【作者】 刘桂东；

【导师】 吴礼树； 姜存仓；

【作者基本信息】 华中农业大学 ， 植物营养学， 2014， 博士

【摘要】 硼是植物必需的微量元素之一,而赣南脐橙产区位于我国土壤有效硼含量极度缺乏的区域。近年来,缺硼导致该产区主栽品种纽荷尔脐橙出现大量叶片黄化现象,严重制约了脐橙产业的发展。砧木在脐橙的生长方面起着重要的作用,因此开展不同砧木脐橙硼营养机理以及纽荷尔脐橙缺硼响应机制的研究,有助于为下一步针对不同砧木采取相配套的施肥措施或着是培育优良砧木以扩大脐橙种植面积并保证高产优质提供依据。本论文采用营养液培养试验,研究了纽荷尔脐橙在硼的利用能力、吸收分配以及移动性方面的砧木效应,探讨了枳橙砧木与枳壳砧木缺硼反应差异及其机理；利用化学测定的方法研究了纽荷尔脐橙叶片缺硼后细胞壁组成的变化,借助电镜(SEM和TEM)、能谱(XPS和XRD)和光谱(FTIR)等生物物理方法分析了缺硼对纽荷尔脐橙叶片表面结构、细胞结构和细胞壁结构的影响,并运用代谢组学技术(GC-MS)比较了不同缺硼程度叶片代谢谱的变化差异。得到的主要结果如下：1.硼充足供应条件下,嫁接于枳橙砧木的纽荷尔脐橙植株各部位硼含量显著低于枳壳砧木纽荷尔脐橙相应各部位的硼含量,而两种砧木脐橙各部位干物重间无显著差异,说明枳橙砧木脐橙需硼量较低,其植株水平上硼的利用能力更高。通过进一步分析体内不同形态硼的相对含量,结果表明枳橙砧木脐橙的R值(半束缚态硼/自由态硼)显著高于枳壳砧木的,即硼在枳橙砧木脐橙中的跨膜能力更强,在细胞水平上解释了枳橙砧木纽荷尔脐橙硼利用能力更强的原因。2.新吸收的硼优先往新叶中转运,且在不同砧木脐橙老叶中的分配模式存在差异,新吸收的硼往枳橙砧木纽荷尔脐橙老叶中的分配远高于往枳壳砧木脐橙老叶中的分配,这些结果可以在一定程度上解释为什么缺硼条件下嫁接于枳壳砧木的纽荷尔脐橙老叶更容易出现缺硼症状。3.脐橙叶片施硼可以发生转运,下部老叶施硼后往新叶中的转运大于往其他老叶中的转运,枳橙与枳壳砧木纽荷尔脐橙中分别至少有吸收总硼的15.8%和17.6%转运到其他部位,但两种砧木之间硼的移动性并没有显著差异；叶片施硼不能转运到根中,反而还会降低根的硼含量和积累量,在缺硼的土壤中如果只采取叶片施硼的方式,反而可能会加重根的缺硼症状,从而影响根对其它元素的吸收。因此,农业生产中在缺硼的柑橘园施硼方式应以土施为主,叶施为辅。4.枳橙与枳壳实生幼苗在缺硼反应方面存在明显的基因型差异。缺硼后枳壳幼苗地上部的生长受到明显的抑制且出现黄化叶片,而对枳橙幼苗地上部的生长没有显著的影响。与处理之前各自植株的硼含量相比,缺硼35天后枳橙各部位硼含量都下降,但枳壳下降的程度更明显。硼正常供应条件下枳壳幼苗叶片及其细胞壁硼含量都明显高于枳橙幼苗相应的硼含量,但缺硼后枳橙叶片细胞壁硼含量增加而枳壳叶片细胞壁硼含量却降低。缺硼使枳橙与枳壳叶片细胞壁中共价结合态果胶含量都下降,而离子结合态果胶组分的增加只发生在枳橙幼苗叶片细胞壁中,枳壳中没有明显的变化。上述结果表明,枳橙砧木苗需硼量较枳壳苗更低,但在缺硼条件下又具有更强的硼吸收能力以及往细胞壁中分配硼的能力,再加上离子结合态果胶(CDTA可溶性果胶组分)的增加,对于维持细胞壁的完整性产生了重要的影响,从而增加了枳橙砧木对缺硼的耐受性。5.枳橙砧木纽荷尔脐橙上部叶(处理后形成)的缺硼程度远大于下部叶(处理时已存在),因此叶片黄化、卷曲等症状只出现在上部叶。缺硼提高了叶片细胞壁的提取率,改变了细胞壁的组成。缺硼使木质素和离子结合态果胶相对含量没有发生变化,共价结合态果胶含量下降,而半纤维素和纤维素相对含量显著增加,推测缺硼加剧了次生细胞壁的合成。缺硼显著降低了上部叶细胞壁中离子结合态果胶硼含量而对下部叶的无明显影响,共价结合态果胶和半纤维素硼含量缺硼后上部叶和下部叶都显著下降,而纤维素硼含量则都没有明显变化。上述结果表明缺硼后硼会优先分配到细胞壁离子结合态果胶中。6.严重缺硼破坏了脐橙叶片表皮结构,角质和蜡质等物质减少；细胞壁蛋白及果胶含量减少而纤维素和半纤维素含量增加；缺硼降低了纤维素结晶度并使细胞壁多糖糖苷键的构型方式发生变化；缺硼使细胞壁中的氢键及多糖交联方式发生改变；缺硼程度决定了细胞壁结构的破坏程度从而决定了缺硼症状的出现。7.上部叶和下部叶在正常生长条件下自身的代谢谱之间存在较大的差异。轻度缺硼的下部叶中蔗糖、葡萄糖和果糖含量都没有发生显著变化而淀粉含量显著增加,严重缺硼的上部叶除蔗糖含量无明显变化外,葡萄糖、果糖和淀粉含量都显著增加,而且缺硼处理的根系中可溶性总糖、蔗糖和淀粉含量也都显著增加。三羧酸循环中的柠檬酸和琥珀酸含量在缺硼处理的上部叶和下部叶中均显著下降,而苹果酸含量在上部叶中无明显变化但在下部叶中显著增加。与糖代谢相关性较高的肌醇和奎尼酸含量均显著增加。上述结果说明脐橙缺硼后会抑制其体内糖的利用并使三羧酸循环模式发生改变。更多还原

【Abstract】 Boron (B) is an essential micronutrient element required for growth and development of higher plants. Ganzhou city in Jiangxi province in south China is a predominant region of navel orange production, where the hot water soluble B concentration is extremely low. In recent years, leaf chloresis a symptom of B deficiency occurs in the main local cultivar Newhall navel orange (Citrus sinensis Osb.), meanwhile the tree vigor declines rapidly after fruit set, and therefore affects fruit yield and quality in the coming years. Rootstock can greatly affect the growth and development of navel orange. Therefore, it is significant to carry out research works on the mechanism of B efficiency in navel orange grafted on different rootstocks and on the response mechanism of B deficiency on the cell wall structure and metabolism in navel orange plants. Furthermore, the study could provide a scientific basis for the study on perennial citrus rootstock-scion interaction of B nutrition and the development of citrus industry. In the present study, we compared the capacity of boron utilization, boron absorption and distribution, and boron mobility in Newhall navel orange plants grafted on citrange (Citrus sinensis (L.) Osb.×Poncirus trifoliate (L.) Raf.) and trifoliate orange (Poncirus trifoliate (L.) Raf), investigated the changes in cell wall composition and the structure of leaf, cell, and cell wall using the chemical extraction and biophysical methods (SEM, TEM, XPS, XRD, and FTIR) under B limitation, and analyzed the changes in metabolism profiling of leaves in Newhall navel orange plants due to B deficiency using metabolomics method (GC-MS) by hydroponic experiment. The main results were summarized as following:1. The relatively high B concentration observed in plants grafted on trifoliate orange suggested higher demand for B than those grafted on citrange, suggesting that the utilization efficiency of B in citrange-grafted plants was greater than that in trifoliate orange-grafted plants at whole plant level. There existed a balance for the B forms within plants to maintain normal growth. R value (R=semi-bound B/free B) was much higher in citrange-grafted plants than in trifoliate orange-grafted plants under same B supply level, suggesting that B transmembrane transport is easier in citrange than in trifoliate orange. This explained why citrange-grafted plants had less B but produced relatively more dry mass at cellular level.2. The newly absorbed B in the new leaves was much higher than that in the lower-old leaves and the upper-old leaves in both grafted plants. Citrange-grafted plants showed higher newly acquired total B content and B concentration in both lower-old and upper-old leaves than those in trifoliate-orange-grafted plants. These results suggest that different rootstocks affect the pattern of B distribution in the old leaves between citrange and trifoliate orange, resulting in greater proportion of B partitioning into the old leaves of citrange than trifoliate orange. This difference of B distribution between the two grafted plants may result in the good performance in the old leaves in citrange-grafted plants with limited-B conditions.3. Foliar application of10B to the lower-old leaves resulted in B translocation to the upper-old leaves and the new leaves with preference mainly to the new leaves in both citrange and trifoliate orange when root B supply was relatively low. However,10B sprayed to the lower-old leaves not only did not increase the abundance percentage of10B in the root, but also reduced B concentration and the total B content in the root. At least15.8%and17.6%of the absorbed10B was translocated to other plant parts in citrange-and trifoliate-orange-grafted plants, respectively. There was no significant difference for the B mobility between citrange and trifoliate orange.4. After35days under B deprivation, shoot dry mass in trifoliate orange decreased by28%, but shoot dry mass of citrange was not significantly affected. Root growth of both types of rootstock seedlings was inhibited, but the trifoliate orange was affected more than the citrange. In comparison with B concentrations in plants prior to the commencement of B treatments, B deprivation for35days decreased B concentration in various parts of citrange plants, and the reduction was much greater in trifoliate orange plants. Trifoliate orange seedlings contained higher B concentration and total B in cell wall on a dry leaf basis than citrange subject to5μM B treatment. However, the proportion of leaf B allocated in cell wall was higher in citrange than trifoliate orange when B supply was deficient in the nutrient. The changes in pectin composition in cell wall due to B deprivation differed between citrange and trifoliate orange. The decreased uronic acid (UA) content in the Na2CO3-soluble pectin was observed in both rootstock, but the increased UA content in CDTA-soluble pectin was observed only in citrange. These results demonstrated that a combination of greater B uptake ability, greater B accumulation in cell walls, as well as the increased CDTA-soluble pectin, under limited external B supply, contribute to the integrity of cell walls in citrange and therefore increased tolerance to B deficiency.5. The extent of B deficiency in the upper leaves was more serious than that in the lower leaves. Therefore, the symptoms of curling of the leaves and leaf chlorosis were observed only in the upper leaves of B-deficient plants. Boron deficiency increased cell wall content and changed the cell wall composition. Boron deficiency had no significant effect on the lignin concentration in both upper leaves and lower leaves, and hardly affected the ionically bound pectin. However, boron deficiency significantly increased the relative hemicellulose and cellulose concentrations, and decreased covalently bound pectin in both upper and lower leaves. These results suggested that B deficiency probably enhanced the synthesis of secondary cell wall. Boron deficiency significantly decreased the B concentration in ionically bound pectin in the upper leaves but not in the lower leaves. The B concentrations in covalently bound pectin and hemicellulose was both decreased in the upper-and lower-leaves due to B deficiency, while there was no significant difference in the cellulose B concentration between the control and deficient-B plants. The results indicated that B seemed to be preferentially translocated to the ionically bound pectin of cell walls under B limitation.6. Seriously B deficiency destroyed the epidermis structure of navel orange leaves, and decreased the content of cutin and wax. Boron deficiency reduced the content of cell wall proteins and pectin but increased the content of cellulose and hemicellulose. The crystallinity of the cellulose and the anomeric configuration of carbohydrates in cell walls were largely destroyed by B deficiency. Furthermore, the mode of hydrogen bonding and the linkage pattern among cell wall components were also destroyed by B deficiency. Therefore, we speculated that the amount of wall components is not decisive for B deficiency symptoms, but that rather structural changes within these fractions are important.7. The metabolite profiling of the upper and lower leaves in orange plants showed remarkable differences under adequate B conditions. Boron deficiency increased the contents of glucose and fructose in upper leaves, but did not change the concentration of sucrose. In contrast, there were no significant differences in sucrose and hexose (glucose and fructose) contents of lower leaves between the B-deficient and control plants. The starch content was higher in both upper and lower leaves in B-deficient than in control plants. In addition, the contents of soluble sugars, sucrose, and starch in roots on a dry weight basis were higher in B-deficient than in control plants. Citrate and succinate decreased very strongly in both the upper and lower leaves under B deficiency. The increased level of malate occurred only in the lower leaves of B-deficient plants. The concentrations of inositol and quinate, which were highly associated with sugar metabolism, increased due to B deficiency. All these results suggested that boron deficiency inhibited sugar usage and altered TCA cyclic flux modes in citrus plants.更多还原