Effects of mycorrhizal fungi on plant growth, nutrient absorption and phytohormones levels in tea under shading condition

High temperature and strong light could induce bitterness and astringency of tea (Camellia sinensis (L.) O. Kuntze) in summer. Arbuscular mycorrhizal (AM) fungus and shading could change tea growth surroundings and improve its quality. The present study evaluated the inoculated effects of an arbuscular mycorrhizal fungus (AMF), Glomus etunicatum, on plant growth, root morphology, leaf nutrient status, phytohormones and the relative expression of root CsCPC, CsTTG1, CsAUX1, CsYUCCA1, CsNCED2, CsGA3OX1, CsDWF4 and CsAOS genes in Camellia sinensis ‘Xinyang population’ seedlings in sands under shading conditions. After 14 weeks of AMF inoculation, root mycorrhizal colonization ranged from 18.5% to 48.00%. AMF inoculation and shading heavily increased plant height, shoot and root biomass, total root length and volume, leaf nutrients content (except Fe), respectively. Both mycorrhizal inoculation and shading significantly increased root hair growth respectively, in company with up-regulation gene CsCPC and downregulation gene CsTTG1. Root auxin level and its transport gene CsAUX1 was both up-regulated by mycorrhizal inoculation and shading. Interestingly, auxin biosynthesis gene CsYUCCA1 has not been affected, which suggested that both mycorrhizal and shading mainly regulate auxin transport but not biosynthesis pathway. The contents of gibberellin (GA) and brassinosteroid (BR) in root were notably increased by mycorrhizal inoculation and shading, accompanied with up-regulation of its biosynthesis genes, CsGA3OX1 and CsDWF4. Regarding the growth inhibiting phytohormones abscisic acid (ABA) and jasmonic acid (JA), mycorrhizal inoculation and shading significantly decreased their levels in root, in company with downregulation of biosynthesis genes, CsNCED2 and CsAOS. These results implied that both AMF inoculation and shading could enhance the tea plant stress resistance and increase nutrient absorption, root biomass and the contents of root phytohormones by up-regulating its transport and biosynthesis pathway.


Introduction
Tea (Camellia sinensis (L.) O. Kuntze) is an important commercial crop consumed in the world, primarily as a beverage made from its leaves after processed (Lin et al., 2010;Shao et al., 2018). As ombrophyte, tea plant growth is often affected by sunlight, temperature, and other environmental factors (Lagad et al., 2013;Sharma and Kayang, 2017). High temperature and strong sunlight could decline tea production and leaves quality during summer (Li et al., 2018b). Arbuscular mycorrhizal are the symbiotic associations formed between soil mycorrhizal fungi and plant roots, can extend their well-developed extraradical hyphae from the roots into growth substrates for water and nutrient acquisition (Hashem et al., 2018;. A complex feedback between host plants and AMF is controlled by their nutrition and physiology, which aims to keep the balance between fungal demands for energy and the plant's need for nutrients (Mathur et al., 2019). Wu et al. (2011a) earlier reported that root traits of trifoliate orange seedlings were significantly improved by AMF inoculation with Glomus mosseae, G. versiforme and Paraglomus occultum. Zou et al. (2017) reported that inoculation with AMF (Diversispora versiformis) significantly increased root hair growth under drought stress. Mathur et al. (2018) has proved that AMF could improve plants' photosynthetic efficacy to resist high temperature stress. These results showed that AMF could regulate root growth, which is very important factor affecting nutrient absorption and plant stress resistance. A study in the past observed that cultivated tea plant existed in AMF, dominated by Acaulospora, Gigaspora, Glomus and Scutellospora (Singh et al., 2008). As reported by Kahneh et al. (2006), inoculation with G. etunicatum, G. intraradices and G. versiforme significantly increased tea plant growth and leaf nutrient content. AMF-inoculated tea plants exhibited a significantly greater growth performance and higher level of leaf nutrient, as compared with non-AMF control (Shao et al., 2018). It concludes that tea plant is relatively dependent on AMF, whereas the mechanism regarding the AMF improve its growth and enhance resistance is not fully known. Shade-cloth materials are used for protection of ombrophyte against strong sunlight and high temperature damage, such as tea (Tang et al., 2008). However, it isn't yet clear whether AMF present similar effect to shade-cloth materials on tea plant and shade stimulate the soil mycorrhizal hyphal length and subsequent mycorrhizal development.
In addition, the synthetical effects of field climatic factors, physiology, growth and production after using sunshade measures on tea plants have been studied. Tang et al. (2008) reported that field temperature, light intensity and max-temperature were significantly lower under shading treatment. So, field climate improved significantly through shading measures. In practical application, it should choose appropriate shading degrees and shading time according to shade-tolerant characteristics of tea varieties, otherwise it would affect the tea photosynthesis and tea yields. It seems that shading has capacity to improve tea plant growth and the capacity to stand up to high temperature stress. According to the plant genome sequencing technology and gene identification, root hair initiation and elongation genes, phytohormones transport and biosynthesis genes in plants are well studied. CPC is positive regulation with root hair growth while TTG1 negatively regulate root hair formation, which has been sufficiently studied in Arabidopsis (Tominaga- Wada and Wada, 2016;Long and Schiefelbein, 2020). Indole-3-acetic acid (IAA), the predominant auxin in plants, plays a critical role in plant growth and developmental processes, its biosynthesis and transport has been clearly elucidated by YUCCA (encodes a flavin monooxygenase-like enzyme) and AUX genes (Bennett et al., 1996;Zhao et al., 2001;Hoyerova et al., 2018;Kyoko et al., 2019). The GA3OX2 could catalyse the final step in gibberellin (GA) biosynthesis, that convert inactive forms of GA into active GA1 and GA4 which has been extensively studied in planta (Schomburg et al., 2003;Roumeliotis et al., 2013). DWF4, encodes a cytochrome P450 that mediates multiple 22alphahydroxylation steps in brassinosteroid (BR) biosynthesis, which are essential plant-specific steroidal hormones and refers to the growth-promoting steroids found in plants (Choe et al., 1998;Li et al., 2018a). Abscisic acid (ABA), the growth inhibitor, is another plant hormone that has regulatory roles during plant growth and development which biosynthesis requires the cleavage of C40 carotenoids by 9-cis-epoxycarotenoid dehydrogenase (NCED) to form its direct precursor (xanthoxin) and regulated by NCED1 (Leng et al., 2014;Estrada-Melo et al., 2015). Jasmonic acid (JA), as another plant growth inhibitor, plays a regulatory role in plant responses to environmental and developmental cues (Kato-Noguchi and Kobayashi, 2009;Martin et al., 2009;Gutierrez et al., 2012;Lian et al., 2013). Allene oxide synthase (AOS), is the key enzymes involved in jasmonic acid biosynthesis in plants have been separation and identification. Lian et al. (2013) has isloated GhAOS and confirmed that it is the key gene in regulating jasmonic acid biosynthesis in Gladiolus hybridus.
Although these genes have been fully studied in plant kingdom, they involve in regulation of the interaction between inoculation with AMF and shading on tea growth to resist environment stress has not been reported. Furthermore, the physiological and molecular mechanisms of the interaction between AMF and shading on tea growth along with increase nutrient absorption and phytohormones levels to resist environment stress (high temperature) are stills unknown. The aim of this study was to evaluate the effect of AMF on tea growth under shade condition in terms of analyzing changes in root hair growth, root morphology, leaf nutrient concentration, root phytohormones levels and the expression of CsCPC, CsTTG1, CsAUX1, CsYUCCA1, CsNCED2, CsGA3OX1, CsDWF4 and CsAOS.

Experimental design
The experiment was arranged in a 2 2 factorial completely randomized blocked design: inoculation with or without AMF (Glomus etunicatum) and shade or un-shade. So, there were 4 treatments: Glomus etunicatum and shade (Ge+Z), only Glomus etunicatum (Ge-Z), only shade (CK+Z), none treated (CK-Z) Each treatment was replicated 6 times, and each replicate had 2 seedlings, for a total of 48 seedlings.

Plant culture
The AMF strain Glomus etunicatum was provided by the Bank of Glomeromycota in China (BGC) and propagated with the identified fungal spores and white clover (Trifolium repens) for 16 weeks in pots, thereby, containing spores, mycorrhizal hyphae, and infected root segments. This AMF strain had shown greater positive effects on growth responses of tea plants than other AMF (Shao et al., 2018).
Seeds of C. sinensis 'Xinyang population', which were provided by the Henan Key Laboratory of Tea Plant Comprehensive Utilization in South Henan, Xinyang Agriculture and Forestry University, were sterilized with 70% alcohol solutions for 20 min and then germinated in autoclaved (0.11 MPa, 121 °C, 1 h) sands under the conditions of 26/20 °C day/night temperature and 90% relative air humidity. After 5 weeks, two-leaf-old tea seedlings with the uniform size were transplanted into a 3.0-L pot containing 3.0 kg of autoclaved (0.11 MPa, 121 °C, 1 h) sands. The sands collected from the Yangtze River side were sieved through 4 mm, rinsed with distilled water, and autoclaved (0.11 MPa and 121 °C) for 1 h. At transplanting, 200 g (corresponding to 2200 spores) of mycorrhizal inoculum was applied into the inoculated pot. The non-AMFinoculated treatments were supplied with the same amount of sterilized (0.11 MPa, 121 °C, 1 h) inoculum plus 2 mL filtrate (25 μm filter) of mycorrhizal inoculum to maintain similar microbial communities, except the AMF spores. All seedlings were transplanted to a greenhouse in the Campus of Yangtze University for 14 weeks. When seedlings transplanting, shading with black net treatments were begun (none-shade treatment: photosynthetic photon flux density was 1010 μmol/m 2 /s, day/night temperature 36/23 °C, and relative air humidity 60%; shading with black net: photosynthetic photon flux density was 320 μmol/m 2 /s, day/night temperature 30/23 °C, and relative air humidity 60%). Each pot was irrigated with 100 mL Hoagland solutions (pH 4.2) every day. The basic culture Hoagland solution  was as follows: 4.00 mmol/L (mM) Ca (NO3)2·4H2O, 6.00 mM KNO3, 2.00 mM MgSO4·7H2O, 1.00 mM NH4H2PO4, 46.00 μM H3BO3, 9.20 μmol/L (μM) MnCl2·4H2O, 0.77 μM ZnSO4·7H2O, 0.32 μM CuSO4·3H2O, 0.12 μM H2MoO4, and 50 mM EDTA-Fe.

Variable determinations
At harvesting, plant height was determined by a ruler. The seedlings were divided into the shoot and the root, whose biomass were measured. Root systems were collected to scan by an Epson Perfection V700 Photo Dual Lens System (J221A, Indonesia), and analyzed by a professional WinRHIZO software in 2007b (Regent Instruments Inc., Quebec, Canada) for morphological traits. The collected roots were frozen immediately at -80°C for the analysis of biochemical variables (the levels of phytohormones and the expression of genes). The concentrations of P, K, Ca, Mg, and Fe in leaves were measured by the inductively complied plasma-atomic emission spectrometry based on the protocol of Shao et al. (2018). Nitrogen (N) concentration of leaf was determined by the Smartchem 200 . The levels of phytohormones were determined by liquid chromatography-mass spectrometry (LC-MS) based on the protocol of Kojima (2012).
Root mycorrhizas were stained according to the protocol of Wu et al. (2019) with staining of 0.05 % trypan blue in lactoglycerol. The root mycorrhizal colonization was calculated as the percentage of infected root lengths against total observed root lengths.
The 1-cm-long root hair zones (2-3 cm from the root tip) of lateral roots were chosen to measure root hair morphology by Scanning Electron Microscope (SEM, JSM-6391LV, JEOL Co., Japan). These root segments were postfixed overnight in 2.5% glutaraldehyde with 0.1 M sodium cacodylate buffer (pH 7.4) and postfixed in 1% osmium tetroxide for 1 h . Fixed specimens were dehydrated with increasing concentrations of alcohol. Immediately after dehydration, samples were dried with the critical-point drying (CPD) method (Bray et al., 1993;Zhang et al., 2016Zhang et al., , 2018. For each treatment, 9 pictures at 100× were randomly chosen for the measurement of the root hair density, and 9 pictures at 400× were also randomly chosen for the measurement of the root hair length and diameter using ImageJ (National Institutes of Health, Maryland, USA, http://rsb.info.nih.gov/ij/).
The relative expression of genes in roots was analyzed by real-time quantitative PCR (qRT-PCR). Root total RNA was extracted in 0.5 g fresh sample (root hair zone: 2-4 cm from root tip) using a TaKaRa MiniBEST Plant RNA Extraction Kit (9769, Takara Bio. Inc, Japan). RNA samples were reverse-transcribed using the PrimeScript TM RT reagent kit with gDNA eraser (PK02006, Takara Bio. Inc, Japan). These primers for selected genes were designed based on the Tea Plant Information Archive (http://tpia.teaplant.org/index.html) and shown in Table 1. The qRT-PCR system was as follows: 8.8 μL ddH2O, 0.4 μL cDNA, 10 μL AceQ qPCR SYBR Green Master Mix, 0.4 μL forward prime, and 0.4 μL reverse prime. qRT-PCR were run on a CFX96 Real Time PCR Detection System (BIO-RAD, USA) under the following conditions: 95°C for 5 min, 40 cycles with 95°C for 10 s, and 60°C for 30 s. qRT-PCR determinations were performed on 3 independent biological samples with 2 technical replications for each sample were examined. Quantification of the gene expression was done with the 2 -∆∆Ct method (Livak and Schmittgen, 2001) in which the housekeeping gene (GADPH) acted as the control. The measured transcripts were normalized to the relative expression value in non-AMFcolonized plants grown without shade.

Statistical analysis
The data were statistical analyzed by the two-factor variance (ANOVA) (SAS software 8.1), and the significant differences between treatments were compared with the Duncan's multiple range tests at P < 0.05.

Results
Plant growth performance As shown in Figure 1, shade had avoided tea plants' sunburn damage observably. Furthermore, Glomus etunicatum was beneficial to tea plant growth especially in shade condition (Figure 1). Plant height, shoot and root biomass were significantly increased in the shade treatments compared to the non-shade treatments, irrespective of AMF status (Table 2). Compared with non-AMF treatments, G. etunicatum treatments significantly increased plant height by 38.9% and 40.2%, shoot biomass by 40.2% and 50.4%, and root biomass by 36.9% and 143% under shade and non-shade conditions ( Table 2).

Root mycorrhizal colonization
There was mycorrhizal colonization found in roots of the AMF-treated tea seedlings. Also, there was observed to vary in response to both types of treatments, mycorrhization and shade. As shown in Table 2   With regard to root hair, AMF and shade seedlings displayed remarkably greater root hair growth ( Figure 2). As shown in Table 3, the root hair length and number of AMF-inoculation (50.21 μm and 52.97 ×10 4 /plant) were significantly higher than non-AMF control (38.89 μm and 40.01 ×10 4 /plant) under shade treatments. However, root hair diameter had no significant difference between AMF and non-AMF treatments with shade treated (Table 3). These situations were as same as under non-shade condition (Figure 2, Table 3). Relative to non-shade control, root hair length and number were significantly increased in shade treatments irrespective of AMF status (Table 3). Root hair diameter, however, had no significant difference between shade and non-shade treatments under both AMF and non-AMF conditions (Table 3).  (Table 4). The promoting effects of AMF on tea leaf nutrient concentrations were as same as under non-shade condition (Table 4). However, no significant difference of leaf Fe levels was observed between AMF-and non-AMF-inoculated tea seedlings exposed to non-shade condition. Except Fe level in leaf, the concentration of N, P, K, Ca and Mg were significantly increased in the shade treatment, compared to the non-shade treatments, irrespective of AMF status (Table 4). Root phytohormones levels The tea seedlings' roots have been harvested for measuring the levels of phytohormones, such as auxin (IAA), abscisic acid (ABA), gibberellin (GA), Brassinolide (BR) and jasmonic acid (JA).
Compared with non-mycorrhizal seedlings, mycorrhizal seedlings had significantly higher levels of IAA, GA and BR under shade and non-shade treatments, but ABA and JA concentrations had no difference under shade condition (Table 5). However, the levels of ABA and JA of non-AMF-inoculated tea root were dramatically higher than AMF-inoculated root under non-shade condition (Table 5). Compared with nonshade treatment, shade treatment observably increased IAA level by 28.0% and 20.4%, GA level by 39.9% and 34.4% and BR level by 21.8% and 13.0% under AMF and non-AMF treatments (Table 5). Furthermore, the levels of ABA and JA of non-AMF-inoculated under non-shade condition were the highest among 4 treatments. Relative expression of genes in root The response of mycorrhized tea seedlings under shade and non-shade treatments were associated with diverse changes in expression of root CsCPC, CsTTG1, CsAUX1, CsYUCCA1, CsNCED2, CsGA3OX1, CsDWF3 and CsAOS.  'Xinyang population' seedlings Data (means ± SD, n = 6) are significantly different (P < 0.05) followed by different letters above the bars.

Discussion
In the present study, the shade treatment significantly increased root AMF colonization than the nonshade treatment, which is in line with the earlier result as reported by Muleta et al. (2007) that notably higher mean counts of AMF spores were found under leguminous shade trees. In addition, AMF significantly increased tea plant growth (plant height, shoot and root biomass, leaf nutrient element content, total root length and volume, root hair growth, etc.) irrespective of shade status, which is in accord with the earlier results as reported by Shao et al. (2018). As is well-known, AMF can produce the hyphae network, which can help host plants to absorb nutrient and water from soil (Wu et al., 2013). AMF could also increase host plants' root biomass which enhanced the ability to absorb nutrient and water from soil (Wu et al., 2012(Wu et al., , 2016Upreti et al., 2016. Furthermore, root mycorrhizal colonization, total root length and volume were significantly positively correlated with leaf nutrient element content in this study. It can be seen from this, AMF helps the tea plant to absorb N, P, K, Ca and Mg by means of extraradical hyphae which generate from AMF, which was consistent with previous studies (Shao et al., 2018).
Besides, this study has confirmed that shade could avoid tea plants' sunburn damage (photosynthetic photon flux density was 1010 μmol/m 2 /s, day/night temperature 36/23°C and relative air humidity 60%). Plant height, leaf nutrient content, shoot and root biomass, total root length and volume were all significantly increased in the shade plants compared to the non-shade plants, which is in line with the earlier results as reported by Kalcsits et al. (2018) in apple. Root hair is tip-growing extensions from the root epidermis, which plays important roles in nutrient and water absorption (Zhang et al., 2013. Based on SEM photos, both of AMF and shade could stimulate root hair growth which was consistent with . This study suggested that AMF and shade may stimulate the number of trihoblasts or the proportion of the bulges of root epidermis to root hairs which vastly increases root surface area and nutrient acquisition . So, their results suggest that AMF and shade-net could use in tea culture especially during summer for enhancing plants growth to against high temperature stress and sunburn damage. To explore the mechanisms of AMF and shade enhance tea plants growth, this study analyzed the root endogenous phytohormones levels. Phytohormones including auxin (IAA), gibberellin (GA), brassinosteroid (BR), abscisic acid (ABA) and jasmonic acid (JA), regulate plant growth and development that could be affected by AMF and shading Li et al., 2019).
AMF-inoculation tea root had significantly higher levels of IAA, GA and BR than none-AMF treated plants' root, which is in line with the earlier results as reported by Zhang et al. (2019b) in trifoliate orange seedlings colonized by AMF (Funneliformis mosseae). Furthermore, shading root also has higher levels of IAA, GA and BR no matter in AMF or none-AMF condition. IAA, one of the best-known phytohormones, have an extremely wide spectrum of activity, and are particularly important in plant growth, such as root hair growth, root and shoot biomass (Zbigniew et al., 2018;Zhang et al., 2018). So, AMF and shading treated tea seedlings has vigorous growth may cause by increasing of endogenous IAA. GA and BR could promote stem elongation, which caused higher tea plant in AMF and shading treatments in this study (Yin et al., 2002;Li et al., 2018c).
But there has no effect of AMF on ABA and JA concentrations under shade condition while AMF reduce its levels when treated with non-shade in this study. ABA and JA, as growth inhibitors that slows the growth of plant, which levels were negatively correlated with plant growth (such as plant height, root and shoot biomass, root hair growth, leaf nutrient concentrations, etc.) (Li et al., 2018d;Yang et al., 2018;Zhang et al., 2020).
In order to understand the molecular mechanism of these changes in tea plants roots, expression of root hair regulated genes and endogenous phytohormones biosynthesis and transport genes were measured. CPC and TTG1 expression are related with root hair growth (Wada et al., 1997;Tominaga-Wada et al., 2016;Long and Schiefelbein, 2020). In the present study, mycorrhizal inoculation and shading significantly up-regulated the expression level of root CsCPC, which is positive regulation with root hair growth (Wada et al., 1997;Savage et al., 2013;Tominaga-Wada et al., 2016). It is in line with the result of the root hair's growth that mycorrhizal inoculation and shading notably increased the number and length of root hair in this study. With regards to the TTG1, Long and Schiefelbein (2020) has reported that it negatively regulates root hair formation in Arabidopsis. It is similar to tea plant in this study that higher expression of CsTTG1 in the treatments of non-mycorrhizal inoculation and non-shade accompanied by less and short root hairs. So, higher expression of CsCPC and lower expression of CsTTG1 potentially promoted the initiation and elongation of root hair in tea plants.
Indole-3-acetic acid (IAA), the predominant auxin in plants, plays a critical role in plant growth and developmental processes, its biosynthesis and transport has been clearly elucidated by YUCCA (encodes a flavin monooxygenase-like enzyme) and AUX genes (Bennett et al., 1996;Zhao et al., 2001;Hoyerova et al., 2018;Kyoko et al., 2019). In the present study, mycorrhizal inoculation and shading notably up regulated the expression level of root CsAUX1, which regulate IAA transport (Bennett et al., 1996). It is in line with the result of the root IAA level in this study that mycorrhizal inoculation and shading significantly accelerated the transportation of IAA in root. However, mycorrhizal inoculation and shading have no significance differentiation in root CsYUCCA1 expression, which regulate IAA biosynthesis. Sieberer and Leyser (2006) have indicated that auxin synthesized in shoot apices then moved along specific transport routes through the plant by unique polar transport machinery. Jia et al. (2019) introduced the IAA reporter DR5ver2:GUS into wild strawberry (Fragaria vesca) to reveal auxin distribution in the seed and fruit receptacle pre-and postfertilization as well as in the root. Based on their results, it considered that both mycorrhizal inoculation and shading have no effect on IAA synthesize in root of tea seedlings. Further studies will be needed to identify the synthesize of IAA in tea plant. The genes that participate in GA biosynthesis and affect plant growth and development has been extensively studied in planta (Schomburg et al., 2003). GA 3-oxidase, catalyse the final step of active GA biosynthesis, converting inactive forms of GA such as GA3ox catalyzes the breakdown of bioactive GAs (Roumeliotis et al., 2013). Roumeliotis et al. (2013) has cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato (Solanum tuberosum), which confirmed that GA3ox2 could catalysed the final step in GA biosynthesis, that convert inactive forms of GA into active GA1 and GA4. In this study, AMF and shading significantly up-regulated the relative expression of root CsGA3OX1 and increased GA content, respectively. It concludes that both AMF and shading could up-regulate CsGA3OX1 to induce GA accumulation in tea root, which results in diverse phenotypes, such as longer plant height, dense root hairs and higher biomasses of root and shoot (Huang et al., 1998).
Brassinosteroid (BR) is essential plant-specific steroidal hormones, which refers to the growthpromoting steroids found in plants (Choe et al., 1998). DWF4, encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis (Choe et al., 1998;Li et al., 2018a). In the present study, AMF and shading significantly up-regulated the relative expression of root CsDWF4 and increased the level of BRs in tea root. Therefore, it concludes that AMF and shading up-regulated CsDWF4 to induce BR accumulation in root respectively, which results in same phenotypes to GAs. It is in line with the result reported by Shahnejat-Bushehri et al. (2016) in Arabidopsis.
Abscisic acid (ABA), the growth inhibitor, is another plant hormone that has regulatory roles during plant growth and development (Leng et al., 2014). ABA biosynthesis requires the cleavage of C40 carotenoids by 9-cis-epoxycarotenoid dehydrogenase (NCED) to form its direct precursor (xanthoxin), which process is a key rate-limiting step in ABA biosynthesis (Estrada-Melo et al., 2015). Transgenic tomato (Solanum lycopersicum) to silence SlNCED1 exhibited reduced endogenous ABA level while PpNCED2/3 positively regulates ABA biosynthesis in peach (Prunus persica) (Sun et al., 2012;Wang et al., 2019). In the present study, mycorrhizal inoculation and shading significantly down-regulated the relative expression of root CsNCED2. It concludes that both AMF and shading down-regulated CsNCED2 to reduce ABA accumulation. So, ABA levels of tea plants were lower whether in mycorrhizal inoculation or shading treated in this study, compared with non-mycorrhizal seedlings under non-shading condition. Jasmonic acid (JA), as another plant growth inhibitor, plays a regulatory role in plant responses to environmental and developmental cues such as blocked root elongation and pericarp development, inhibited protein phosphatase activity and epicotyl growth (Kato-Noguchi et al., 2009;Martin et al., 2009;Gutierrez et al., 2012;Lian et al., 2013). Allene oxide synthase (AOS), is the key enzymes involved in jasmonic acid biosynthesis in plants, leading to an unstable allene oxide (Lian et al., 2013). Lian et al. (2013) isloated GhAOS and confirmed that it is the key gene in regulating jasmonic acid biosynthesis in Gladiolus hybridus. In this study, AMF and shading significantly down-regulated the relative expression of root CsAOS and decreased JA level, respectively. Therefore, it could be speculated that AMF and shading down-regulated CsAOS to suppress JA biosynthesis, which results in promoting tea plants growth.

Conclusions
AMF and shading seedlings recorded higher plant growth performance (plant height, shoot biomass and leaf nutrients content except Fe) and root morphology (root biomass, total root length and volume) than non-AMF or non-shading seedlings. AMF and shading significantly increased root hair growth, in company with up-regulation of positive regulation root hair growth gene CsCPC and down-regulation of negative regulation root hair growth gene CsTTG1. Root auxin level and transport gene CsAUX1 was up-regulated by AMF and shading. Interestingly, auxin biosynthesis gene CsYUCCA1 has not been affected, which suggested that both AMF and shading mainly regulate auxin transport but not biosynthesis pathway. The contents of GA and BR in root were notably increased by AMF and shading, accompanied with up-regulation of its biosynthesis genes, CsGA3OX1 and CsDWF4. With regard to the growth inhibiting phytohormones ABA and JA, our results showed that AMF and shading significantly decreased its levels in root, in company with down-regulation of its biosynthesis genes, CsNCED2 and CsAOS. These results implied that AMF and shading could resist high temperature and strong light stress that had positive effects on tea plant growth, nutrient absorption, especially in roots through improving root hair growth and increasing the contents of root positively phytohormones by up-regulating its transport and biosynthesis pathway. Such results will provide a clear path to manage tea plants in field for roots and mycorrhizas in shading condition.

Authors' Contributions
Conceptualization: DJZ and YYL; Data curation: MFS, DY and XCH; Formal analysis: MFS and XCH; Funding acquisition: DJZ and YYL; Investigation: MFS and DY; Project administration: DJZ and YYL; Supervision: DJZ; Writing -original draft: MFS and DY; Writing -review and editing: DJZ and YYL. All authors read and approved the final manuscript.