Table_4_Screening of Key Proteins Affecting Floral Initiation of Saffron Under Cold Stress Using iTRAQ-Based Proteomics.xls

BackgroundSaffron crocus (Crocus sativus) is an expensive and valuable species that presents preventive and curative effects. This study aimed to screen the key proteins affecting the floral initiation of saffron under cold stress and thus increasing yield by regulating the temperature.ResultsProtein expression profiles in flowering and non-flowering saffron buds were established using isobaric tags for relative or absolute quantitation (iTRAQ). A total of 5,624 proteins were identified, and 201 differentially abundant protein species (DAPs) were further obtained between the flowering and non-flowering groups. The most important functions of the upregulated DAPs were “sucrose metabolic process,” “lipid transport,” “glutathione metabolic process,” and “gene silencing by RNA.” Downregulated DAPs were significantly enriched in “starch biosynthetic process” and several oxidative stress response pathways. Three new flower-related proteins, CsFLK, CseIF4a, and CsHUA1, were identified in this study. The following eight key genes were validated by real-time qPCR in flowering and non-flowering top buds from five different growth phases: floral induction- and floral organ development-related genes CsFLK, CseIF4A, CsHUA1, and CsGSTU7; sucrose synthase activity-related genes CsSUS1 and CsSUS2; and starch synthase activity-related genes CsGBSS1 and CsPU1. These findings demonstrate the important roles played by sucrose/starch biosynthesis pathways in floral development at the mRNA level. During normal floral organ development, the sucrose contents in the top buds of saffron increased, and the starch contents decreased. In contrast, non-flowering buds showed significantly decreased sucrose contents under cold stress and no significant changes in starch contents compared with those in the dormancy stage.ConclusionIn this report, the protein profiles of saffron under cold stress and a normal environment were revealed for the first time by iTRAQ. A possible “reactive oxygen species–antioxidant system–starch/sugar interconversion flowering pathway” was established to explain the phenomenon that saffron does not bloom due to low temperature treatment.

背景：番红花（Crocus sativus）是一种珍贵且具有预防及治疗效果的物种。本研究旨在筛选在低温胁迫下影响番红花花芽起始的关键蛋白，通过调节温度来提高产量。结果：采用同位素标记相对或绝对定量（iTRAQ）技术，建立了开花与非开花番红花花芽的蛋白质表达谱。共鉴定出5,624种蛋白，并在开花组与非开花组之间获得了201种差异丰度蛋白（DAPs）。上调的DAPs的主要功能包括“蔗糖代谢过程”、“脂质运输”、“谷胱甘肽代谢过程”以及“RNA介导的基因沉默”。下调的DAPs在“淀粉合成过程”和多个氧化应激响应途径中显著富集。本研究新鉴定出三种与开花相关的蛋白，分别为CsFLK、CseIF4a和CsHUA1。通过对五个不同生长阶段的开花与非开花顶芽进行实时定量PCR验证，确认了以下八个关键基因：与花诱导和花器官发育相关的基因CsFLK、CseIF4A、CsHUA1和CsGSTU7；与蔗糖合酶活性相关的基因CsSUS1和CsSUS2；以及与淀粉合酶活性相关的基因CsGBSS1和CsPU1。这些发现证实了蔗糖/淀粉生物合成途径在花发育的mRNA水平上的重要作用。在正常的器官发育过程中，番红花顶芽中的蔗糖含量增加，而淀粉含量减少。相反，在低温胁迫下，非开花芽的蔗糖含量显著降低，与休眠阶段相比，淀粉含量没有显著变化。结论：本研究首次通过iTRAQ技术揭示了番红花在低温胁迫和正常环境下的蛋白质谱。建立了一种可能的“活性氧-抗氧化系统-淀粉/糖相互转化开花途径”，以解释番红花因低温处理而未能开花的现象。