氮形态对小白菜根系生长、根区pH及细胞壁组分的影响

doi:10.3864/j.issn.0578-1752.2024.11.013

摘要/Abstract

摘要：

【目的】氮素（N）是调控蔬菜作物生长的重要因子，探究氮形态对小白菜根系生长及细胞壁组分的影响，明确根区pH变化与质膜（PM）H⁺-ATPase的关系，为进一步研究氮素调控植物根系生长发育提供理论基础。【方法】以小白菜（‘华王’）为试材，设置3个N素形态（硝态氮（NO₃^--N）、铵态氮（NH₄⁺-N）、甘氨酸态氮（Gly-N）），采用营养液水培法，探究无机氮和有机氮调控小白菜根区pH和根细胞壁组分对根系生长的影响，同时探讨氮营养对小白菜根系生长素（IAA）和PM H⁺-ATPase酶的影响。用根系扫描法和生理试验等方法，测定小白菜的总根长、总根体积、总根表面积和根尖数等根系形态指标以及介质pH、PM H⁺-ATPase酶活性、IAA含量、细胞壁及其主要组分含量、果胶甲酯酶（PME）、扩张素（EXP）含量等生理指标，并分析根系形态和各生理指标之间的相关性。【结果】Gly-N和NO₃^--N均碱化介质pH，且Gly-N碱化介质pH的能力高于NO₃^--N处理，NH₄⁺-N酸化介质pH。与NO₃^--N处理相比，Gly-N处理显著降低小白菜地上部（40.23%）和根系干物质含量（21.74%）、主根长（55.15%）、总根长（58.63%）、总根表面积（53.12%）、总根尖数（73.07%）、根分叉数（65.39%）、内部链接数（40.91%）和外部链接数（73.94%），提高了根系细胞壁提取率（22.00%）、果胶（47.74%）、纤维素（25.30%）、EXP（109.53%）、PM H⁺-ATPase酶活性（15.42%）、根冠比（30.33%）、拓扑指数（11.43%）和分形维数（4.32%）；NH₄⁺-N处理植株地上部干物质含量、主根长、根分叉数、内部链接数、外部链接数、根系PME活性和IAA含量分别显著降低22.98%、34.30%、35.32%、26.22%、29.02%、36.38%和8.74%，根系PM H⁺-ATPase酶活性、细胞壁提取率、果胶和半纤维素分别显著提高14.60%、19.38%、22.98%、74.33%。根系形态指标与生理指标之间的相关分析结果表明，总根表面积与总根长、总根尖数、外部链接数、根分叉数呈极显著正相关（P<0.01），与主根长、内部链接数、叶干重呈显著正相关（P<0.05）；总根表面积与根直径、扩张素、拓扑指数呈极显著负相关（P<0.01），与果胶、纤维素和分形维数呈显著负相关（P<0.05）。根直径与扩张素、纤维素、分形维数、拓扑指数呈极显著正相关（P<0.01），与果胶呈显著正相关（P<0.05）。【结论】Gly-N和NH₄⁺-N均增加了小白菜根系细胞壁含量，抑制了小白菜根系的伸长生长；Gly-N更有利于果胶和纤维素的积累，NH₄⁺-N有利于半纤维素的积累。

关键词: 小白菜, 氮形态, pH, 根系生长, 细胞壁

Abstract:

【Objective】Nitrogen (N) is a critical factor in regulating the growth of vegetable crops. The purpose of this study was to explore how different N forms affect root growth and cell wall components in pakchoi, and to identify the relationship between root zone pH changes and PM H⁺-ATPase, so as to provide a theoretical basis for further research on nitrogen regulation of plant root growth and development.【Method】Pakchoi (Brassica campestris ssp. Chinensis L.) was used as the test material. Three N forms, including nitrate nitrogen (NO₃^--N), ammonium nitrogen (NH₄⁺-N), and glycine nitrogen (Gly-N), were utilized in a hydroponic nutrient solution experiment. The intrinsic mechanisms by which inorganic and organic nitrogen regulate the pH in the root zone and the cell wall components on the root growth of pakchoi were explored, and the effects of nitrogen nutrition on root auxin (IAA) and plasma membrane (PM) H⁺-ATPase were investigated. The root scanning and physiological experiments were used to determine the root morphology indicators, including total root length, volume, surface area, and tip number; along with physiological indicators including medium pH, PM H⁺- ATPase activity, IAA content, the main components content of cell wall, pectin methylesterase (PME) and vasodilator (EXP) content, and finally the correlation between root morphology and various physiological indicators were analyzed.【Result】Both Gly-N and NO₃^--N treatments increased the medium pH, and even Gly-N showed a greater capacity for alkalization than the NO₃^--N treatment, while NH₄⁺-N acidified the medium pH. Compared with NO₃^--N treatment, Gly-N treatment significantly decreased the dry matter content in shoots and roots, primary root length, total root length, surface area, tip number, root forks, number of internal, and external links of pakchoi by 40.23%, 21.74%, 55.15%, 58.63%, 53.12%, 73.07%, 65.39%, 40.91%, and 73.94%, respectively, and increased the root cell wall extraction rate, pectin, cellulose, EXP content, PM H⁺-ATPase activity, root/shoot ratio, topological index, and fractal dimension by 22.00%, 47.74%, 25.30%, 109.53%, 15.42%, 30.33%, 11.43%, and 4.32%, respectively. Compared with NO₃^--N treatment, NH₄⁺-N treatment significantly reduced the plant’s shoot dry matter content, primary root length, root forks, number of internal and external links, root PME activity, and IAA content by 22.98%, 34.30%, 35.32%, 26.22%, 29.02%, 36.38%, and 8.74%, respectively, while enhancing root PM H⁺-ATPase activity, cell wall extraction rate, pectin, and hemicellulose content by 14.60%, 19.38%, 22.98%, and 74.33%, respectively. The correlation analysis between root morphology indicators and physiological indicators revealed that total root surface area was highly significantly positively correlated with total root length, the total number of root tips and root forks (P<0.01), and also positively with primary root length, and negatively correlated with root diameter, expansin, and topological index (P<0.01), as well as with pectin, cellulose, and fractal dimension (P<0.05). The root diameter showed a strongly significant positive correlation with expansin, cellulose, fractal dimension, and topological index (P<0.01), while significantly positively correlated with pectin (P<0.05).【Conclusion】Both Gly-N and NH₄⁺-N treatments enhanced cell wall content of the root system, thereby inhibited the elongation and growth of pakchoi roots. Among them, Gly-N facilitated the accumulation of pectin and cellulose, and NH₄⁺-N promoted the accumulation of hemicellulose.

Key words: pakchoi, nitrogen form, pH, root growth, cell wall

韩瑞锋, 郭雨琴, 王玉琢, 成永三, 侯雷平, 张毅. 氮形态对小白菜根系生长、根区pH及细胞壁组分的影响[J]. 中国农业科学, 2024, 57(11): 2215-2226.

HAN RuiFeng, GUO YuQin, WANG YuZhuo, CHENG YongSan, HOU LeiPing, ZHANG Yi. Effect of Nitrogen Form on Root Growth, pH in Root Zones and Cell Wall Components of Pakchoi[J]. Scientia Agricultura Sinica, 2024, 57(11): 2215-2226.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.11.013

https://www.chinaagrisci.com/CN/Y2024/V57/I11/2215

图/表 8

图1

表1

表2

图2

图3

表3

图4

图5

参考文献 41

[1]	吴召林, 祁娟, 刘文辉, 金鑫, 杨航, 宿敬龙, 李明. 氮素形态及其配比对老芒麦生长及生理特性的影响. 草业科学, 2020, 37(5): 942-951.
	WU Z L, QI J, LIU W H, JIN X, YANG H, SU J L, LI M. Effects of nitrogen forms and proportions of nitrogen forms on the growth and physiological characteristics of Elymus sibiricus. Pratacultural Science, 2020, 37(5): 942-951. (in Chinese)
[2]	LEGHARI S J, WAHOCHO N A, LAGHARI G M, HAFEEZLAGHARI A, MUSTAFABHABHAN G, HUSSAINTALPUR K, BHUTTO T A, WAHOCHO S A, LASHARI A A. Role of nitrogen for plant growth and development: A review. Advances in Environmental Biology, 2016, 10(9): 209-218.
[3]	刘晓嵩. 硝态氮和甘氨酸态氮供应下菠菜氮素吸收与代谢差异研究[D]. 上海: 上海交通大学, 2016.
	LIU X S. Nitrogen uptake and metabolism of spinach (Spinacia oleracea L.) under nitrate or Glycine supply[D]. Shanghai: Shanghai Jiao Tong University, 2016. (in Chinese)
[4]	王小丽, 周倩, 黄丹枫. 甘氨酸浓度对普通白菜幼苗生长及氮代谢关键酶活性的影响. 中国蔬菜, 2016(7): 68-74.
	WANG X L, ZHOU Q, HUANG D F. Effects of glycine nitrogen concentration on pakchoi seedling growth and key enzyme activity involved in nitrogen metabolism. China Vegetables, 2016(7): 68-74. (in Chinese)
[5]	陈鹏, 张茂星, 张明超, 刘赣, 朱毅勇. 不同氮素营养形态对香蕉生长及其根系质子泵活性的影响. 南京农业大学学报, 2015, 38(1): 101-106.
	CHEN P, ZHANG M X, ZHANG M C, LIU G, ZHU Y Y. Influence of nitrate/ammonium ratio on the growth and root plasma membrane H⁺-ATPase activity of banana. Journal of Nanjing Agricultural University, 2015, 38(1): 101-106. (in Chinese)
[6]	苑婧娴. 氨基酸对小麦幼苗生长及生理特性的影响[D]. 南京: 南京农业大学, 2013.
	YUAN J X. Effects of amino acid on growth and physiological traita in wheat seeding[D]. Nanjing: Nanjing Agricultural University, 2013. (in Chinese)
[7]	宋世威. 有机生产系统中甜瓜氮素营养生理研究[D]. 上海: 上海交通大学, 2008.
	SONG S W. Study on nutritional physiology of nitrogen in muskmelon under organic farming system[D]. Shanghai: Shanghai Jiao Tong University, 2008. (in Chinese)
[8]	马庆旭. 植物对氨基酸的吸收及pH和Cd胁迫对其吸收的影响机制[D]. 杭州: 浙江大学, 2019.
	MA Q X. The uptake of amino acids and the effects of pH and Cd stress on plants absorption[D]. Hangzhou: Zhejiang University, 2019. (in Chinese)
[9]	周小华, 李昆志. 质膜H⁺-ATPase对2种湿地植物吸收NO₃^-的影响. 环境工程, 2019, 37(7): 104-107, 158.
	ZHOU X H, LI K Z. Effect of plasma membrane H⁺-ATPase on NO₃^- uptake in two wetland plants. Environmental Engineering, 2019, 37(7): 104-107, 158. (in Chinese)
[10]	ZHANG M X, WANG Y, CHEN X, XU F Y, DING M, YE W X, KAWAI Y Y, TODA Y, HAYASHI Y, SUZUKI T, ZENG H Q, XIAO L, XIAO X, XU J, GUO S W, YAN F, SHEN Q R, XU G H, KINOSHITA T, ZHU Y Y. Plasma membrane H⁺-ATPase overexpression increases rice yield via simultaneous enhancement of nutrient uptake and photosynthesis. Nature Communications, 2021, 12: 735.
[11]	FENG L, YANG T Y, ZHANG Z L, LI F D, CHEN Q, SUN J, SHI C Y, DENG W W, TAO M M, TAI Y L, YANG H, CAO Q, WAN X C. Identification and characterization of cationic amino acid transporters (CATs) in tea plant (Camellia sinensis). Plant Growth Regulation, 2018, 84(1): 57-69.
[12]	曹小闯. 土壤氨基酸态氮对植物的氮营养贡献及其地带性分布规律[D]. 杭州: 浙江大学, 2014.
	CAO X C. Contribution of amino acid to plant nitrogen nutrition and zonal distribution of soil amino acids[D]. Hangzhou: Zhejiang University, 2014. (in Chinese)
[13]	许飞云, 张茂星, 曾后清, 朱毅勇. 水稻根系细胞膜质子泵在氮磷钾养分吸收中的作用. 中国水稻科学, 2016, 30(1): 106-110. doi: 10.16819/j.1001-7216.2016.5115
	XU F Y, ZHANG M X, ZENG H Q, ZHU Y Y. Involvement of plasma membrane H+-ATPase in uptake of nitrogen, phosphorus and potassium by rice root. Chinese Journal of Rice Science, 2016, 30(1): 106-110. (in Chinese)
[14]	刘朝阳, 李贞霞, 于丹. 小白菜营养成分测定分析. 中国园艺文摘, 2014, 30(4): 29-31.
	LIU C Y, LI Z X, YU D. Analysis and determination on nutrients of Chinese cabbage. Chinese Horticulture Abstracts, 2014, 30(4): 29-31. (in Chinese)
[15]	韩瑞锋, 梁韵, 黄丹枫. 氮素形态对小白菜根系和硝酸盐含量的影响. 上海交通大学学报(农业科学版), 2017, 35(3): 37-44.
	HAN R F, LIANG Y, HUANG D F. Effects of nitrogen form on root morphology and nitrate concentrations of pakchoi. Journal of Shanghai Jiao Tong University (Agricultural Science), 2017, 35(3): 37-44. (in Chinese)
[16]	BARBEZ E, DÜNSER K, GAIDORA A, LENDL T, BUSCH W. Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(24): E4884-E4893.
[17]	田辉, 宋海星, 吴秀文, 张振华. 低氮条件下油菜根系伸长与细胞壁组分的变化. 湖南农业大学学报(自然科学版), 2022, 48(4): 386-393.
	TIAN H, SONG H X, WU X W, ZHANG Z H. The root elongation and the changes of cell wall components of rapeseed root under low nitrogen conditions. Journal of Hunan Agricultural University (Natural Sciences), 2022, 48(4): 386-393. (in Chinese)
[18]	RAO T P, YANO K, YAMAUCHI A, TATSUMI J. A simple method for quantitative estimation of rhizosphere pH along root axes through visualization. Plant Production Science, 2000, 3(2): 94-100.
[19]	岳小红, 曹靖, 耿杰, 李瑾, 张宗菊, 张琳捷. 盐分胁迫对啤酒大麦幼苗生长、离子平衡和根际pH变化的影响. 生态学报, 2018, 38(20): 7373-7380.
	YUE X H, CAO J, GENG J, LI J, ZHANG Z J, ZHANG L J. Effects of different types of salt stress on growth, ion balance and rhizosphere pH changes in beer barley seedlings. Acta Ecologica Sinica, 2018, 38(20): 7373-7380. (in Chinese)
[20]	朱晓芳. 拟南芥细胞壁半纤维素结合铝的机制及其调控[D]. 杭州: 浙江大学, 2014.
	ZHU X F. The binding of aluminum by cell wall hemicelluloses and its regulation in Arabidopsis[D]. Hangzhou: Zhejiang University, 2014. (in Chinese)
[21]	LU F C, WANG C, CHEN M J, YUE F X, RALPH J. A facile spectroscopic method for measuring lignin content in lignocellulosic biomass. Green Chemistry, 2021, 23(14): 5106-5112.
[22]	NACRY P, BOUGUYON E, GOJON A. Nitrogen acquisition by roots: physiological and developmental mechanisms ensuring plant adaptation to a fluctuating resource. Plant and Soil, 2013, 370(1): 1-29.
[23]	马玉峰, 周忠雄, 李雨桐, 高雪琴, 乔亚丽, 张文斌, 颉建明, 胡琳莉, 郁继华. 氮素水平及形态对娃娃菜根系特征及生理指标的影响. 中国农业科学, 2022, 55(2): 378-389. doi: 10.3864/j.issn.0578-1752.2022.02.012.
	MA Y F, ZHOU Z X, LI Y T, GAO X Q, QIAO Y L, ZHANG W B, XIE J M, HU L L, YU J H. Effects of nitrogen level and form on root morphology of mini Chinese cabbage and its physiological index. Scientia Agricultura Sinica, 2022, 55(2): 378-389. doi: 10.3864/j.issn.0578-1752.2022.02.012. (in Chinese)
[24]	MEIER M, LIU Y, LAY-PRUITT K S, TAKAHASHI H, VON WIRÉN N. Auxin-mediated root branching is determined by the form of available nitrogen. Nature Plants, 2020, 6: 1136-1145. doi: 10.1038/s41477-020-00756-2 pmid: 32917974
[25]	黄秀, 叶昌, 燕金香, 李福明, 褚光, 徐春梅, 陈松, 章秀福, 王丹英. 不同氮吸收效率水稻品种的苗期铵吸收特性及生长差异分析. 中国农业科学, 2021, 54(7): 1455-1468. doi: 10.3864/j.issn.0578-1752.2021.07.011.
	HUANG X, YE C, YAN J X, LI F M, CHU G, XU C M, CHEN S, ZHANG X F, WANG D Y. Analysis of ammonium uptake and growth differences of rice varieties with different nitrogen recovery efficiency at seedling stage. Scientia Agricultura Sinica, 2021, 54(7): 1455-1468. doi: 10.3864/j.issn.0578-1752.2021.07.011. (in Chinese)
[26]	丁明. 铵态氮营养下水稻根系质子泵的作用机制[D]. 南京: 南京农业大学, 2019.
	DING M. Involvement of plasma membrane H⁺ATPase of rice roots in the ammonium-nutrition[D]. Nanjing: Nanjing Agricultural University, 2019. (in Chinese)
[27]	HAN R F, KHALID M, JUAN J X, HUANG D F. Exogenous glycine inhibits root elongation and reduces nitrate-N uptake in pak choi (Brassica campestris ssp. Chinensis L.). PLoS ONE, 2018, 13(9): e0204488.
[28]	曹小闯, 李晓艳, 朱练峰, 张均华, 禹盛苗, 金千瑜, 吴良欢. 外源甘氨酸态氮、硝态氮和铵态氮的浓度配比对小白菜生长和品质的影响. 农业环境科学学报, 2015, 34(10): 1846-1852.
	CAO X C, LI X Y, ZHU L F, ZHANG J H, YU S M, JIN Q Y, WU L H. Effects of different ratios of exogenous glycine, nitrate and ammonium on growth and quality of pakchoi (Brassica chinensis L.). Journal of Agro-Environment Science, 2015, 34(10): 1846-1852. (in Chinese)
[29]	李宏斌. 基于三维可视化模型的植物根系构型和拓扑参数提取[D]. 杨凌: 西北农林科技大学, 2023.
	LI H B. Extraction of plant root architecture and topological parameters based on 3D visualization model[D]. Yangling: Northwest A & F University, 2023. (in Chinese)
[30]	ZHU Y N, QI B F, HAO Y W, LIU H C, SUN G W, CHEN R Y, SONG S W. Appropriate NH₄⁺/NO₃^- ratio triggers plant growth and nutrient uptake of flowering Chinese cabbage by optimizing the pH value of nutrient solution. Frontiers in Plant Science, 2021, 12: 656144.
[31]	ZHANG X C, CHEN L M, WU H H, LIU L L, WAN X C. Root plasma membrane H⁺-ATPase is involved in low pH-inhibited nitrogen accumulation in tea plants (Camellia sinensis L.). Plant Growth Regulation, 2018, 86(3): 423-432.
[32]	AFZAL M R, ZHANG M X, JIN H Y, WANG G M, ZHANG M C, DING M, RAZA S, HU J, ZENG H Q, GAO X, SUBBARAO G V, ZHU Y Y. Post-translational regulation of plasma membrane H⁺-ATPase is involved in the release of biological nitrification inhibitors from sorghum roots. Plant and Soil, 2020, 450(1): 357-372.
[33]	MŁODZIŃSKA E, KŁOBUS G, CHRISTENSEN M D, FUGLSANG A T. The plasma membrane H⁺-ATPase AHA2 contributes to the root architecture in response to different nitrogen supply. Physiologia Plantarum, 2015, 154(2): 270-282.
[34]	孟永娇. 黄瓜扩张素蛋白基因CsEXPb1的克隆及功能研究[D]. 南京: 南京农业大学, 2016.
	MENG Y J. Cloning and functional research of a cucumber expansin gene CsEXPb1[D]. Nanjing: Nanjing Agricultural University, 2016. (in Chinese)
[35]	宋科. 铵硝混合营养对烤烟苗期根系生长的影响[D]. 北京: 中国农业科学院, 2017.
	SONG K. Effects of ammonium and nitrate nutrition on root growth of flue cured tobacco[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. (in Chinese)
[36]	LI L X, VERSTRAETEN I, ROOSJEN M, TAKAHASHI K, RODRIGUEZ L, MERRIN J, CHEN J, SHABALA L, SMET W, REN H, VANNESTE S, SHABALA S, DE RYBEL B, WEIJERS D, KINOSHITA T, GRAY W M, FRIML J. Cell surface and intracellular auxin signalling for H⁺ fluxes in root growth. Nature, 2021, 599: 273-277.
[37]	王生银. 生长素缓解紫花苜蓿铝毒害的作用机制研究[D]. 上海: 上海交通大学, 2017.
	WANG S Y. The mechanism of auxin alleviate aluminum toxicity in alfalfa[D]. Shanghai: Shanghai Jiao Tong University, 2017. (in Chinese)
[38]	MAROWA P, DING A M, KONG Y Z. Expansins: Roles in plant growth and potential applications in crop improvement. Plant Cell Reports, 2016, 35(5): 949-965. doi: 10.1007/s00299-016-1948-4 pmid: 26888755
[39]	PODGÓRSKA A, BURIAN M, GIECZEWSKA K, OSTASZEWSKA- BUGAJSKA M, ZEBROWSKI J, SOLECKA D, SZAL B. Altered cell wall plasticity can restrict plant growth under ammonium nutrition. Frontiers in Plant Science, 2017, 8: 1344. doi: 10.3389/fpls.2017.01344 pmid: 28848567
[40]	GŁAZOWSKA S, BALDWIN L, MRAVEC J, BUKH C, FANGEL J U, WILLATS W G, SCHJOERRING J K. The source of inorganic nitrogen has distinct effects on cell wall composition in Brachypodium distachyon. Journal of Experimental Botany, 2019, 70(21): 6461-6473.
[41]	RIVAI R R, MIYAMOTO T, AWANO T, TAKADA R, TOBIMATSU Y, UMEZAWA T, KOBAYASHI M. Nitrogen deficiency results in changes to cell wall composition of sorghum seedlings. Scientific Reports, 2021, 11: 23309. doi: 10.1038/s41598-021-02570-y pmid: 34857783

处理 Treatment	主根长 Primordial root length (cm)	总根长 Total root length (cm)	总根系表面积 Total root surface area (cm²)	平均根系直径 Average root diameter (mm)	总根系体积 Total root volume (cm³)	根系活力 Root activity (μg∙g^-1∙h^-1 FW)
N	26.53±3.24a	320.50±3.17a	21.65±0.26a	0.23±0.00b	0.26±0.02a	335.25±21.18a
A	17.43±0.49b	315.74±10.88a	21.08±0.73a	0.21±0.01b	0.16±0.02b	194.34±10.91b
G	11.90±0.38c	132.60±12.66b	10.15±0.91b	0.31±0.00a	0.15±0.01b	143.33±13.23b

处理 Treatment	根尖数 Root tip number	根分叉数 Root fork number	内部链接数 Altitude	外部链接数 Magtitude	拓扑指数 Topological index	分形维数 Fractal dimension
N	1267.67±137.12a	3502.33±160.68a	95.33±4.26a	698.33±54.72a	0.70±0.00b	1.39±0.01b
A	966.33±132.17a	2265.33±266.83b	70.33±3.71b	495.67±48.07b	0.69±0.01b	1.36±0.02b
G	341.33±45.41b	1212.00±111.54c	56.33±2.96c	182.00±16.92c	0.78±0.01a	1.45±0.00a

处理 Treatment	细胞壁含量 Cell wall content (mg∙g^-1 FW)	果胶含量 Pectin content (μg∙mg^-1DW)	半纤维素含量 Hemicellulose content (μg∙mg^-1DW)	纤维素含量 Cellulose content (μg∙mg^-1 DW)	细胞壁提取率 Cell wall extraction rate
N	30.47±0.12b	22.54±0.21c	14.88±0.85b	209.89±14.75b	6.09±0.02b
A	36.33±1.92a	27.72±0.95b	25.94±2.02a	191.41±16.34b	7.27±0.38a
G	37.17±1.15a	33.30±2.18a	19.24±0.61b	262.99±6.75a	7.43±0.23a