Response mechanisms of millet and its rhizosphere soil microbial communities to chromium stress

doi:10.17521/cjpe.2022.0049

Abstract

Abstract:

Aims Heavy metal chromium (Cr) contamination has toxic effects on crops and will disrupt soil microbial community homeostasis in agricultural soils. Nevertheless, the mechanisms underlying the responses of different crops and their rhizosphere soil microbial communities to Cr stress were different. By using time series data, this study analyzed the effects of Cr stress on ‘Jingu 21’ growth, functional pathways of differentially expressed genes (DEGs) in cereals and soil microbial community structure and function. Our objective was to elucidate the response mechanism of millet (Setaria italica) and soil microbial community, and provide a theoretical basis for the growth of cereals under Cr stress and the ecological restoration of the contaminated soil.

Methods We collected millet seedlings and soil samples from the pot experiments with planted millet before (CK) and 6-hour and 6-day after Cr stress (Cr_6h, Cr_6d), and determined the physiological traits of seedlings and soil physicochemical properties. The gene expression and the functional pathways enriched in the seedlings were investigated by transcriptome analysis; the dynamics of microbial community composition, structure, diversity as well as function in time series and their correlations with soil physicochemical properties were studied by high-throughput sequencing analysis.

Important findings 1) Transcriptome analysis showed that Cr stress induced up-regulation of gene expression (54% for up-regulated DEGs); GO enrichment analysis showed that DEGs significantly down-regulated the expression of photosynthesis-related genes in CK & Cr_6h, Cr_6h & Cr_6d samples, and also significantly up-regulated the expression of defense and damage regulation-related genes, and down-regulated the expression of cell wall and cell membrane and cell division-related genes in Cr_6h & Cr_6d samples. 2) High-throughput sequencing revealed a significant change in the composition of soil bacterial and fungal communities at the phylum and genus level during the time series of Cr stress. The α diversity of bacterial communities showed a phase change from stress to stability (Shannon-Wiener diversity index for CK, Cr_6h, Cr_6d were 6.09, 5.93, 6.05, respectively. Simpson diversity index for CK, Cr_6h, Cr_6d were 0.006 8, 0.007 8, 0.006 8, respectively; Chao diversity index for CK, Cr_6h, Cr_6d were 2 818.49, 2 630.73, 2 769.38, respectively), while the α diversity of fungal community decreased significantly (Shannon-Wiener diversity index for CK, Cr_6h, Cr_6d were 4.17, 3.81, 3.23, respectively). The distribution of β diversity of bacterial community in the Cr stress time series was significantly different from that of fungal community. 3) Correlation analysis between soil physicochemical properties and microbial communities showed that soil physicochemical factors were significantly correlated with a variety of fungal flora, but weakly correlated with bacterial flora. Cr stress significantly inhibited the photosynthesis of millet seedlings by reducing chlorophyll content, photosystem activity and affecting structural components such as thylakoid, and inhibited the proliferation and differentiation of leaf cells by down-regulating the expression of cell wall and microtubule-related components. At the same time, Cr stress also activated the plant defense system to overcome toxicity. Meanwhile, soil bacterial and fungal communities adapted to Cr stress through changing community composition and diversity, and their response levels and strategies differed in the stress time series.

Key words: heavy metal stress, time series, ‘Jingu 21’, microbial community, high-throughput sequencing, transcriptome analysis

BAI Xue, LI Yu-Jing, JING Xiu-Qing, ZHAO Xiao-Dong, CHANG Sha-Sha, JING Tao-Yu, LIU Jin-Ru, ZHAO Peng-Yu. Response mechanisms of millet and its rhizosphere soil microbial communities to chromium stress[J]. Chin J Plant Ecol, 2023, 47(3): 418-433.

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URL: https://www.plant-ecology.com/EN/10.17521/cjpe.2022.0049

https://www.plant-ecology.com/EN/Y2023/V47/I3/418

Figures/Tables 9

Table 1 Soil physical and chemical properties under chromium (Cr) stress (mean ± SD)

指标 Index	对照 Control	Cr胁迫6 h Cr stress for 6 h	Cr胁迫6 d Cr stress for 6 d
氮含量 Nitrogen content (mg·kg^-1)	38.89 ± 8.27^b	34.28 ± 3.43^b	80.83 ± 6.56^a
磷含量 Phosphorus content (mg·kg^-1)	53.83 ± 12.17^b	48.17 ± 5.50^b	111.28 ± 10.45^a
钾含量 Potassium content (mg·kg^-1)	117.34 ± 28.51^b	111.33 ± 11.71^b	262.00 ± 25.32^a
电导率 Electrical conductivity (us·cm^-1)	743.61 ± 127.88^b	663.89 ± 79.75^b	1628.78 ± 113.72^a
pH	6.90 ± 0.08^b	7.04 ± 0.12^b	7.29 ± 0.13^a

Fig. 1 Morphology changes of Setaria italica seedlings after chromium (Cr) stress. CK, control; Cr_6d, chromium stress for 6 d; Cr_6h, Cr stress for 6 h.

Table 2 Setaria italica growth and biomass determination in the chromium (Cr) stress time series (mean ± SD)

指标 Index	对照 Control	Cr胁迫6 h Cr stress for 6 h	Cr胁迫6 d Cr stress for 6 d
茎长 Stem length (cm)	5.86 ± 0.55^b	5.52 ± 0.55^b	7.73 ± 1.12^a
根长 Root length (cm)	4.18 ± 0.81^a	3.64 ± 0.23^a	3.83 ± 0.75^a
干质量 Dry mass (g)	0.003 1 ± 0.000 5^b	0.003 4 ± 0.000 5^b	0.005 0 ± 0.000 6^a
鲜质量 Fresh mass (g)	0.028 3 ± 0.005 3^b	0.029 4 ± 0.002 6^b	0.037 8 ± 0.005 1^a
叶绿素含量 Chlorophyll content (SPAD)	24.68 ± 1.70^a	18.84 ± 1.77^b	16.34 ± 2.00^b
氮含量 Nitrogen content (mg·g^-1)	7.49 ± 0.51^a	6.19 ± 0.52^b	5.45 ± 0.58^b

Fig. 2 Differentially expressed genes (DEGs) in leaves of Setaria italica in the chromium (Cr) stress time series. A-C, Volcano plots of DEGs in leaves of S. italica from three sample pairs. The blue, red and gray dots represent down-regulated, up-regulated and no significant change genes, respectively. D, Venn diagram showing the effect of different samples on DEGs in leaves of S. italica. E, GO annotation analysis of DEGs in leaves of S. italica under Cr treatment. CK, control; Cr_6d, Cr stress for 6 d; Cr_6h, Cr stress for 6 h. FC, differential expression multiple; pa, the adjusted p value.

Fig. 3 Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in chromium (Cr) stressed Setaria italica leaves. pa, the adjusted p value. CK, control; Cr_6d, Cr stress for 6 d; Cr_6h, Cr stress for 6 h.

Fig. 4 Community structure and inter-community differences between bacterial and fungal communities in the rhizosphere soil of Setaria italica at phylum level and genus level in the chromium (Cr) stress time series. A, B, Bacterial and fungal Venn diagrams. Numbers in these figures represent the number of operational taxonomic units in different groups. C, D, Bacterial and fungal phylum-level community composition. E, F, Bacterial and fungal genus-level community composition. CK, control; Cr_6d, Cr stress for 6 d ; Cr_6h, Cr stress for 6 h. Different lowercase letters indicate significant differences among treatments (p < 0.05).

Fig. 5 Analysis of α-diversity and β-diversity of bacterial (A, C) and fungal (B, D) communities in the chromium (Cr) stress time series. A, B, α-diversity of bacterial and fungal communities. Different lowercase letters mean significant differences among treatments (p < 0.05). C, D, β-diversity of bacterial chromium fungal community. The closer the points of two samples, the more similar the species composition of the two samples. CK, control; Cr_6h, Cr stress for 6 h; Cr_6d, Cr stress for 6 d. Stress is a metric reflecting the suitability of the model.

Fig. 6 Correlation analysis of soil physicochemical factors with bacterial (A) and fungal (B) communities。EC, electrical conductivity; K, potassium content, N, nitrogen content; P, phosphorus content. The size of the circular dots indicates the magnitude of the correlation coefficient, the color indicates the positive or negative correlation coefficient, and the square color block indicates the p value of the correlation test.

Table 3 Functional prediction analysis of soil microbial community in the chromium (Cr) stress time series (mean ± SD)

功能分组 Guild		对照 Control	Cr胁迫6 h Cr stress for 6 h	Cr胁迫6 d Cr stress for 6 d
细菌 Bacterial	全局和概述 Global and overview maps	34 369 841.00 ± 4 720 050.73^a	34 518 331.33 ± 3 830 827.80^a	35 436 018.33 ± 2 851 467.46^a
	碳水化合物代谢 Carbohydrate metabolism	7 915 005.25 ± 1 088 978.09^a	7 977 677.58 ± 893 493.00^a	8 182 307.00 ± 66 8193.19^a
	氨基酸代谢 Amino acid metabolism	7 063 478.92 ± 966 823.00^a	7 092 711.17 ± 792 150.10^a	7 279 350.08 ± 586 179.57^a
	能量代谢 Energy metabolism	3 723 520.25 ± 510 073.28^a	3 742 113.67 ± 409 484.99^a	3 833 272.92 ± 303 226.45^a
	辅助因子和维生素代谢 Metabolism of cofactors and vitamins	3 596 104.96 ± 488 259.88^a	3 580 709.88 ± 393 862.62^a	3 674 852.42 ± 301 407.93^a
真菌 Fungal	未定义的腐生菌 Undefined saprotroph	11 919.33 ± 1 237.78^a	9 633.50 ± 2 636.32^a	18 537.33 ± 10 095.58^a
	动物病原体-内生菌-植物病原体-未定义腐生菌 Animal pathogen-endophyte-plant pathogen- undefined saprotroph	10 675.17 ± 1 585.46^ab	12 060.50 ± 1 946.59^a	7 946.33 ± 3 433.26^b
	动物病原体-粪便腐生菌-内生菌-附生植物- 植物腐生菌-木材腐生菌 Animal pathogen-dungsaprotroph-endophyte- epiphytelant saprotroph-wood saprotroph	7 631.33 ± 1 345.52^a	9 334.50 ± 2 107.57^a	6 545.83 ± 4 562.01^a
	植物病原体 Plant pathogen	10 021.17 ± 2 090.68^a	5 757.83 ± 1763.85^b	4 860.67 ± 2 198.03^b
	动物病原体-内生菌-地衣寄生虫-植物病原体- 土壤腐生菌-木材腐生菌 Animal pathogen-endophyte-lichen parasite-plant pathogen-soil saprotroph-wood saprotroph	1 255.17 ± 416.48^a	1 119.50 ± 217.63^a	11 740.17 ± 17 952.63^a

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