Elsevier

Scientia Horticulturae

Volume 272, 15 October 2020, 109491
Scientia Horticulturae

Comprehensive transcriptome profiling of tea leaves (Camellia sinensis) in response to simulated acid rain

https://doi.org/10.1016/j.scienta.2020.109491Get rights and content

Highlights

  • No negative effects occurred for SAR (pH 3.5 and pH 4.5).

  • SAR (pH 2.5) reduced chloroplasts and damaged the outer leaf layers.

  • SAR (pH 2.5) altered genes related to photosynthesis and various metabolic pathways.

  • Plants respond to SAR stress by mechanisms of resistance, avoidance, and escape.

Abstract

Tea plants (Camellia sinensis (L.) O. Kuntze), which are typically cultivated in high-elevation mountainous regions in southern China, are severely affected by acid rain. Our recent study indicated that simulated acid rain (SAR) at pH 2.5 produced necrotic spots on the leaf surface, impaired plant development by inhibiting photosynthesis and the antioxidant defense system, and caused metabolic disorders. In contrast, a pH of 4.5 had no toxic effects on tea seedlings. However, molecular-level evidence for these conclusions is still lacking. In this study, we explored the changes in leaf micromorphology under different SAR treatments using electron microscopy, and presented the first case study on the systemic responses of the tea leaf transcriptome to SAR. An examination of leaves using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that SAR (pH 4.5 and pH 3.5) did not impair the epidermis or chloroplast structures, but SAR (pH 2.5) reduced the number of chloroplasts, corroded epicuticular wax and trichomes, and altered stomatal density and size. Transcriptomic analysis revealed that the expression of multiple genes related to photosynthesis and carbohydrate, nitrogen, and sulfur metabolism were altered under SAR. Additionally, epicuticle wax biosynthesis and plant hormone signal transduction pathways were found to significantly influenced by high-acidity SAR. Taken together, our results suggest that tea plant responses to intense SAR stress incorporate three aspects: (1) stress resistance via altering metabolic pathways to enable survival; (2) stress avoidance via restraining uncontrolled non-stomatal gas exchange; and (3) stress escape via redistributing nitrogen from stress damaged leaves. These findings provide valuable insights into plant responses to acid rain stress.

Introduction

Acid rain has become a critical global environmental issue during recent decades due to rapid industrial progress (Li et al., 2019b). Sulfur dioxide (SO2) and nitrogen oxides (NOx) released into the air by fossil-fuel power plants, vehicles, and oil refineries are the primary sources of acid rain (Shu et al., 2019). Along with Europe and North America, China has become one of the world’s regions most severely affected by acid rain due to fossil fuel overconsumption (Zheng et al., 2019). According to the China Ecological Environment Bulletin of 2018 (http://www.mee.gov.cn/), 5.5% of China’s land is affected by acid rain, primarily south of the Yangtze River, and most vulnerable areas in southern China were polluted by acid rain with pH < 5.0 (Liu et al., 2017; Ma et al., 2019). China has made extensive efforts to reduce acid deposition, including shutting high-emission factories, innovating cleaner production technologies, and implementing industrial emission stint (Chan and Yao, 2008). Despite these efforts, negative consequences continue to accrue. For instance, continuous acid rain pollution south of the Yangtze River has caused river acidification, wildlife depletion, and forest degradation (Sun et al., 2016). Researchers have also observed acid rain caused branch dieback in the upper canopy of Masson pine forests in southern China (Liu et al., 2013). Therefore, there is a pressing need to determine the ecological impacts of acid rain, especially its effects on plants.

As sessile organisms, plants as a group can be viewed as the largest victim of acid precipitation (Ramlall et al., 2015). In recent years, several studies have reavealed that acid rain can directly or indirectly cause a series of injuries to plants, including leaf necrosis (Macaulay and Enahoro, 2015), branch dieback (Fan and Wang, 2000), altered respiration and photosynthesis (Huang et al., 2019; Shu et al., 2019), and decreased crop yield (Dolatabadian et al., 2013). The leaf surface forms the interface between plants and a deteriorating atmospheric environment. Cuticles furnish the first line of defense by protecting and waterproofing the plant surface; the cuticular wax serves as a barrier that restrains uncontrolled non-stomatal gas exchange, sheds rainwater from the plant surface, and limits nonstomatal water loss (Xue et al., 2017). However, at high concentrations and under prolonged exposure, acid rain can degrade the epicuticular wax and impair stomatal function (Shepherd and Wynne Griffiths, 2006). This not only increases epidermis permeability and imposes chronic water and nutrient loss (Post-Beittenmiller, 1996), but also seriously threatens the mesophyll cell structure (Sun et al., 2016). The chloroplast is the essential site for plant photosynthesis; its structural integrity is integral to its biological function (Zhang et al., 2014). In soybean (Sun et al., 2012) and rice (Sun et al., 2016), SAR (pH < 4.0) causes granum thylakoid thinning and loosening of the lamellar structure of the thylakoid. This is probably associated with excessive accumulation of reactive oxygen species (ROS) and induced peroxidation of chloroplast membrane lipids. Numerous studies have used chlorophyll fluorescence and photosynthetic parameters as sensitive indicators to evaluate a plant’s photosynthetic performance (Liu et al., 2019a, b; Neves et al., 2009). However, comprehensive research on photosynthesis is still required at the molecular level.

Multiple high-throughput technologies which have been developed in the past few years, such as transcriptomic, proteomics, and metabolomics, are effective in revealing the complicated mechanisms of many biological processes in living organisms (Colebatch et al., 2002). Among these, use of the transcriptomic approaches for obtaining comprehensive transcript changes has already common in recent years, especially in applications concerning the mechanisms underlying plant responses to abiotic stress (Parmar et al., 2019; Wang et al., 2016). A previous study using DNA array analysis on Arabidopsis revealed that SAR altered the expression of genes related to primary metabolic pathways, including nitrogen, sulfur, amino acids, photosynthesis, and ROS. Also, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play essential roles in the response of Arabidopsis to SAR stress (Liu et al., 2013). Recently, through the next-generation sequencing platform (Illumina Hiseq 2500), researchers have identified 416 genes in soybean seedlings, related to the regulation of sulfur and nitrogen metabolism, carbohydrate metabolism, photosynthesis, and ROS, all of which were differentially expressed under SAR (Yang et al., 2018). However, up to now, investigation into the response of tea plants to acid rain are has been largely overlooked.

Tea is one of the three most widely consumed non-alcoholic beverages worldwide, and the major cash crop in developing countries such as China, India, Sri Lanka, and Kenya (Yan et al., 2020). It is typically cultivated in high-elevation mountainous regions in southern China, and is severely affected by acid rain (Hu et al., 2019). Therefore, it is urgent to study acid rain’s mechanisms of toxicity on tea plants and find corresponding countermeasures. A previous study indicated that SAR at pH 5.0 and pH 4.0 promoted the growth of tea plant, while pH 3.0 decreased chlorophyll content and photosynthesis (Duan et al., 2013). In the recent physiological and biochemical study we conducted, we preliminarily concluded that SAR (pH 2.5) could restrict photosynthesis and the antioxidant defense system, causing metabolic disorders and ultimately affecting plant development and growth; in contrast, SAR (pH 4.5) had no toxic effects on tea seedlings (Zhang et al., 2020). Nevertheless, the specific threshold of tea plant response to SAR concentration and the exhaustive molecular mechanisms underlying tea plant tolerance to SAR remain largely unknown. The relatively recent deciphering of the tea genome (Wei et al., 2018; Xia et al., 2017b) has provided an effective means of monitoring the expression of key genes in tea plants under stress, which is vital for promoting the development of the tea industry (Zhang et al., 2019).

In this study, we explored the micromorphological changes of tea leaves under three SAR treatments (pH 4.5, pH 3.5, pH 2.5) by applying SEM and TEM, together with next-generation RNA-sequencing analysis, to plants that have undergone intense SAR (pH 2.5) exposure. Moreover, we interpreted the changes to plant pathways in response to SAR treatment by analyzing the simultaneous responses of gene networks form a systems biology perspective. Our results will provide a basis for developing strategies to reduce the risks associated with acid rain and maintain sustainable tea production.

Section snippets

Plant materials and SAR treatment

One-year-old healthy tea (Camellia sinensis cv. Xiangfeicui) seedlings from the Yunyuan experimental base of Hunan Agricultural University (28°10′N, 113°04′E), Hunan Province, China, were nurtured in polyethylene pots (23 × 18 × 25 cm) holding 9.0 kg of yellow-red soil. The soil composition, plant growth conditions, and SAR treatment method were consistent with our previous study (Zhang et al., 2020). One part of the young shoot (one bud with two leaves) was used for electron microscopy

Effects of SAR on the mesophyll cells and epidermal structure

Fig. 1A shows that mesophyll cells of the control are intact and contain chloroplasts, mitochondria, starch granules, and osmiophilic granules. Chloroplasts in the control samples are profuse and arranged around the plasma membrane. Under the SAR (pH 4.5) treatment, the mesophyll cell structure remained unchanged (Fig. 1B). When the pH value of SAR decreased to 3.5, chloroplasts appeared slightly loose but were organized in an orderly arrangement (Fig. 1C). However, chloroplasts were

SAR impair chloroplast structure and suppress photosynthesis

As one of the most fundamental biological process expressed in plants by which they convert light energy into chemical energy, photosynthesis is highly dependent on the structural integrity of the chloroplasts (Ma et al., 2019). In soybean, the chloroplast lamellae are densely arranged, and no apparent changes to chloroplast structure were observed after treatment with low-acidity SAR (pH 4.5 and pH 3.5); however, high-acidity SAR (pH 2.5) significantly increased the acidity in the cells, which

Conclusions

In this work, we used electron microscopy to determine that SAR (pH 2.5) significantly changes the micromorphology of leaves, indicating that photosynthesis and the outer protective layers of leaves are affected. However, SAR (pH 3.5 and pH 4.5) did not have a similar effect. The results of our research further confirmed that the response mechanism of tea to SAR stress mainly includes three aspects: resistance, avoidance, and escape. SAR is resisted via altered metabolic paths, which enable

Funding

This work was supported by Joint Funds of National Natural Science Foundation of China [grant number U19A2030], the National Natural Science Foundation of China [grant number 31271789], Central Committee Guides Local Science and Technology Development Program [grant number 2019XF5041], and the Special Project for the Construction of Modern Agricultural Industrial Technology System in Hunan Province [Xiangcai Nongzhi; grant number 20190047].

CRediT authorship contribution statement

Chenyu Zhang: Conceptualization, Investigation, Writing - original draft. Xiaoqin Yi: Writing - review & editing, Supervision. Fang Zhou: Resources, Formal analysis. Xizhi Gao: Data curation. Minhan Wang: Software. Jianjiao Chen: Validation. Jianan Huang: Project administration. Chengwen Shen: Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The research team thanks Professor Huaqin Xu for the experimental site.

References (85)

  • X. Liu et al.

    Comparative effects of sulfuric and nitric acid rain on litter decomposition and soil microbial community in subtropical plantation of Yangtze River Delta region

    Sci. Total Environ.

    (2017)
  • M. Liu et al.

    Physiological responses of Elaeocarpus glabripetalus seedlings exposed to simulated acid rain and cadmium

    Ecotoxicol. Environ. Saf.

    (2019)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method

    Methods

    (2001)
  • Y. Ma et al.

    Initial simulated acid rain impacts reactive oxygen species metabolism and photosynthetic abilities in Cinnamonum camphora undergoing high temperature

    Ind. Crops Prod.

    (2019)
  • N.R. Neves et al.

    Photosynthesis and oxidative stress in the restinga plant species Eugenia uniflora L. exposed to simulated acid rain and iron ore dust deposition: Potential use in environmental risk assessment

    Sci. Total Environ.

    (2009)
  • M. Noji et al.

    Isoform-dependent differences in feedback regulation and subcellular localization of serine acetyltransferase involved in cysteine biosynthesis from Arabidopsis thaliana

    J. Biol. Chem.

    (1998)
  • J. Pelloux et al.

    New insights into pectin methylesterase structure and function

    Trends Plant Sci.

    (2007)
  • F. Qiao et al.

    Elevated nitrogen metabolism and nitric oxide production are involved in Arabidopsis resistance to acid rain

    Plant Physiol. Biochem.

    (2018)
  • F. Sami et al.

    Role of sugars under abiotic stress

    Plant Physiol. Biochem.

    (2016)
  • X. Shu et al.

    Ecophysiological responses of Jatropha curcas L. Seedlings to simulated acid rain under different soil types

    Ecotoxicol. Environ. Saf.

    (2019)
  • M. Singh et al.

    Responses of photosynthesis, nitrogen and proline metabolism to salinity stress in Solanum lycopersicum under different levels of nitrogen supplementation

    Plant Physiol. Biochem.

    (2016)
  • Z. Sun et al.

    Interactive effects of cadmium and acid rain on photosynthetic light reaction in soybean seedlings

    Ecotoxicol. Environ. Saf.

    (2012)
  • M.A. Troncoso-Ponce et al.

    Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.)

    Plant Physiol. Biochem.

    (2010)
  • W. Wang et al.

    Transcriptomic analysis reveals the molecular mechanisms of drought-stress-Induced decreases in Camellia sinensis leaf quality

    Front. Plant Sci.

    (2016)
  • L.-X. Wei et al.

    Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings

    Plant Physiol. Biochem.

    (2015)
  • B. Xia et al.

    Analysis of the combined effects of lanthanum and acid rain, and their mechanisms, on nitrate reductase transcription in plants

    Ecotoxicol. Environ. Saf.

    (2017)
  • E.-H. Xia et al.

    The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis

    Mol. Plant

    (2017)
  • P. Yan et al.

    Soil acidification in Chinese tea plantations

    Sci. Total Environ.

    (2020)
  • L. Yang et al.

    Comprehensive transcriptome profiling of soybean leaves in response to simulated acid rain

    Ecotoxicol. Environ. Saf.

    (2018)
  • X. Yi et al.

    The PsbP protein, but not the PsbQ protein, is required for normal thylakoid architecture in Arabidopsis thaliana

    FEBS Lett.

    (2009)
  • R. Zahoor et al.

    Title: potassium application regulates nitrogen metabolism and osmotic adjustment in cotton (Gossypium hirsutum L.) functional leaf under drought stress

    J. Plant Physiol.

    (2017)
  • Z. Zhang et al.

    Advances in research on functional genes of tea plant

    Gene

    (2019)
  • C. Zhang et al.

    Physiological and biochemical responses of tea seedlings (Camellia sinensis) to simulated acid rain conditions

    Ecotoxicol. Environ. Saf.

    (2020)
  • S. Anders et al.

    HTSeq—a Python framework to work with high-throughput sequencing data

    Bioinformatics

    (2014)
  • R.M. Benstein et al.

    Arabidopsis Phosphoglycerate dehydrogenase1 of the phosphoserine pathway is essential for development and required for ammonium assimilation and tryptophan biosynthesis

    Plant Cell

    (2013)
  • K.X. Chan et al.

    Secondary sulfur metabolism in cellular signalling and oxidative stress responses

    J. Exp. Bot.

    (2019)
  • P.R. Chitnis

    Photosystem I: function and physiology

    Annu. Rev. Plant Physiol. Plant Mol. Biol.

    (2001)
  • G. Colebatch et al.

    Functional genomics: tools of the trade

    New Phytol.

    (2002)
  • S. de Bianchi et al.

    Arabidopsis mutants deleted in the light-harvesting protein Lhcb4 have a disrupted photosystem II macrostructure and are defective in Photoprotection

    Plant Cell

    (2011)
  • A. Dolatabadian et al.

    The role of calcium in improving photosynthesis and related physiological and biochemical attributes of spring wheat subjected to simulated acid rain

    Physiol. Mol. Biol. Plants

    (2013)
  • X.H. Duan et al.

    Effects of simulated acid rain and aluminum enrichment on growth and photosynthesis of tea seedlings

    Adv. Mat. Res.

    (2013)
  • C.H. Foyer et al.

    Understanding oxidative stress and antioxidant functions to enhance photosynthesis

    Plant Physiol.

    (2011)
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