磷素对小麦(Triticum aestivum L.)淀粉粒微观特性的影响及其形成机理

Changes of Micro-Structural Characteristics of Starch Granules and the Mechanisms Under Different Phosphorus Application Rates in Wheat(Triticum aestivum L.)

【目的】磷是小麦生长发育必需的三大营养元素之一。小麦淀粉粒表面存在微孔和微通道结构,对淀粉的生物合成和理化特性有重要影响。探索磷素对淀粉粒微观特性的影响及其形成机理,可为研究不同磷素条件下小麦淀粉的生物合成及品质变化机理提供新途径。【方法】采用新疆冬小麦主栽品种新冬23号为参试材料,设置3种施磷水平,不施磷(CK:P_2O_5 0 kg·hm^(-2))、常规施磷(CP:P_2O_5 105 kg·hm^(-2))和高量施磷(HP:P_2O_5 210 kg·hm^(-2)),所用肥料为重过磷酸钙,于小麦播种后160 d(大约5%的植株已返青)开沟条施,并于花后7、14、21、28和35 d取样。通过扫描电镜观察不同磷素水平成熟期淀粉粒微观结构以及淀粉粒在内源(种子萌发)和外源淀粉酶(淀粉葡萄糖苷酶)酶解条件下的形态变化,同时测定淀粉粒经淀粉葡萄糖苷酶水解后产生的还原糖浓度;通过激光共焦显微镜观察淀粉粒微通道结构的变化;通过实时荧光定量PCR研究籽粒发育过程中淀粉合成酶与降解酶基因表达量,并通过原位杂交技术对淀粉降解酶关键基因进行定位。【结果】不同磷素条件下小麦胚乳淀粉粒形态未发生明显改变,但CP处理条件下更容易观察到淀粉粒的微孔结构,且其内部显示出较多荧光;另外,籽粒发芽6 d时,CP处理条件下淀粉粒表面的孔洞最多。经外源淀粉葡萄糖苷酶处理后,HP和CP处理条件下更容易观察到被水解成两半的A型淀粉粒,同时其产生的还原糖浓度也显著提高,这说明不同磷素条件下淀粉粒表面和高部微观结构发生了变化。常规施磷条件下,小麦胚乳淀粉合成与降解相关酶基因的表达量显著高于不施磷和高量施磷。施磷条件下胚乳外缘amy4、bam1和bam5转录水平提高,且常规施磷条件下转录水平更高。【结论】常规施磷条件下胚乳边缘淀粉酶基因转录水平的提高可能影响了淀粉合成酶和降解酶之间的平衡,进而影响淀粉粒的合成和微观特性变化。

【Objective】Phosphorus is one of the three essential nutrients for crops. Pores and channels in starch granules play an important role in starch biosynthesis and physicochemical characteristics of wheat（Triticum aestivum L.）. This study was conducted to reveal the changes of micro-structural characteristics of starch granules and the mechanisms under different phosphorus application rates in wheat. It may provide more information to improve the pathway of starch biosynthesis. 【Method】The wheat cultivar Xindong 23 was used in this study. On 160 d after sowing（about 5% plants returning green）, three levels of phosphorus treatments, i.e., control（CK, P2O5, 0 kg·hm-2）, conventional phosphorus（CP, P2O5, 105 kg·hm-2） and high phosphorus（HP, P2O5, 210 kg·hm-2）, were applied, and the samples were collected on the 7, 14, 21, 28 and 35 days post anthesis（DPA）. The micro-structural morphology of mature starch granules and the morphological changes of starch granules digested by endogenous（germination） and exogenous（amyloglucosidase） enzymes were examined using scanning electron microscopy. The concentration of reducing sugars after amyloglucosidase digestion of starches was studied, and the channel structure within starch granules was observed using confocal laser scanning microscopy. The expression patterns of the genes involved in starch biosynthesis and degradation were investigated using real-time q PCR and in situ hybridization.【Result】The shape of the starch granules in different phosphorus treatments did not show a significant difference. However, in response to CP treatment, the ＂pinholes＂ on the granules were easier to find, and the fluorescence from the starch granules was stronger and clearly visible. At 6 days post germination（DPG）, the number of ＂pinholes＂ and pits on the starch granules in CP treatment was most abundant. After exogenous amyloglucosidase digestion, the A-type starch granules developed under CP or HP conditions were more prone to be broken into halves than those formed under CK conditions; the concentration of reducing sugars was also higher after amyloglucosidase digestion of the starch granules developed under CP or HP conditions. These data suggest dissimilar micro-structural changes of starch granules induced by different phosphorus application rates. Under conventional phosphorus application rate, the expression levels of the genes involved in starch biosynthesis and degradation were significantly higher than those under the control and high phosphorus conditions during wheat grain filling. In addition, phosphorus application promoted the transcripts of amy4, bam1 and bam5 genes accumulated in the peripheral cells of the endosperm, and this effect was more pronounced in CP treatment. 【Conclusion】Different phosphorus application rates did not significantly affect the shape of starch granules, but the micro-pores and channels in the starch granules and the relative expression levels of the genes involved in starch biosynthesis and degradation were significantly increased by CP application. Furthermore, CP treatment increased the transcript levels of amy4, bam1 and bam5 in the peripheral cells of the endosperm. These transcriptional changes may alter the balance between starch synthase and hydrolase activities, which may have an impact on the micro-structural characteristics of starch granules.