小麦花后干旱胁迫下不同磷素运筹对胚乳细胞程序性死亡的调控

Regulation of programmed cell death in endosperm by different phosphorus managements under post-anthesis drought stress in wheat

【目的】探明花后干旱胁迫下不同磷素运筹对小麦胚乳细胞程序性死亡(PCD)的影响,验证不同磷素运筹延缓胚乳PCD进程,减轻花后干旱胁迫危害的效果。【方法】选用冬小麦‘新冬20号’为材料进行裂区试验,主因素为水分,副因素为磷肥施用方法。两个水分处理为:干旱胁迫(开花后停止灌水)和适水条件(开花至成熟,每12天进行一次灌水)。在P2O5施用量105 kg/hm2下,设置3种磷肥运筹方式:P1(返青期一次性施用)、P2(返青和拔节期各追施50%)、P3(返青、拔节和灌浆期分别追施40%、30%和30%的磷肥)。花后每7天取小麦穗样一次,共取样5次。观察了穗中部籽粒胚乳细胞超微结构、酸性磷酸酶(ACPase)化学定位、胚乳细胞核数以及胚乳细胞基因组中的DNA片段,分析了胚乳细胞ACPase活性和胚乳PCD相关基因的相对表达量。【结果】1)适水和干旱胁迫条件下,P3处理小麦的核变形和核膜凹陷较P1、P2轻,P3处理细胞核变形较少,TUNEL检测P3处理细胞核被标记数目较少,P3延长了胚乳细胞核变形至解体的进程。2)干旱胁迫下,P3处理DNA片段化出现时期较晚;适水条件下,P3处理DNA片段化出现时期同样较晚,且干旱胁迫下DNA片段化出现的时期较适水条件下提前。3)适水和干旱胁迫下,P3处理细胞核计数显著高于P1、P2处理,核酸酶活性同样显著高于P1、P2处理,P3处理的胚乳细胞ACPase活性较P1、P2处理高。4)dad1和Bcl2基因负向调控PCD进程,干旱胁迫下,dad1基因在花后7天的相对表达量较适水条件下增加,花后14天相对表达量有所下降,P3处理下dad1的相对表达量在花后28天达到峰值,Bcl2基因在花后7天P3处理的相对表达量较适水条件下增加,花后28天P3处理Bcl2的相对表达量达到峰值;适水条件下,dad1基因的表达集中在花后14和28天,Bcl2基因的表达集中在花后21和28天,都表现为P1处理高于P2、P3处理。5)干旱胁迫下,P1处理最大灌浆速率和平均灌浆速率最高;P3处理最大灌浆速率出现时期最晚,籽粒灌浆增重持续时间较长,理论最大千粒重较P1、P2处理高。适水条件下,最大灌浆速率和平均灌浆速率以P2处理最高;P3处理最大灌浆速率出现时期最晚,籽粒灌浆增重持续时间较长,理论最大千粒重较P1、P2处理高。【结论】花后干旱胁迫下,通过3次施磷减少了花后小麦籽粒胚乳细胞DNA的断裂,延缓了细胞核变形、解体的速度,维持了较高的细胞数,有效延缓了胚乳PCD的进程。干旱加剧了胚乳细胞的程序化死亡进程,磷肥分3次追施延缓胚乳细胞程序性死亡的效果较适水条件下更突出,因此,生产上水分亏缺时,磷素运筹可根据小麦生长需要,采取多次施磷方式调控植株营养状况,延缓PCD进程,减轻花后干旱胁迫危害损失程度。

【Objectives】The effects of different phosphorus(P)managements on programmed cell death(PCD)of wheat endosperm cells under post-anthesis drought stress were investigated,and it was verified that different phosphorus managements could delay the process of endosperm PCD and reduce the harm of post-anthesis drought stress.【Methods】The winter wheat Xindong 20 was selected as the material to carry out the regional test.The main factor was water,and the secondary factor was the application method of phosphate fertilizer.The two water treatments were drought stress(DT,stop irrigation after flowering)and suitable water irrigation(WT,flowering to maturity,irrigation every 12 days).Under the P2O5 application rate of 105 kg/hm2,3 kinds of phosphate fertilizer managements were setup,as:P1(applying all P fertilizer at wheat regreening stage),P2(applying 50%P fertilizer at regreening stage and 50%at jointing stage),and P3(applying 40%at regreening stage,30%at jointing stage and 30%at grain filling stage).Wheat spike samples were taken every 7 days after flowering,a total of 5 times.The ultrastructure of endosperm cells,the chemical localization of acid phosphatase(ACPase),the number of endosperm nuclei,and the DNA fragments in the endosperm cell genome were observed.The ACPase activity of endosperm cells and the relative expression of endosperm PCD-related genes were analyzed.【Results】1)Under noremal water and drought stress,the nuclear deformation and nuclear membrane depression of wheat treated with P3 were lighter than those of P1 and P2,the nuclear deformation of P3 was less,and the number of P3 cell nuclei labeled by TUNEL was less,P3 prolonged the process of endosperm nucleus deformation to disintegration.2)Under drought stress,DNA fragmentation occurred late at P3;under suitable water condition,DNA fragmentation appeared later at P3,and the time of DNA fragmentation under drought stress was earlier than that under suitable water condition.3)Under suitable water treatment and drought stress,the number of nuclei in P3 was significantly higher than that in P1 and P2,and the nuclease activity was also significantly higher than that in P1 and P2,ACPase activity in endosperm cells of P3 was higher than that of P1 and P2.4)dad1 negatively regulate PCD process,under drought stress,the relative expression of dad1 genes increased at 7 days post anthesis,and then decreased at 14 days post anthesis.The relative expression of dad1 reached the peak at 28 days post anthesis under P3 treatment.Bcl2 negatively regulate PCD process,At 7 days post anthesis,the relative expression of P3 was higher than that of suitable water,The relative expression of dad1 reached the peak at 28 days post anthesis under P3 treatment.Under suitable water conditions,the expression of dad1 gene was concentrated at 14 and 28 days post anthesis,and the expression of Bcl2 gene was concentrated at 21 and 28 days post anthesis of P1 was higher than that of P2 and P3.5)Under drought conditions,the maximum filling rate and average filling rate P1 were the highest,the maximum filling rate P3 was the latest,the duration of grain filling weight gain P3 was longer,and the theoretical maximum 1000-grain weight of P3 was higher than that of P1 and P2.Under the suitable water condition,the maximum filling rate and average filling rate P2 were the highest,the maximum filling rate P3 appeared the latest,the grain filling weight gain duration P3 was longer,and the theoretical maximum 1000-grain weight of P3 was higher than that of P1 and P2.【Conclusions】Under post-anthesis drought stress,three times of phosphorus application reduced the DNA breakage of endosperm cells in post-anthesis wheat grains,delayed the speed of nuclear deformation and disintegration,maintained a high number of cells,and effectively delayed the process of endosperm PCD.Drought aggravated the programmed cell death process of endosperm cells,and the effect of three times of phosphate fertilizer on delaying the programmed cell death of endosperm cells was more prominent than that under suitable water conditions,therefore.When water is deficient in production,phosphorus management can regulate the nutritional status of plants by multiple phosphorus application according to the growth needs of wheat,delay the PCD process,and reduce the damage degree of drought stress post anthesis.