Physiological response and transcriptome analysis of the desert steppe dominant plant Lespedeza potaninii to drought stress

doi:10.11686/cyxb2022326

Abstract

Abstract:

Drought is the greatest threat to grassland plants， and research on drought-tolerant grassland plants will contribute to a better understanding of the regulatory mechanisms behind the adaptive responses of plants to drought. Lespedeza potaninii is a high quality， perennial， strongly drought tolerant forage species that is widely distributed in desert grassland areas in some parts of China. Current research on drought resistance in L. potaninii focuses on changes in osmoregulatory substances and functional gene sequence analysis， but the underlying mechanism of its drought resistance is still unclear. In this study， a single-factor water control experiment was conducted with soil moisture content of 70%-80% of the field water holding capacity for the control group （CK） and 20%-30% of the field water holding capacity for the severely drought stressed group （Tr）. After 4 weeks of treatment， tests on physiological and biochemical indicators such as biomass distribution， water use， osmoregulation and root distribution were carried out on L. potaninii， while leaves and young roots were collected for transcriptome sequencing analysis. The results showed that under drought stress conditions， L. potaninii exhibited increased content of proline （Pro）， a higher soluble protein content （SP）， increased soluble sugar， increased K⁺ ions for osmoregulation to maintain hydration of the plant tissues， increased δ¹³C indicating improved water use efficiency and increased malondialdehyde indicating oxidant stress， reduced relative water content， reduced leaf internal CO₂ concentration and stomatal conductance， reduced biomass and increased root to crown ratio， among other changes， in response to drought stress. By RNA-Seq differential gene expression analysis， 4058 differential genes were found in leaves and 2172 differential genes in roots， making a total of 744 differential genes in leaves and roots. These differential genes （DEGs） include those responding positively （up-regulated） or negatively （down-regulated） to drought. Up-regulated differentially expressed genes in leaves are mainly related to plant-pathogen interactions and phytohormone signaling， while down-regulated differentially expressed genes are mainly related to photosynthetic metabolism including carbon fixation， photosynthetic antenna protein synthesis， and photosynthetic process product metabolism. In roots， up-regulated differential genes were mainly related to arginine and proline metabolism， protein processing in the endoplasmic reticulum， and down-regulated differential genes were mainly related to starch and sucrose metabolism， isoflavone biosynthesis， and flavonoid biosynthesis. The main transcription factors differentially expressed in L. potaninii leaves were AP₂/ERF-ERF， NAC， bHLH， WRKY， C₂H₂， and in roots were HSF， MYB， AP₂/ERF-ERF， WRKY. The transcription factor bHLH was specifically down-regulated and HSF was specifically up-regulated. The expression of P5CR， PLD and P4H was up-regulated in roots and leaves. In leaves， ProDH was down-regulated， which will produce more proline and 4-hydroxy-proline， and AST was up-regulated which produces more 4-hydroxy-ketoglutarate and enhances the osmoregulatory ability and ensures water uptake and use by L. potaninii. Therefore， the main response to drought stress in L. potaninii was to initiate various physiological and metabolic activities through differential expression of genes related to hormone signal transduction， osmoregulation and gas exchange.

Key words: drought stress, Lespedeza potaninii, physiological, transcriptome analysis

Hao ZHANG, Hai-ying HU, Hui-xia LI, Hai-ming HE, Shuang MA, Feng-hua MA, Ke-chen SONG. Physiological response and transcriptome analysis of the desert steppe dominant plant Lespedeza potaninii to drought stress[J]. Acta Prataculturae Sinica, 2023, 32(7): 188-205.

Figures/Tables 11

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叶Leaf		根Root
基因 Gene ID	引物序列 Primer sequences （5′→3′）	基因 Gene ID	引物序列 Primer sequences （5′→3′）
BMK_Unigene_037155	GTGGTGATGACAAGGAAGAGAA ACTGGCAACTTCTCCTTAACC	BMK_Unigene_039577	ATACCGTCCCATAGCAAGAATG TCTCCAGCCTCCACTCAATA
BMK_Unigene_111723	CTCACACAGATGCAGGGTATG CCTCCTGGAAGCTTCTCTTTAAT	BMK_Unigene_040501	AACCCTACCTTCCTTCACAATC CACTCTTCACTTCCACCATCA
BMK_Unigene_009299	CGCTTTGGTGTTGGAGATTG CAATTGCGATGGGAGCAATAG	BMK_Unigene_016501	GATGTCGGGTCTTGGGATAAA GCTCCTCCATTGATAGGTCATAA
BMK_Unigene_035523	GGTGTGGAATGAGGAAGAGAAA CCACTCCCGAAAGCCAATAA	BMK_Unigene_276197	ACTGGTGTGAAAGGTCTTGTAG GTCTATGACAGGAGCACTCAAC
BMK_Unigene_035906	GACCTTCAACACTCCTGCTATG CATCTCCAGAGTCCAGAACAATAC	BMK_Unigene_029018	GCCATTCATTTGTTCCCATCC GTGTTTCTGTGTTCTGGGAATTT

叶Leaf		根Root
基因 Gene ID	引物序列 Primer sequences （5′→3′）	基因 Gene ID	引物序列 Primer sequences （5′→3′）
BMK_Unigene_037155	GTGGTGATGACAAGGAAGAGAA ACTGGCAACTTCTCCTTAACC	BMK_Unigene_039577	ATACCGTCCCATAGCAAGAATG TCTCCAGCCTCCACTCAATA
BMK_Unigene_111723	CTCACACAGATGCAGGGTATG CCTCCTGGAAGCTTCTCTTTAAT	BMK_Unigene_040501	AACCCTACCTTCCTTCACAATC CACTCTTCACTTCCACCATCA
BMK_Unigene_009299	CGCTTTGGTGTTGGAGATTG CAATTGCGATGGGAGCAATAG	BMK_Unigene_016501	GATGTCGGGTCTTGGGATAAA GCTCCTCCATTGATAGGTCATAA
BMK_Unigene_035523	GGTGTGGAATGAGGAAGAGAAA CCACTCCCGAAAGCCAATAA	BMK_Unigene_276197	ACTGGTGTGAAAGGTCTTGTAG GTCTATGACAGGAGCACTCAAC
BMK_Unigene_035906	GACCTTCAACACTCCTGCTATG CATCTCCAGAGTCCAGAACAATAC	BMK_Unigene_029018	GCCATTCATTTGTTCCCATCC GTGTTTCTGTGTTCTGGGAATTT

指标Index	对照CK	处理Tr	F检验F-test	P
总根长Total root length （cm）	1265.98±24.87a	933.40±6.64b	166.87	0.00
总根表面积Total root area （cm²）	230.64±44.39a	92.15±2.97b	9.69	0.04
总根体积Total root volume （cm³）	1.98±0.16a	0.81±0.04b	51.15	0.00
根尖数Root tips number	3702.00±329.57a	2578.00±64.73b	11.20	0.03
根分枝数Root forks number	4309.67±127.96a	3384.33±240.77b	14.69	0.02
根生物量Root biomass （g·plant^-1）	1.40±0.10a	1.32±0.24a	0.27	0.63
茎生物量Stem biomass （g·plant^-1）	1.50±0.16a	1.16±0.12b	8.61	0.04
叶生物量Leaf biomass （g·plant^-1）	1.39±0.06a	1.27±0.24a	0.76	0.43
根冠比Root-shoot ratio	0.48±0.02b	0.54±0.02a	10.13	0.03

指标Index	对照CK	处理Tr	F检验F-test	P
总根长Total root length （cm）	1265.98±24.87a	933.40±6.64b	166.87	0.00
总根表面积Total root area （cm²）	230.64±44.39a	92.15±2.97b	9.69	0.04
总根体积Total root volume （cm³）	1.98±0.16a	0.81±0.04b	51.15	0.00
根尖数Root tips number	3702.00±329.57a	2578.00±64.73b	11.20	0.03
根分枝数Root forks number	4309.67±127.96a	3384.33±240.77b	14.69	0.02
根生物量Root biomass （g·plant^-1）	1.40±0.10a	1.32±0.24a	0.27	0.63
茎生物量Stem biomass （g·plant^-1）	1.50±0.16a	1.16±0.12b	8.61	0.04
叶生物量Leaf biomass （g·plant^-1）	1.39±0.06a	1.27±0.24a	0.76	0.43
根冠比Root-shoot ratio	0.48±0.02b	0.54±0.02a	10.13	0.03

样品名称 Sample		基本数据 Base number	GC占比 GC content （%）	Q₂₀ （%）	Q₃₀ （%）	过滤后的数据 Clean reads	映射读取 Mapped reads	映射比率 Mapped ratio （%）
CK	L₁	6186621368	44.54	98.60	95.43	20723703	12840214	61.96
	L₂	6234665288	45.00	98.58	95.41	20868074	12951855	62.07
	L₃	6225191496	44.71	98.47	95.09	20846238	12614651	60.51
	R₁	5786751212	43.89	98.25	94.59	19357392	11709354	60.49
	R₂	6370728902	43.86	98.08	94.12	21294316	13130824	61.66
	R₃	6138978110	44.20	98.34	94.85	20547009	12650760	61.57
Tr	L₁	6225600476	44.79	98.49	95.15	20816470	12764270	61.32
	L₂	5816270068	44.10	98.56	95.34	19465715	11695609	60.08
	L₃	6358918316	44.00	98.55	95.32	21287781	12910160	60.65
	R₁	5959686196	44.03	98.50	95.26	19944009	12394261	62.15
	R₂	6326554166	44.16	98.54	95.38	21198666	13124275	61.91
	R₃	6560622132	43.93	98.68	96.02	21970365	13775359	62.70