Nitrogen Stress: Effect on Physiology and Relative NRTs Genes Expression in Beta vulgaris Seedlings

doi:10.11924/j.issn.1000-6850.casb2020-0044

Abstract

Abstract:

This research aims at further studying the regulatory mechanism of N utilization in sugar beet (Beta vulgaris L.) in N deficient environment, and laying a foundation for improving plants for NUE by molecular breeding and genetic engineering approaches. In this study, sugar beet seedlings of ‘780016b/12you’ were used as material, the physiological and molecular response mechanism was studied under low nitrogen and nitrogen deficiency stress, through observation of phenotype, determination of chlorophyll content and responsive changes of related genes and enzyme activity. The results showed that sugar beet leaves turned yellow partially due to nitrogen deficiency, and the SPAD value exhibited decreased chlorophyll content with the increase of stress time. qPCR showed that both BvNRT2.1 and BvNRT3.2 were induced by low N or N deficiency, and the expression of the two genes in roots was higher than that in leaves, and BvNRT2.1 responded to stress more obviously. Meanwhile, with the increase of stress time, nitrate reductase (NR) activity in beet decreased gradually, but the activity of NR in leaves was always higher than that in roots. Hence, it is concluded that low nitrogen or nitrogen deficiency stress could cause a serious impact on the photosynthesis and nitrate reductases activity of sugar beet seedlings, and lead to the inhibition of growth to some extent. It is inferred that, in order to adapt to the N stress environment, sugar beet itself might compensate for the lack of nutrition caused by N deficiency or low nitrogen through up regulating the expression of nitrate transporter genes directly or indirectly.

Key words: Beta vulgaris, nitrogen stress, NRTs, NR activity, chlorophyll

CLC Number:

S566.3

Liu Dali, Wei Duo, Gao Zhuo, Wang Qiuhong, Ma Longbiao, Zhou Jianchao. Nitrogen Stress: Effect on Physiology and Relative NRTs Genes Expression in Beta vulgaris Seedlings[J]. Chinese Agricultural Science Bulletin, 2020, 36(32): 23-29.

Figures/Tables 6

References 29

[1]	Tabuchi M, Abiko T, Yamaya T. Assimilation of ammonium ions and reutilization of nitrogen in rice (O. sativa L.)[J]. Journal of Experimental Botany, 2007,58:2319-2327. doi: 10.1093/jxb/erm016 URL pmid: 17350935
[2]	Lawlor D W. Limitation of photosynjournal in water-stressed leaves: Stomata vs. metabolism and the role of ATP[J]. Annals of Botany, 2002,89:871-885. doi: 10.1093/aob/mcf110 URL pmid: 12102513
[3]	Xu G, Fan X, Miller A J. Plant nitrogen assimilation and use efficiency[J]. Annual Review of Plant Biology, 2012,63(1):153-182. doi: 10.1146/annurev-arplant-042811-105532 URL
[4]	Matas A J, Gapper N E, Chung M Y, et al. Biology and genetic engineering of fruit maturation for enhanced quality and shelf-life[J]. Current Opinion in Biotechnology, 2009,20:197-203. doi: 10.1016/j.copbio.2009.02.015 URL pmid: 19339169
[5]	Wong S C, Cowan I R, Farquhar G D. Leaf conductance in relation to rate of CO₂ assimilation. I. Influence of nitrogen nutrition, phosphorus nutrition, photon flux density, and ambient partial pressure of CO₂ during ontogeny[J]. Plant Physiology, 1985,78:821-825. URL pmid: 16664333
[6]	Dai T B, Cao W X, Jing Q. Effects of nitrogen form on nitrogen absorption and photosynjournal of different wheat genotypes[J]. Chinese Journal of Applied Ecology, 2001,12:849-852.
[7]	Kant S, Bi Y M, Weretilnyk E, et al. The Arabidopsis halophytic relative Thellungiella halophila tolerates nitrogen-limiting conditions by maintaining growth, nitrogen uptake, and assimilation[J]. Plant Physiology, 2008,147:1168-1180. doi: 10.1104/pp.108.118125 URL pmid: 18467466
[8]	Wingler A, Purdy S, MacLean J, et al. The role of sugars in integrating environmental signals during the regulation of leaf senescence[J]. Journal of Experimental Botany, 2006,57:391-399. doi: 10.1093/jxb/eri279 URL pmid: 16157653
[9]	Walker R L, Burns I G, Moorby J. Responses of plant growth rate to nitrogen supply: A comparison of relative addition and N interruption treatments[J]. Journal of Experimental Botany, 2001,52:309-317. URL pmid: 11283176
[10]	Diaz C, Saliba-Colombani V, Loudet O, et al. Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana[J]. Plant Cell Physiology, 2006,47:74-83. URL pmid: 16284408
[11]	Bi Y M, Wang R L, Zhu T, et al. Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis[J]. BMC Genomics, 2007,8:281. doi: 10.1186/1471-2164-8-281 URL pmid: 17705847
[12]	Miller A J, Fan X R, Orsel M, et al. Nitrate transport and signaling[J]. Journal of Experimental Botany, 2007,58:2297-2306. doi: 10.1093/jxb/erm066 URL pmid: 17519352
[13]	Tsay Y F, Chiu C C, Tsai C B, et al. Nitrate transporters and peptide transporters[J]. FEBS Letters, 2007,581:2290-2300. doi: 10.1016/j.febslet.2007.04.047 URL pmid: 17481610
[14]	Crawford NM, Forde BG. Molecular and developmental biology of inorganic nitrogen nutrition[M]. In: Meyerowitz EM, ed. The Arabidopsis book. Rockville, 2002, MD: American Society of Plant Biologists.
[15]	Kant S, Bi Y M, Rothstein S J. Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency[J]. Journal of Experimental Botany, 2011,62(4):1499-1509. URL pmid: 20926552
[16]	Remans T, Nacry P, Pervent M, et al. The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches[J]. Proceedings of the National Academy of Sciences, 2006,103:19206-19211. doi: 10.1073/pnas.0605275103 URL
[17]	Ho C H, Lin S H, Hu H C, et al. CHL1 functions as a nitrate sensor in plants[J]. Cell, 2009,138:1184-1194. doi: 10.1016/j.cell.2009.07.004 URL pmid: 19766570
[18]	Hakeem K R, Ahmad A, Iqbal M, et al. Nitrogen-efficient rice cultivars can reduce nitrate pollution[J]. Environmental Science and Pollution Research, 2011,18:1184-1193. doi: 10.1007/s11356-010-0434-8 URL
[19]	Fraisier V, Gojon A, Tillard P, et al. Constitutive expression of a putative high affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for a post transcriptional regulation by a reduced nitrogen source[J]. Plant Journal, 2000,23:489-496. doi: 10.1046/j.1365-313x.2000.00813.x URL pmid: 10972875
[20]	Pathak R R, Ahmad A, Lochab S, et al. Molecular physiology of plant nitrogen use efficiency and biotechnological options for its enhancement[J]. Current Science, 2008,94(11):1394-1403.
[21]	王秋红, 周建朝, 王孝纯. 采用SPAD仪进行甜菜氮素营养诊断技术研究[J]. 中国农学通报, 2015,31(36):92-98.
[22]	Ding L, Wang K J, Jiang G M, et al. Effects of nitrogen deficiency on photosynthetic traits of maize hybrids released in different years[J]. Annals of Botany, 2005,96:925-930. doi: 10.1093/aob/mci244 URL pmid: 16103036
[23]	Wang R, Okamoto M, Xing X, et al. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism[J]. Plant Physiology, 2003,132:556-567. doi: 10.1104/pp.103.021253 URL pmid: 12805587
[24]	Yong Z, Kotur Z, Glass A D M. Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots[J]. Plant Journal, 2010,63:739-748. doi: 10.1111/j.1365-313X.2010.04278.x URL pmid: 20561257
[25]	Schofield R A, Bi Y M, Kant S, et al. Over-expression of STP13, a hexose transporter, improves plant growth and nitrogen use in Arabidopsis thaliana seedlings[J]. Plant, Cell and Environment, 2009,32:271-285. doi: 10.1111/j.1365-3040.2008.01919.x URL pmid: 19054349
[26]	Feng H, Yan M, Fan X, et al. Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status[J]. Journal of Experimental Botany, 2011,62:2319-2332. URL pmid: 21220781
[27]	Wei M. Growth and physiological response to nitrogen deficiency and re-supply in leaf-vegetable sweetpotato (Ipomoea batatas Lam.). HortScience, 2015,50(5):754-758
[28]	Foyer C H, Valadier M H, Migge A, et al. Drought-induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves[J]. Plant Physiology, 1998,117(1):283-292. doi: 10.1104/pp.117.1.283 URL pmid: 9576798
[29]	Hernández-Cruz A E, Sánchez E, Preciado-Rangel P, et al. Nitrate reductase activity, biomass, yield, and quality in cotton in response to nitrogen fertilization[J]. International Journal of Experimental Botany, 2015,84:454-460.

基因名称	序列号		引物序列(5’-3’)	扩增长度
BvNRT2.1	XM_010687027	Forward	CGGACATGGGTCTTTGTGCT	143 bp
BvNRT2.1	XM_010687027	Reverse	GCCATCCCAAAACTTGCTGC	143 bp
BvNRT3.2	XM_010669198	Forward	AAAGGGGTGTTTGCCGATCT	147 bp
BvNRT3.2	XM_010669198	Reverse	ACTGTATGTGGCAGAATCTGTGT	147 bp
18s rRNA	FJ669720	Forward	CCTCCAATGGATCCTCGTTA	153 bp
18s rRNA	FJ669720	Reverse	AAACGGCTACCACATCCAAG	153 bp

基因名称	序列号		引物序列(5’-3’)	扩增长度
BvNRT2.1	XM_010687027	Forward	CGGACATGGGTCTTTGTGCT	143 bp
BvNRT2.1	XM_010687027	Reverse	GCCATCCCAAAACTTGCTGC	143 bp
BvNRT3.2	XM_010669198	Forward	AAAGGGGTGTTTGCCGATCT	147 bp
BvNRT3.2	XM_010669198	Reverse	ACTGTATGTGGCAGAATCTGTGT	147 bp
18s rRNA	FJ669720	Forward	CCTCCAATGGATCCTCGTTA	153 bp
18s rRNA	FJ669720	Reverse	AAACGGCTACCACATCCAAG	153 bp

部位	N 5.0			N 0.0
部位	0 h	24 h	96 h	0 h	24 h	96 h	0 h	24 h	96 h
叶	134.75±6.23	136.44±6.44	137.42±7.58	136.54±7.64	54.28±6.32	33.26±8.22	136.98±7.38	89.06±6.96	77.69±8.29
根	94.23±3.21	95.63±4.68	97.32±4.91	93.26±4.37	30.64±5.62	26.79±6.87	94.51±5.24	40.82±5.33	35.51±4.25

部位	N 5.0			N 0.0
部位	0 h	24 h	96 h	0 h	24 h	96 h	0 h	24 h	96 h
叶	134.75±6.23	136.44±6.44	137.42±7.58	136.54±7.64	54.28±6.32	33.26±8.22	136.98±7.38	89.06±6.96	77.69±8.29
根	94.23±3.21	95.63±4.68	97.32±4.91	93.26±4.37	30.64±5.62	26.79±6.87	94.51±5.24	40.82±5.33	35.51±4.25