不同柑橘砧木对缺硼的响应差异及结构变化与代谢机制

发布时间：2018-08-25 11:57

【摘要】：硼是植物必需的微量营养元素之一,对植物的生长发育起着十分重要的作用。柑橘是我国南方的第一大水果,而其大多数产区位于我国土壤有效硼缺乏或严重缺乏的红壤区域。近年来,缺硼造成田间大量柑橘叶片出现黄化现象,严重制约了柑橘的产量和品质。砧木作为提供根系的材料,对柑橘的生长、果实的品质和产量及对其逆境胁迫的适应性和对抗性等方面都有重要的影响。因此研究不同柑橘砧木对缺硼响应的差异及机制,能够为选择优良砧木进行柑橘高效优质生产及对不同砧木采取相配套的施肥措施提供科学理论依据。本研究以枳橙砧木(拟推广)和枳壳砧木(主栽)为材料,采用营养液试验,研究了缺硼胁迫下不同砧木在硼利用能力、硼的吸收转运以及细胞壁特性等方面的差异及机理;利用核磁共振(NMR)和傅里叶红外光谱(FTIR)技术分析了细胞壁有机碳结构及其组分化学结构在缺硼后的变化;借助石蜡组织切片、SEM(扫描电镜)和TEM(透射电镜)等手段观察了缺硼后叶片和根系微观结构上的变化情况;并运用基于GC-MS技术的代谢组学手段深入探讨了枳壳砧木叶片和根系代谢产物与代谢通路对缺硼胁迫的响应及差异。得到的主要结果如下:(1)缺硼胁迫下枳橙砧木与枳壳砧木在植株生长及硼利用能力上的差异缺硼显著抑制了两种砧木的根系生长,明显降低了根系参数(总根长、根总表面积、根体总积和根尖数);而缺硼对枳壳砧木根系参数的抑制程度要大于枳橙。缺硼对枳壳砧木叶片光合色素的破坏程度大于枳橙砧木,且缺硼显著降低了枳壳的净光合速率,对枳橙则没有显著影响,说明枳壳砧木的光合作用更易受到缺硼的影响。这些结果表明枳橙砧木与枳壳砧木对缺硼的反应存在着差异。无论是正常硼处理还是缺硼处理,枳壳砧木的硼含量和硼积累量都高于枳橙砧木;其叶片和根系中提取的细胞壁物质含量也高于枳橙砧木,且原生质体硼占器官总硼的比例显著高于枳橙,说明枳壳砧木的需硼量高于枳橙;低硼胁迫下枳橙砧木的硼利用效率明显高于枳壳砧木,比枳壳高了29.13%,表明枳橙砧木能以较低的硼水平产生更多的干物质。缺硼显著提高了两种砧木叶片和根细胞壁硼占总硼的比例,枳橙分别增加了192.08%和131.03%,高于枳壳砧木的166.67%和112.70%,说明缺硼条件下枳橙砧木将硼优先分配到细胞壁中的能力更强于枳壳砧木。(2)缺硼胁迫下枳橙砧木与枳壳砧木在硼吸收转运上的差异实时荧光定量PCR结果显示,所选择的硼相关基因更多的在枳橙砧木根和老茎中上调表达,而上调表达的基因在枳壳砧木的地上部相对更多。基因Ci NIP5在枳橙和枳壳根中都显著上调表达,促进了低硼条件下根系能吸收更多的硼;在硼积累量上表现为缺硼引起的枳橙砧木根中硼积累量所占植株总硼百分比的上升幅度显著大于枳壳,说明低硼条件下枳橙砧木根的吸收能力可能更强于枳壳。Cm BOR1在枳橙根中上调表达,使得枳橙砧木根中硼相对更多地通过木质部装载运输到茎中,枳壳的这种能力更弱一些,枳橙砧木的茎中硼积累量所占比例上升了近一倍,远大于枳壳的54.65%,也证明了这一点。枳壳更弱的木质部装载能力导致根中的硼不能较好地向地上部运输,只能通过Ci NIP6、Cm BOR1、Ci NIP5和TIP4;1在叶和/或茎中上调表达,将地上部有限的硼分配到需要硼的部位,以满足枳壳砧木地上部对硼更高的需求。综上所述,低硼条件下枳橙砧木根系对硼的持续吸收能力及通过木质部装载持续运往地上部的能力均强于枳壳,而缺硼条件下枳壳砧木地上部的持续再分配能力略强于枳橙,这可能与其高需硼量及由根往地上部的硼转运能力相对较低有关。(3)缺硼胁迫改变了枳橙砧木叶片和根系的膜透性及抗氧化酶活性缺硼抑制了枳橙砧木地上部和根系的生长,根系受缺硼的抑制程度大于地上部;枳橙砧木各部位的硼含量和硼积累量在缺硼后也明显降低。缺硼处理显著提高了叶片和根系的MDA含量,根系提高了55.21%,远高于叶片的23.85%,表明缺硼使得根系的膜系统受损害更加严重。缺硼后,根系中SOD的活性显著上升;叶片和根系中的CAT活性均明显降低,而APX和POD活性则明显升高,其中变化幅度最大的是根系中的POD活性,增幅高达117%,这与POD还参与其他代谢途径有关。(4)缺硼胁迫影响了枳橙砧木叶片和根系的微观结构且抑制了细根的生长发育叶片的石蜡切片显示缺硼处理导致叶片栅栏组织的细胞排列紊乱,海绵组织的细胞数目明显增多,其在叶肉中所占的比例明显高于对照组,且抑制了维管束结构的发育;叶片的透射电镜(TEM)观察显示缺硼植株的叶绿体内积累了较多的淀粉粒,细胞壁有明显的增厚现象,且细胞形态也发生了改变。这些结果表明叶肉组织和维管束结构在短期缺硼后的变化程度还不足以破坏叶片的表皮结构,所以我们在叶片上并没有观察到典型的缺硼症状;然而这种变化程度已然影响到了光合作用的正常进行,造成了淀粉的积累。缺硼条件下,枳橙砧木细根各根系参数的下降幅度均明显大于中根和粗根,降幅均达一半以上,说明缺硼主要抑制了细根的生长发育;中根和粗根总根长和根尖数所占比例显著上升,表明缺硼造成枳橙砧木根尖变粗膨大。根系的石蜡切片显示缺硼导致根尖根冠脱落、分生区细胞增大及其圆锥形结构消失,同时造成了疏导组织发育差,维管束分化不完善;根系的扫描(SEM)和透射(TEM)电镜观察表明,缺硼破坏了细胞内部结构的完整性,液泡肿胀破裂,抑制细胞内物质的合成与维管束的发育,导管口径变小,并伴有堵塞现象,韧皮部筛管口堵塞严重,使细胞壁明显增厚,以上结果都解释了根尖出现短粗膨大的原因。(5)缺硼胁迫破坏了枳橙砧木根系细胞壁的正常化学结构缺硼使枳橙砧木新根的生长受到了明显的抑制;新生部位(新根、新叶、新梢)硼含量和硼积累量都显著降低;缺硼提高了每单位根系鲜重细胞壁物质的含量,且细胞壁硼含量占根总硼的比例显著增加了143.2%。从细胞壁傅里叶红外光谱(FTIR)图谱来看,缺硼降低了细胞壁中果胶及蛋白的相对含量,并影响了细胞壁的正常结构:使细胞壁中的氢键发生改变,破坏了细胞壁大分子之间的连接模式;降低了细胞壁中果胶酯化交联的程度,破坏了细胞壁中果胶的网络结构;且改变了细胞壁蛋白的结构。(6)缺硼胁迫改变了枳壳砧木叶片和根系的代谢产物和代谢途径利用基于气相色谱-质谱联用技术(GC-MS)的代谢组学手段,研究了柑橘枳壳砧木幼苗叶片和根系对缺硼的代谢响应,结果表明缺硼后叶片和根系代谢物和代谢途径的变化存在较大的差异,说明植物不同器官对缺硼的代谢响应是不同的。缺硼会引起叶片和根系中很多代谢物的变化,叶片以糖类和芳香族化合物的变化为主,而根系氨基酸和糖类变化更大。缺硼后在叶片中变化较大代谢物有:蜜二糖和核糖显著增加;半乳糖酸、景天庚酮糖、4-羟基肉桂酸、阿魏酸、棕榈酸、芥子酸显著降低;根系中显著增加的代谢物有:天冬酰胺、苏氨酸、色氨酸、阿魏酸、芥子酸。其中天冬酰胺在根系和叶片中都增加,而半乳糖酸则都减少;阿魏酸和芥子酸的变化趋势在根系和叶片中相反。缺硼导致了磷酸戊糖途径(PPP)的下调、抑制了糖的利用,进而使得叶片中的糖类过量积累;根中大多数氨基酸水平显著增高与蛋白生物合成的降低、回补途径的增强和根尖组织损伤有关。另外,缺硼也对叶片和根系中的莽草酸途径(Shikimate pathway)产生了显著的影响,由于缺硼抑制了体内糖的利用而引起叶片中莽草酸途径中4-羟基肉桂酸、阿魏酸和芥子酸的含量降低,而根中莽草酸途径的整体上调与根尖缺硼症状(根尖木栓化、膨大)密切相关。
[Abstract]:Boron is one of the essential micronutrients for plants and plays an important role in the growth and development of plants. Citrus is the largest fruit in the south of China. Most of its producing areas are located in the red soil areas where the available boron in soil is deficient or seriously deficient. Rootstocks, as root materials, play an important role in citrus growth, fruit quality and yield, adaptability to stress and resistance. Therefore, studying the differences and mechanisms of Different Citrus Rootstocks in response to boron deficiency can help to select good rootstocks for citrus high quality and efficiency. In this study, the difference and mechanism of boron utilization ability, boron uptake and transport, and cell wall characteristics of different rootstocks under boron deficiency stress were studied by nutrient solution test with orange rootstock (to be popularized) and orange shell rootstock (to be planted). Nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the structural and chemical changes of cell wall organic carbon (OC) after boron deficiency. The main results were as follows: (1) Boron deficiency significantly inhibited the root growth of orange rootstocks and trifoliate rootstocks, and significantly reduced the root growth of the two rootstocks. The root parameters (total root length, total root surface area, total root volume and number of root tips) were determined, while the inhibition of boron deficiency on root parameters of trifoliate orange rootstock was greater than that of trifoliate orange. The results showed that the reaction of Citrus aurantii Rootstock to boron deficiency was different from that of Citrus aurantii rootstock. The ratio of protoplast boron to total organ boron of orange rootstock was significantly higher than that of orange, indicating that the boron requirement of orange rootstock was higher than that of orange. The boron utilization efficiency of orange rootstock under low boron stress was significantly higher than that of orange rootstock, 29.13% higher than that of orange rootstock, indicating that orange rootstock could produce more dry matter at lower boron level. The ratio of boron to total boron in leaves and cell walls of Rootstocks increased by 192.08% and 131.03% respectively, which was higher than that of trifoliate rootstocks by 166.67% and 112.70%. This indicated that the boron preferential allocation ability of trifoliate Rootstocks to cell walls was stronger than that of trifoliate rootstocks under boron deficiency stress. (2) The difference of Boron Absorption and transport between trifoliate rootstocks and trifoliate rootstocks under boron defici The results of hetero-real-time fluorescence quantitative PCR showed that the selected boron-related genes were up-regulated in the roots and old stems of orange rootstocks, and the up-regulated genes were up-regulated in the shoots of orange rootstocks. The accumulation of boron in Rootstock Roots of orange caused by boron deficiency increased significantly more than that of Fructus aurantii, suggesting that the absorption capacity of Rootstock Roots of orange under low boron condition might be stronger than that of Fructus aurantii. The boron accumulation in the stem of the rootstock was nearly doubled, which was much higher than that of the Fructus aurantii (54.65%). In summary, the ability of root system to absorb boron and transport boron to the shoot through the xylem of orange rootstock under low boron condition was stronger than that of orange rootstock under boron deficiency condition. The above-ground redistribution ability of Rootstocks was slightly stronger than that of oranges, which might be related to their high boron requirement and relatively low boron transport capacity from root to shoot. (3) Boron deficiency stress changed the membrane permeability and antioxidant enzyme activity of leaves and roots of orange rootstocks. Boron deficiency inhibited the above-ground and root growth of orange rootstocks. The content of MDA in leaves and roots was increased by 55.21%, which was much higher than that in leaves by 23.85%. It indicated that boron deficiency caused more serious damage to the membrane system of roots. The activity of CAT in leaves and roots decreased significantly, while the activity of APX and POD increased significantly. The most significant change was that POD activity in roots increased by 117%, which was related to the participation of POD in other metabolic pathways. (4) Boron deficiency stress affected the microstructure of leaves and roots and inhibited the growth of fine roots. Paraffin section showed that boron deficiency caused disorder of cell arrangement in palisade tissues and increased the number of cells in spongy tissues. The proportion of cells in mesophyll was significantly higher than that in control group, and inhibited the development of vascular bundle structure. These results indicate that the changes of mesophyll and vascular bundle structure after short-term boron deficiency are not enough to destroy the epidermal structure of leaves, so we have not observed the typical symptoms of boron deficiency in leaves; however, the degree of such changes has already been observed. Under the condition of boron deficiency, the decrease of root parameters of fine roots of orange rootstock was more than half of that of medium roots and coarse roots, which indicated that boron deficiency mainly inhibited the growth and development of fine roots, and the proportion of total root length and number of root tips of middle roots and coarse roots increased significantly. The paraffin section of roots showed that boron deficiency caused the root apex shedding, the cell growth in meristematic zone and the disappearance of conical structure. At the same time, it caused the poor development of drainage tissue and the imperfect differentiation of vascular bundles. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observation of roots showed that boron deficiency destroyed the inner structure of cells. The intactness, the swelling and rupture of vacuoles, the inhibition of the synthesis of intracellular substances and the development of vascular bundles, the smaller diameter of ducts, accompanied by blockage, the serious blockage of phloem sieve canal orifice, and the obvious thickening of cell wall, all these results explained why the root tips were short and bulky. (5) Boron deficiency stress destroyed the normal root cell wall of orange rootstocks. Boron deficiency in chemical structure significantly inhibited the growth of new roots of orange rootstocks; boron content and accumulation in new parts (new roots, new leaves, new shoots) decreased significantly; boron deficiency increased the content of cell wall substances per unit root weight, and the ratio of cell wall boron content to total root boron increased significantly by 143.2%. According to FTIR spectra, boron deficiency decreased the relative content of pectin and protein in cell wall, and affected the normal structure of cell wall. (6) Boron deficiency stress altered metabolic products and pathways in leaves and roots of Citrus aurantium rootstocks. The metabolic responses of leaves and roots of citrus aurantium Rootstocks to boron deficiency were studied by GC-MS. The metabolites and metabolic pathways varied greatly, suggesting that different organs of plants responded differently to boron deficiency. Boron deficiency could cause changes in many metabolites in leaves and roots. The changes of carbohydrates and aromatic compounds in leaves were dominant, while the changes of amino acids and carbohydrates in roots were greater. Metabolites were: significant increases in melittose and ribose; significant decreases in galactic acid, Sedum heptanone sugar, 4-hydroxycinnamic acid, ferulic acid, palmitic acid and sinapic acid; and significant increases in root metabolism were asparagine, threonine, tryptophan, ferulic acid and sinapic acid, in which asparagine increased in roots and leaves, while galactic acid decreased Boron deficiency led to the down-regulation of pentose phosphate pathway (PPP), inhibited the utilization of sugar, and consequently led to the excessive accumulation of carbohydrates in leaves. The significant increase of most amino acids in roots was related to the decrease of protein biosynthesis, the enhancement of replenishment pathway and the damage of root tips. In addition, boron deficiency also had a significant effect on the Shikimate pathway in leaves and roots. As boron deficiency inhibited the utilization of sugar in vivo, the contents of 4-hydroxycinnamic acid, ferulic acid and sinapic acid in the shikimate pathway decreased, while the shikimate pathway in roots was up-regulated and the symptoms of boron deficiency in root tips were observed. Cork and expansion are closely related.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S666

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