Physiological Process and Mechanism of Senescence in Mutant Rice with Functional Deletion on OsVHA-A1 Gene
-
摘要: 负责编码水稻液泡膜ATP酶A亚基的OsVHA-A1基因在水稻生长发育和抵抗环境压力等诸多方面发挥重要作用。本试验以筛选纯合的OsVHA-A1基因缺失突变体株系及其野生型为材料，发现OsVHA-A1基因缺失突变会导致该突变体水稻生育后期发生严重的早衰现象与减产。两种基因型水稻叶片与根系中抗氧化保护酶活性随生育期呈现动态变化，孕穗期后突变体根系和叶片的衰老指标（包括H2O2含量、O2-产生率和MDA含量）均明显高于野生型；与野生型对比，突变体根系衰老程度显著高于其叶片。在早衰前后两个时期，即苗期和灌浆高峰期，对OsVHA-A1基因进行qRT-PCR定量，发现灌浆高峰期时的突变体与野生型叶片和根系中的OsVHA-A1基因相对于苗期均下调表达，V-ATPase酶活性也显著下降，突变体下降幅度大于野生型。利用16S扩增子分析根际土壤微生物组成时发现，灌浆高峰期突变体根际土壤中植物病原菌的含量显著上升。同时，HPLC技术测得突变体根际土壤中对羟基苯甲酸和对香豆酸两种酚酸类物质极显著低于野生型。说明不同基因型水稻根际土壤微生物存在对根系分泌物的选择性利用并诱导根际病原菌的增长。相关性分析结果表明，孕穗期后两种基因型水稻的根系活力下降变化与其功能叶片叶绿素含量及光合速率降低呈一定的相关关系。综上所述，OsVHA-A1基因缺失突变直接介导了水稻根系和叶片早衰的发生，并通过选择性诱导根际病原菌增长而加剧突变体根系与整体老化。Abstract: Vacuolar H+-ATPases (V-ATPase) is a multi-subunit enzyme complex found in the tonoplast of eukaryotes. V-ATPase subunitA(VHA) encoded by OsVHA-A1 gene is crucial in the development and tolerance to environmental stress of rice. This study applied a wild type and a purified mutant rice with a single cytosine deletion from its OsVHA-A1 gene to compare the differences of their phenotypes during the entire growth period. The mutant rice senesced at late-growth stage resulting a significant yield reduction as compared to the wild type. The activities of antioxidant enzymes including SOD, POD and CAT of the two isogene rice lines showed differential temporal patterns.The senescence-induced increases on O2- production as well as H2O2 and MDA contents in the leaves and roots of the mutant rice after booting stage were generally higher than those of the wild type.The performance of the roots tended to be poorer than that of the leaves in the mutant type in comparison with that of the wild type. OsVHA-A1 was significantly down-regulated and the V-ATPase activity significantly declined at grain-filling stage after senescence from seeding stage before senescence for both lines. But the down-regulated OsVHA-A1 expression and reduced V-ATPase activity in the roots and leaves were greater in the mutant rice than those in the wild type. It appeared that OsVHA-A1 played a crucial role in regulating rice senescence.Aside from genetic changes, environmental factors also contributed to the heightened root senescence, as the 16S rDNA sequencing showed that the pathogenic fungi population in the rhizosphere soil increased significantly when the mutant type at its grain-filling stage. In the soil, HPLC analysis identified 5 phenolic acids, i.e., phydroxybenzoic, vanillin, syringate, pcoumaric acid, and ferulic acid. The contents of phydroxybenzoic and pcoumaric acid were extremely significantly lower associated with the mutant than the wild type. It suggested that the pathogens might participate in the process of root senescence. Furthermore, the declined root activity was found correlating with the chlorophyll or photosynthesis decrease in the isogen rice lines after booting stage.It was concluded that the absence of functional OsVHA-A1 directly affected the senescence, whereas the altered underground microbial community further hastened the aging process of the rice plant.
-
Key words:
- senescence /
- antioxidant enzymes /
- V-ATPase /
- rhizosphere microorganism /
- phenolic acid
-
图 1 不同生育期OsVHA-A1基因缺失突变体与其野生型形态学特征
注：WT代表野生型，Mut代表突变体；A为分蘖期，B抽穗期。
Figure 1. Morphological phenotypes of OsVHA-A1 mutant and wild type rice at various development stages
图 3 OsVHA-A1突变体与野生型水稻叶片和根系中OsVHA-A1基因表达与V-ATPase酶活性
注：以苗期野生型叶片中OsVHA-A1基因表达量为对照组(CK)，采用2-△△Ct法计算基因相对表达量。
Figure 3. OsVHA-A1 expression and V-ATPase activity in leaves and roots of mutant and wild type rice
图 4 OsVHA-A1基因缺失突变体与其野生型根际土壤中的细菌和真菌(Groups)α多样性
注：A为细菌，B为真菌。
Figure 4. α-diversity indices based on observe species of fungi and bacteria in rhizosphere soil of wild and mutant type rice
图 5 OsVHA-A1基因缺失突变体与其野生型根际土壤中主要细菌与真菌群落结构(TOP 10)分析
注：A为细菌主要组成群落，B为真菌主要组成群落。
Figure 5. Bacterial and fungal communities in rhizosphere soil of wild and mutant type rice (P < 0.05)
图 6 不同浓度梯度酚酸标准品液相色谱
注：A为对羟基苯甲酸, B为香兰素，C为丁香酸, D为对香豆酸, E为阿魏酸。质量浓度梯度：6.250、3.125、1.563、0.781 mg·L-1。
Figure 6. HPLC standards for phenolic acid of varied concentrations
图 7 OsVHA-A1基因缺失突变体与其野生型水稻根际土壤酚酸液相色谱
注：1为对羟基苯甲酸t=11.57, 2为香兰素t=13.21，3为丁香酸t=14.78, 4为对香豆酸t=14.78, 5为阿魏酸t=30.57。
Figure 7. HPLC for phenolic acids in rhizosphere soil of mutant and wild type rice
图 8 灌浆高峰期OsVHA-A1基因缺失突变体与其野生型水稻根际土壤酚酸含量
Figure 8. Phenolic acid contents in rhizosphere soil of mutant and wild type rice
图 9 早衰后(孕穗期，抽穗期，灌浆高峰期)根系活力与叶绿素相关性分析
注：A为野生型，B为OsVHA-A1基因缺失突变体。图 10同。
Figure 9. Correlation between root activity and leaf chlorophyll content after senescence in different stages
图 10 早衰后(孕穗期，抽穗期，灌浆高峰期)根系活力与光合作用相关性分析
Figure 10. Correlation between root activity and leaf photosynthesis after senescence in different stages
表 1 OsVHA-A1基因缺失突变体与其野生型水稻农艺性状
Table 1. Agronomic traits of OsVHA-A1 mutant and wild type rice
水稻
类型株高
/cm根长
/cm地上部
/cm根干重
/g叶干重
/g有效穗
/穗结实率
/%千粒重
/g野生 127.3±2.5A 18.9±1.4a 108.5±2.1A 1.34±0.1A 4.75±1.5A 11.2±2.1A 88.3±4.6A 25.3±1.5A 突变 86.8±3B 15.8±1.2b 67.5±2.5B 0.73±0.1B 1.26±1.2B 5.8±1.5B 37.6±5.6B 13.5±1.4B 注：同列数据后不同大、小写字母分别表示差异达极显著(P＜0.01)和显著水平(P＜0.05)。 
下载: 导出CSV
-
[1] 王复标, 黄福灯, 程方民, 等.水稻生育后期叶片早衰突变体的光合特性与叶绿体超微结构观察[J].作物学报, 2012, 38(5):871-879. http://d.old.wanfangdata.com.cn/Periodical/zuowxb201205014 [2] 白海齐.水稻灌浆期叶片衰老期间miRNA高通量测序及生物信息学分析[D].杭州: 杭州师范大学, 2015. [3] 郭士伟, 夏士健, 赵学强, 等.超级杂交稻两优培九及其亲本生育后期早衰的内源激素和营养生理研究[J].农业科学与技术:英文版, 2014(11):1914-1918. doi: 10.3969/j.issn.1009-4229.2014.11.021 [4] 谢金水.生育后期养分胁迫对水稻衰老进程影响的蛋白质组学研究[D].南昌: 江西农业大学, 2012. [5] LIM P O, KIM H J, NAM HG. Leaf senescence[J]. Annu Rev Plant Biol, 2007, 58:115-136. doi: 10.1146/annurev.arplant.57.032905.105316 [6] YOSHIDA S. Molecular regulation of leaf senescence[J]. Curr Opin Plant Biol, 2003(6):79-84. http://d.old.wanfangdata.com.cn/Periodical/yyxb201606007 [7] WANG P, SUN X, CHANG C, et al. Delay in leaf senescence of Malus hupehensis, by long-term melatonin application is associated with its regulation of metabolic status and protein degradation[J]. Journal of Pineal Research, 2013, 55(4):424-434. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=110e6484f23d5d5b50e5c840c4240890 [8] OZGUR R, UZILDAY B, SEKMEN A H, et al. Reactive oxygen species regulation and antioxidant drfence in halophytes[J]. Functional Plant Biology Fpg, 2013, 40(8-9):832-847. https://www.ncbi.nlm.nih.gov/pubmed/9328571 [9] HUA C, WANG R. Changes of SOD and CAT activities and MDA content during senescence of hybrid rice and three lines leaves[J]. Acta Botanica Boreali-occidentalia Sinica, 2003, 23(3):406-409. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=xbzwxb200303008 [10] LI Z, WANG F, LEI B, et al. Genotypic-dependent alteration in transcriptional expression of various CAT isoenzyme genes in esl, mutant rice and its relation to H2O2-induced leaf senescence[J]. Plant Growth Regulation, 2014, 73(3):237-248. doi: 10.1007/s10725-013-9884-6 [11] UEDA Y, UEHARA N, SASAKI H, et al. Impacts of acute ozone stress on superoxide dismutase (SOD) expression and reactive oxygen species (ROS) formation in rice leaves[J]. Plant Physiology & Biochemistry Ppb, 2013, 70(1):396-402. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=156d9ed6b39060adf4dc146b3c58042f [12] RIBEIRO C W, KORBES A P, GARIGHAN J A, et al. Rice peroxisomal ascorbate peroxidase knockdown affects ROS signaling and triggers early leaf senescence[J]. Plant Science, 2017, 263:55-65. doi: 10.1016/j.plantsci.2017.07.009 [13] KIM J, CHANG C, TUCKER M L. To grow old:regulatory role of ethylene and jasmonic acid in senescence[J]. Frontiers in Plant Science, 2015, 6(20):20. http://www.ncbi.nlm.nih.gov/pubmed/25688252 [14] MAO J J, ZHAO C C, HUANG F D, et al. Physiological Characterization and Gene Fine Mapping of a Leaf Early Senescence and Salt-sensitive Mutant osles in Rice[J]. Acta Agronomica Sinica, 2014, 40(5):769-778. doi: 10.3724/SP.J.1006.2014.00769 [15] HÄFFNER E, KONIETZKI S, DIEDERICHSEN E. Keeping Control:The Role of Senescence and Development in Plant Pathogenesis and Defense[J]. Plants, 2015, 4(3):449-488. doi: 10.3390/plants4030449 [16] WANG F, LIU J, ZHOU L, et al. Senescence-specific change in ROS scavenging enzyme activities and regulation of various SOD isozymes to ROS levels in psf mutant rice leaves[J]. Plant Physiology & Biochemistry Ppb, 2016, 109:248-261. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=6e8e2f8b721ec0aab79aeb8825b13a86 [17] ZHANG J, ZHANG N, LIU Y X, et al. Root microbiota shift in rice correlates with resident time in the field and developmental stage[J]. Science China Life Sciences, 2018, 61(6):1-9. doi: 10.1007/s11427-018-9284-4 [18] BERENDSEN R L, PIETERSE C M, BAKKER P A. The rhizosphere microbiome and plant health[J]. Trends in Plant Science, 2012, 17(8):478-486. doi: 10.1016/j.tplants.2012.04.001 [19] EAST R. Microbiome:Soil science comes to life[J]. Nature, 2013, 501(7468):18-19. doi: 10.1038/501S18a [20] KUNDU P, BLACHER E, ELINAV E, et al. Our gut microbiome:the evolving inner self[J]. Cell, 2017, 171(7):1481-1493. doi: 10.1016/j.cell.2017.11.024 [21] CLAESSON M J, JEFFERY I B, CONDE S, et al. Gut microbiota composition correlates with diet and health in the elderly[J]. Nature, 2012, 488(7410):178-184. doi: 10.1038/nature11319 [22] 张惠莹. OsVHA-A基因生化及生物学功能研究与分析[D].重庆: 重庆大学, 2013. [23] FORSTER C, KANE P M. Cytosolic Ca2+ homeostasis is a constitutive function of the V-ATPase in Saccharomyces cerevisiae[J]. Journal of Biological Chemistry, 2000, 275(49):38245-38253. doi: 10.1074/jbc.M006650200 [24] SHAO E, NISHI T, KAWASAKINISHI S, et al. Mutational analysis of the non-homologous region of subunit A of the yeast V-ATPase[J]. Journal of Biological Chemistry, 2003, 278(15):12985-12991. doi: 10.1074/jbc.M212096200 [25] SHAO E, FORGAC M. Involvement of the nonhomologous region of subunit A of the yeast V-ATPase in coupling and in vivo dissociation[J]. Journal of Biological Chemistry, 2004, 279(47):48663-48670. doi: 10.1074/jbc.M408278200 [26] YANG X, GONG P, LI K, et al. A single cytosine deletion in theOsPLS1gene encoding vacuolar-type H+-ATPase subunit A1 leads to premature leaf senescence and seed dormancy in rice[J]. Journal of Experimental Botany, 2016, 67(9):2761-2776. doi: 10.1093/jxb/erw109 [27] BEAUCHAMP C, FRIDOVICH I. Superoxide dismutase. Improved assays and an assay applicable to acrylamide gel[J]. Anal Biochem, 1971, 44:276-287. doi: 10.1016/0003-2697(71)90370-8 [28] ZHOU W, ZHAO D, LIN X. Effects of waterlogging on nitrogen accumulation and alleviation of waterlogging damage by application of nitrogen fertilizer and mixtalol in winter rape (Brassica napes L.)[J]. Journal of Plant Growth Regulation, 1997, 16:47-53. doi: 10.1007/PL00006974 [29] MUÑOZ-MUÑOZ J L, GARCÍA-MOLINA F, GARCÍA-RUIZ P A, et al. Enzymatic and chemical oxidation of trihydroxylated phenols[J]. Food Chemistry, 2009, 113(2):435-444. doi: 10.1016/j.foodchem.2008.07.076 [30] QUINTANILLA-GUERRERO F, DUARTE-VAZQUEZ M, GARCIA-ALMENDAREZ B, et al. Polyethylene glycol improves phenol removal by immobilized turnip peroxidase[J]. Bioresource Technology, 2008, 99(18):8605-8611. doi: 10.1016/j.biortech.2008.04.031 [31] AEBI H. Catalase in vitro[J]. Methods Enzymol, 1984, 105(105):121-126. http://d.old.wanfangdata.com.cn/OAPaper/oai_doaj-articles_1ba2d838d4c25e398eab33b9f6a6e4a4 [32] NAKANO Y, ASADA K. Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts[J]. Plant & Cell Physiology, 1981, 22(5):867-880. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=HighWire000002692968 [33] CHAKRABARTY D, DATTA S K. Micropropagation of gerbera:lipid peroxidation and antioxidant enzyme activities during acclimatization process[J]. Physiol Plant, 2008, (30):325-331. doi: 10.1007/s11738-007-0125-3 [34] KUMAR G, KNOWLES N R. Changes in Lipid Peroxidation and Lipolytic and Free-Radical Scavenging Enzyme Activities during Aging and Sprouting of Potato (Solanum tuberosum) Seed-Tubers[J]. Plant Physiology, 1993, 102(1):115. doi: 10.1104/pp.102.1.115 [35] KE D, SUN G, WANG Z. Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings[J]. Plant Growth Regulation, 2007, 51(1):83-91. doi: 10.1007/s10725-006-9150-2 [36] BRADFORD M, BRADFORD M M, BRADFORD N. A rapid and sensitive method of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(S 1-2):248-254. doi: 10.1016-0003-2697(76)90527-3/ [37] SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative Crmethod[J]. Nat Protoc, 2008(3):1101-1108. [38] 吴丹, 赵立, 庞文生, 等.太子参根际土壤酚酸类自毒物质的分析测定[J].中国民族民间医药, 2017, 26(24):32-34. http://d.old.wanfangdata.com.cn/Periodical/zgmzmjyyzz201724010 [39] EDGAR R C. UPARSE:highly accurate OTU sequences from microbial amplicon reads[J]. Nature Methods, 2013, 10(10):996-998. doi: 10.1038/nmeth.2604 [40] WANG Q, GARRITY G M, TIEDJE J M, et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Applied & Environmental Microbiology, 2007, 73(16):5261. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=6c141ed427613641a3970c1b595b045f [41] DESANTIS T Z, HUGENHOLTZ P, LARSEN N, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB[J]. Applied & Environmental Microbiology, 2006, 72(7):5069-5072. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=37aa57e221455e0469915ba972b04513 [42] SOGIN M L, MORRISON H G, HUBER J A, et al. Microbial diversity in the deep sea and the underexplored "rare biosphere"[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(32):12115-12120. doi: 10.1073/pnas.0605127103 [43] 石岩, 位东斌.土壤水分胁迫对小麦根系与旗叶衰老的影响[J].西北植物学报, 1998(2):196-201. doi: 10.3321/j.issn:1000-4025.1998.02.008 [44] 邵彩虹, 李瑶, 钱银飞, 等.养分胁迫对威优916生育后期根系衰老影响的蛋白质组学分析[J].华北农学报, 2013, 28(2):12-19. doi: 10.3969/j.issn.1000-7091.2013.02.003 [45] 王占武, 李晓芝, 刘彦利.根际微生物对冬小麦根系发育及产量性状的影响[J].华北农学报, 2000, 15(Z1):51-54. doi: 10.3321/j.issn:1000-7091.2000.Z1.011 [46] LIN S, HUANGPU J J, CHEN T, et al. Allelopathic potential and identification of allelochemicals in Pseudostellariae heterophylla rhizosphere soil in different crop rotations[J]. Allelopathy Journal, 2014, 33(2):151-161. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=6656a99ec212f2bba0468183646f7708 [47] 胡海军, 吴亚男, 鄂洋, 等.设施园艺连作障碍研究进展[J].安徽农业科学, 2016, 44(5):49-51. doi: 10.3969/j.issn.0517-6611.2016.05.019 [48] YUMIN, YUJUN-WO, CAOPEI-GEN, et al. Agrochemical Characteristics of Soil for Continuous Cropping Lily[J]. Chinese Journal of Soil Science, 2004, 35(3):377-379. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=trtb200403032 [49] RICE E L. Allelipathy-an overview[M].Chemically Mediated Interactions Between Plants and Other Organisms. Springer us, 1985:81-105. [50] CHOU C H.The role of Allelopathy in Phytochemical Ecology[J].Phytochemical Ecology, 1989(9):19-37. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=26dfd3d16dec8f77246ed8164088b797 [51] 周军建.不同水稻根系分泌物对根际土壤微生物群落多样性影响的研究[D].福州: 福建农林大学, 2007. [52] 李进春.高产水稻后期长相及提高光合作用的有效措施[J].宁夏农林科技, 2013, 54(5):14-14. doi: 10.3969/j.issn.1002-204X.2013.05.006 [53] 刘道宏.植物叶片的衰老[J].植物生理学报, 1983(2):16-21. http://d.old.wanfangdata.com.cn/Periodical/xbzwxb200106037 [54] 王复标, 黄福灯, 程方民, 等.水稻生育后期叶片早衰突变体的光合特性与叶绿体超微结构观察[J].作物学报, 2012, 38(5):871-879. http://d.old.wanfangdata.com.cn/Periodical/zuowxb201205014 [55] 李木英, 石庆华, 郑伟, 等.杂交稻后期叶片早衰特征及其与叶片N含量和根系活力关系初探[J].江西农业大学学报, 2008, 30(5):000757-765. http://d.old.wanfangdata.com.cn/Periodical/jxnydxxb200805001 [56] WANG Q, GARRITY G M, TIEDJE J M, et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Applied & Environmental Microbiology, 2007, 73(16):5261. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=6c141ed427613641a3970c1b595b045f -
点击查看大图
图(10) / 表(1)
计量
- 文章访问数: 1047
- HTML全文浏览量: 180
- PDF下载量: 18
- 被引次数: 0
