOC107782967、LOC107806749、LOC107775674、LOC107829632、LOC107799198、LOC107768465的表达量分别下调2.74、1.81、49.5、3.5、2.64和2.96倍，而LOC107789301的表达量则上调8.23倍，该表达变化仅发生于主脉，叶片中未出现显著改变。
结论：叶绿素含量降低可能导致碳水化合物合成不足，而碳水化合物是能量与碳骨架的供应来源；由此引发的氮还原、同化与转运能力下降，加之硝酸盐再分配能力不足但积累能力较强，最终可能导致主脉中硝酸盐的积累。本研究结果为降低烟草主脉及其制品中的硝酸盐含量与烟草特异性亚硝胺生成量奠定了理论基础。