Synchrotron X-ray Fluorescence Microtomography Profiling of Malus xiaojinensis Provides Insights into Mechanisms of Divalent Metals Transport Subjected to Iron Deficiency
Synchrotron X-ray Fluorescence Microtomography Profiling of Malus xiaojinensis Provides Insights into Mechanisms of Divalent Metals Transport Subjected to Iron Deficiency
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DOI：
10.21273/hortsci.50.6.801
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发表时间：
2015-06
期刊：
影响因子：
1.9
通讯作者：
Meiling Zhang;Ming Chen;Zhen Wang;Ting Wu;Yi Wang;Xinzhong Zhang;Zhenhai Han
中科院分区：
文献类型：
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作者：
Meiling Zhang;Ming Chen;Zhen Wang;Ting Wu;Yi Wang;Xinzhong Zhang;Zhenhai Han
嫁接已广泛应用于果园管理中，因为砧木可以使树木更能耐受环境胁迫。缺铁是全球苹果生产的主要限制环境因素之一。人们对器官、细胞和亚细胞水平的缺铁适应性反应进行了系统研究，但组织水平上Fe与其他二价阳离子之间的相互作用却鲜为人知。利用同步辐射 X 射线荧光 (SR-mXRF) 绘制了小金海棠纵向和横向根样本中选定元素 Fe、Zn、Mn、Ni 和 Co 的位置图。缺铁导致表皮和皮质中五种微量营养素的相对含量显着增加。木质部距根尖1000～2000mm区间，同一位置缺铁条件下和富铁条件下根系元素含量比值明显增加。根部铁吸收和转运相关基因的表达分析表明，MdNramp3和MxCS1显着增加。这些结果表明缺铁促进了木质部微量营养素的长距离运输，MdNramp3和MxCS1可能在此过程中发挥重要作用。重要的是，这项研究直接提供了组织水平上的可视化二价金属分布，以更好地了解苹果砧木中的金属吸收过程。铁作为重要的微量营养素之一，参与许多重要的生理生化过程。尽管环境中铁含量丰富，但由于碱性和钙质土壤，植物经常缺铁（Guerinot 和 Yi，1994）。在长期的进化过程中，植物已经进化出诸如基于还原的策略I和基于螯合的策略II等紧密机制来吸收、运输、利用或储存铁（Marschner等，1986）。在Fe吸收、细胞内或细胞间以及长距离转运的过程中，涉及许多基因，包括根表皮细胞膜转运蛋白：铁相关转运蛋白1（IRT1）和天然抗性相关巨噬细胞蛋白1（Nramp1）（Curie等，2000；Korshunova等，1999），液泡膜转运蛋白（Nramp3， Nramp4）和液泡铁转运蛋白 1（Kim 等，2006；Lanquar 等，2005）、铁还原酶缺陷 3（FRD3）、柠檬酸合酶 1（CS1）、烟酰胺合酶 1（NAS1）和黄色条纹 1 样（YSL）（Kobayashi 和 Nishizawa，2012）。在这些基因中，ZIP（ZRT，IRT样蛋白）金属转运蛋白家族和NRAMP家族一直引起我们的关注。植物对金属元素的吸收是一个复杂的过程；它们经常使用相同的运输系统进行吸收、运输或储存（Rogers et al., 2000）。研究表明拟南芥对锌和铁的吸收存在竞争（Fukao 等，2011）。缺锰可以有效地增加铁的运输（Yang et al., 2008）。 Ni积累可能作为缺铁信号并诱导缺铁反应，从而上调铁吸收基因的表达（Nishida等人，2012）。此外，之前的所有研究都是针对Fe与其他二价金属之间的相互作用进行的。木本植物缺铁时吸收二价金属离子的机制鲜为人知。小金海棠是中国的原生苹果砧木，具有高效铁吸收的特点（Han et al., 1998；Wu et al., 2012；Zha et al., 2014）。本研究利用SR-mXRF技术检测了策略I植物小金海棠根中二价金属的空间分布以及缺铁处理下根中铁吸收和转运相关基因的表达情况，这将有助于研究植物缺铁时二价金属的组织特异性分布。材料和方法植物栽培和样品制备。小金海棠幼苗在含有0.5 mg/L吲哚-3-丁酸(IBA)和0.5 mg/L 6-苄氨基嘌呤的Murashige和Skoog (MS)培养基上繁殖1个月，然后转移到含有0.5 mg/L IBA的半浓度改良MS培养基上生根1个半月。将生根的幼苗移至一半强度的改良 Hoagland 营养液中 1 周，然后切换至 Hoagland 表 1. 用于通过实时聚合酶链式反应定量转录物的引物序列。基因引物序列 MdIRT1 Fwd 5# TTGACAAGGGAGAAAACGGAGAC 3# Rev 5# AACAACTGAATGGACAATGATACCC 3# MdCS1 Fwd 5# GTTCTCGTCTCGGGCAACTGTC 3# Rev 5# TTCCTCTCATTCCACCAATCACC 3# MdFRD3 Fwd 5# GTGGAAGATGCCTGTTGGTGTT 3# 修订版 5# CAATAGATACTCCTGCTGCCGC 3# MdNAS1 转发 5# CGATGTTTCCAAGTTATGCCAA 3# 修订版 5# TTCTCCAAAAGTCCTTCTGCCT 3# MdNRAMP1 转发 5# CCGAAGTTATTGGCACAAGCGTT 3# 修订版 5# TTCACATAACCCATTTCCCCGA 3# MdNRAMP3 转发 5# GACGACGACTGCCAAACGCCG 3# 修订版 5# CCACAGCACCATCCTCGCCCA 3# MdNRAMP4 转发 5# CGGCCCTAGTGATGGTAATC 3# 修订版 5# GCTACATACCCGGCAGTAAA 3# MdYSL5 转发 5# GTCCATGTTTGTGAGCCAAATC 3# Rev 5#GGATACGCACTTCCAGGTATTC3# b-Actin Fwd 5# TGGTGAGGCTCTATTCCAAC 3# Rev 5# TGGCATATACTCTGGAGGCT 3# 2015 年 2 月 13 日接收出版。2015 年 4 月 16 日接受出版。我们感谢国家自然科学基金会提供的财政支持中国 (No. 31272139和31401840），北京市自然科学基金（6154028）。感谢中国科学院高能物理研究所北京同步辐射装置4W1B终端站授予m-XRF束流时间并进行SR-mXRF实验。 4W1Bare 的工作人员感谢他们对测量和数据缩减的支持。这些作者对这项工作做出了同等的贡献。应向谁提出重印请求；电子邮箱：rschan@cau.edu.cn。园艺科学卷。 50(6) JUNE 2015 801 图 1. 使用 SR-mXRF 技术绘制的小金海棠根部纬向剖面的元素图。上图是显微镜下观察到的根部样品的纵切面图像。以 50 毫米的步长收集地图的 SR-mXRF 信号。 +Fe 和 –Fe 的映射面积分别为 800 mm · 800 mm 和 650 mm · 650 mm。样品在 +Fe 条件 (40 mM FeNaEDTA) 或 –Fe 条件 (0 mM FeNaEDTA) 下处理 3 d。比例尺 = 100 毫米。条形颜色从蓝色到红色表示铁含量从低到高。 SR-mXRF = 同步辐射
Grafting has been widely used in orchard management because the rootstock can make the tree more tolerant to environmental stresses. Iron deficiency is one of the major limiting environmental factor in apple production worldwide. Systematic research has been made about iron-deficiency adaptive responses in the level of organs, cells, and subcells, whereas the interactions between Fe and other divalent cations in tissue level are little known. Synchrotron radiation X-ray fluorescence (SR-mXRF) was used to map the location of selected elements Fe, Zn, Mn, Ni, and Co in the longitudinal and latitudinal root samples of Malus xiaojinensis. Iron deficiency induced a significant increase in the relative contents of five micronutrients in epidermis and cortex. The ratio of element contents of roots under Fe-deficient condition and Fe-sufficient condition at same position increased obviously in the section of 1000to 2000-mm distance from the root tip in xylem. Expression analysis of iron absorptionand transport-related genes in roots showed thatMdNramp3 andMxCS1 increased significantly. These results indicated that iron deficiency promoted the long-distance transport of micronutrients in xylem, and MdNramp3 and MxCS1 might play an important role in this process. Importantly, this study directly provides visual divalent metals distribution in tissue level for an improved understanding of metal absorption process in apple rootstock. As one of the important micronutrient, iron participates in many important physiological and biochemical processes. Although iron is abundant in the environment, plants often suffer from iron deficiency because of the alkaline and calcareous soils (Guerinot and Yi, 1994). In the long-term evolution process, plants have evolved into tight mechanisms such as reduction-based strategy I and chelation-based strategy II for iron uptake, transport, utilization, or storage (Marschner et al., 1986). In the process of Fe absorption, intracellular or intercellular and long-distance transport, many genes are involved including the root epidermal cell membrane transporters: iron-related transporter 1 (IRT1) and natural resistance-associated macrophage protein 1 (Nramp1) (Curie et al., 2000; Korshunova et al., 1999), vacuolar membrane transporter (Nramp3, Nramp4) and vacuolar iron transporter 1 (Kim et al., 2006; Lanquar et al., 2005), ferric reductase defective 3 (FRD3), citrate synthase 1 (CS1), nicotianamine synthase 1 (NAS1), and yellow stripe1-like (YSL) (Kobayashi and Nishizawa, 2012). Among these genes, the ZIP (ZRT, IRT-like proteins) metal transporter family and NRAMP family catch our attention all the time. The absorption of metal elements in plants is a complicated process; they often use the same transport system for absorption, transportation, or storage (Rogers et al., 2000). Research suggests that there is a competition between Zn and Fe absorption in Arabidopsis thaliana (Fukao et al., 2011). Mn deficiency can increase the transport of Fe efficiently (Yang et al., 2008). Ni accumulation may act as an iron-deficiency signal and induce the Fe-deficient response to upregulate Fe absorption genes expression (Nishida et al., 2012). In addition, all of the previous studies were performed with the interaction between Fe and other divalent metals. The mechanism of divalent metal ions absorption under iron deficiency in woody plants is little known. Malus xiaojinensis is a native apple rootstock in China and has been characterized by its high efficiency for iron uptake (Han et al., 1998; Wu et al., 2012; Zha et al., 2014). In this study, we detected the spatial distribution of selected divalent metals in the root of Malus xiaojinensis, which belongs to the strategy I plant by using SR-mXRF as well as the expression of iron absorptionand transport-related genes in roots under irondeficiency treatment, which will help to investigate the tissue-specific distribution of divalent metals when plants are subjected to iron deficiency. Materials and Methods Plant cultivation and sample preparation. The seedlings of Malus xiaojinensis were propagated on Murashige and Skoog (MS) medium with 0.5 mg/L Indole-3-Butytric acid (IBA) and 0.5 mg/L 6-Benzylaminopurine for one month and transferred to one-halfstrength modified MS medium with 0.5 mg/L IBA for rooting for one month and a half. The rooted seedlings were moved to onehalf-strength modified Hoagland nutrient solution for 1 week and switched to Hoagland Table 1. Primer sequences for the quantification of transcripts by real-time polymerase chain reaction. Gene Primer sequences MdIRT1 Fwd 5# TTGACAAGGGAGAAAACGGAGAC 3# Rev 5# AACAACTGAATGGACAATGATACCC 3# MdCS1 Fwd 5# GTTCTCGTCTCGGGCAACTGTC 3# Rev 5# TTCCTCTCATTCCACCAATCACC 3# MdFRD3 Fwd 5# GTGGAAGATGCCTGTTGGTGTT 3# Rev 5# CAATAGATACTCCTGCTGCCGC 3# MdNAS1 Fwd 5# CGATGTTTCCAAGTTATGCCAA 3# Rev 5# TTCTCCAAAAGTCCTTCTGCCT 3# MdNRAMP1 Fwd 5# CCGAAGTTATTGGCACAGCGTT 3# Rev 5# TTCACATAACCCATTTCCCCGA 3# MdNRAMP3 Fwd 5# GACGACGACTGCCAAACGCCG 3# Rev 5# CCACAGCACCATCCTCGCCCA 3# MdNRAMP4 Fwd 5# CGGCCCTAGTGATGGTAATC 3# Rev 5# GCTACATACCCGGCAGTAAA 3# MdYSL5 Fwd 5# GTCCATGTTTGTGAGCCAAATC 3# Rev 5#GGATACGCACTTCCAGGTATTC3# b-Actin Fwd 5# TGGTGAGGCTCTATTCCAAC 3# Rev 5# TGGCATATACTCTGGAGGCT 3# Received for publication 13 Feb. 2015. Accepted for publication 16 Apr. 2015. We acknowledge financial supports which were provided by the National Natural Science Foundation of China (No. 31272139 and No. 31401840), Beijing Natural Science Foundation (No. 6154028). We extend gratitude to 4W1B end station of Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, where the m-XRF beam time was granted and experiment of SR-mXRFproceeded. The staffmembers of 4W1Bare acknowledged for their support in measurements and data reduction. These authors contributed equally to this work. To whom reprint requests should be addressed; e-mail rschan@cau.edu.cn. HORTSCIENCE VOL. 50(6) JUNE 2015 801 Fig. 1. Elemental maps of latitudinal sections of roots of Malus xiaojinens, using the SR-mXRF technique. The top pictures are the images of longitudinal sections of root samples observed by microscope. The SR-mXRF signals for map were collected at 50-mm steps. The areas mapped for +Fe and –Fe were 800 mm · 800 mm and 650 mm · 650 mm, respectively. The samples were treated under +Fe condition (40 mM FeNaEDTA), or –Fe condition (0 mM FeNaEDTA) for 3 d. Scale bars = 100 mm. The color of the bars from blue to red means the iron content from low to high. SR-mXRF = Synchrotron radiation