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Effect of Melatonin and Lighting Regime on Physiological Responses and Haematological Profile of Isa Brown laying Birds

Background: Melatonin is the main neurohormone synthesized and released by the pineal gland. It stimulates several antioxidative enzymes which increase its efficiency as an antioxidant and enhance the maturation of oocytes and the development of follicles in animals. Artificial lighting on the other hand improves bird’s performance especially reproductive performance and behavior. By providing artificial light, growers can manipulate this natural cycle to their advantage and increase the egg laying. Therefore, this current study was aimed to evaluate the effect of melatonin and increased lighting on the welfare of laying birds. Methods: In November 2018 to December 2019, a total 162 sixteen weeks Isa Brown hens were used for the experiment which was grouped into 9 treatments. Melatonin and lighting at three levels (0mg, 5mg, 10mg and 12hrs, 15hrs and 18hrs) were administered orally to the birds four times weekly within a day interval for 30 weeks from 16 weeks to 46 weeks. Data on rectal temperature, respiratory rate and heart rate were collected and analysed. Blood samples were also collected and analysed for haematological parameters Result: Results showed that rectal temperature (RT), respiratory rate (RR) and heart rate (HT) were significantly (p<0.05) influenced by both melatonin and lighting. Melatonin at 5mg significantly (p<0.05) reduced the rectal temperature (40.55  C), respiratory rate (139.44bpm) and heart rate (320bpm) while lighting at 18 hours significantly (p<0.05) increased the rectal temperature, respiratory rate and heart rate due to heat stress among the parameters. The haematological profiles were influenced (p<0.05) by the administration of melatonin. Treatments levels on melatonin administration (5mg and 10mg) had increased circulating packed cell volume of 28.00% and 28.11% respectively compared to the control group which had 25.66%. The results indicates that melatonin at 5mg and 10mg improved the welfare of the birds but increased lighting beyond 12 hours compromised immunity of the

INTRODUCTION
Prolonged heat stress results in dramatic physiological changes in chicken especially in broiler birds which is associated with elevated body temperature, increased respiratory, panting and cannibalism (Abbas et al., 2000). These changes are used as indicators of heat stress (Melesse et al., 2011). In addition to high ambient temperature, relative humidity also plays an important role in bringing about stress in the chickens leading to production and economic losses (Ajakaiye et al., 2011). These losses are heavily incurred in animals like cows, sows, broilers, layers, and turkeys and contribute to heavy much economic loss in livestock production. In the USA, St Pierre et al. (2003) noted that economic losses in the layer sector are very high due to heat stress and when the heat stress was reduced, it was observed that the losses also reduced. W hen environmental temperature exceeds the limits of the thermoneutral zone, body temperature of the animal increases leading to distress.
Melatonin (N-acetyl-5-methoxytryptamine) with a chemical formula C 13 H 16 N 2 O 2 is the main neurohormone synthesized and released by the pineal gland. This hormone appears to be a potent free radical scavenger opposing the most toxic hydroxyl radicals (Webb et al., 1995). Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant (Reiter et al., 2000). It is one of the important hormones that prevent metabolic and physiologic disorders in poultry but does not attract attention by poultry scientists (Suleyman et al., 2018). Melatonin helps regulate feed consumption, energy metabolism and body heat. It provides elimination of free radicals in the body and also stimulates growth hormone secretion and, thus, affects growth performance of poultry positively (Suleyman et al., 2018). Melatonin sets the internal biological clock that regulates different daily and seasonal rhythms in various physiological systems including the cardiopulmonary, reproductive, excretory, thermoregulatory, behavioural, immune and

Effect of Melatonin and Lighting Regime on Physiological Responses and Haematological Profile of Isa Brown laying Birds
neuroendocrine systems in birds (Melamud et al., 2012). Therefore the objective of this research is to evaluate the effect of melatonin and lighting on the physiologic responses and health status of laying birds during hot dry season.

MATERIALS AND METHODS
The experiment was carried out between November 2018 to December 2019 at the Poultry Unit of the Teaching and Research Farm, Ebonyi State University, Abakaliki in Nigeria. A total of one hundred and sixty-two (162) 16 weeks Isa Brown hens were used for the experiment which was grouped into 9 treatments. They were further subdivided into three replicates of six birds each in a 2×3 factorial in a completely randomized design at 20 weeks when the birds had started laying. Anymetre weathermeter which measures both temperature and humidity was used to record the temperature and relative humidity within and outside the pen. The weekly mean temperature and relative humidity index recorded outside the pen ranged from 39.06  C-40.06  C and 60% while inside the poultry house had the value range of 43.06  C-46.02  C and 56% but highest within the treatment area that had highest duration of lighting with the value range of 47.02  C-48.04  C and 55%. Feed and water were served to the birds ad libitum.
The experimental factors in the experiment were melatonin supplement and lighting regime. Melatonin was purchased from Roban Pharmacy in Abakaliki. The melatonin was dissolved in warm water at the rate of 5mg/ 2mls or 10mg/2mls and administered orally after cooling by drenching four times weekly (Monday, Wednesday, Friday and Sunday) while 100 watt bulbs were used to provide lighting whose light intensity were measured using photometer with light intensity of 1700Lux from 06:00pm daily in each experiment according to the treatment specifications in each group. The three levels of melatonin administered were 0mg, 5mg and 10mg while lighting groups were 12 hours, 15 hours and 18 hours daily. A standard digital clinical thermometer was used to measure the rectal temperature (RT) at 07:00h and 15:00h weekly after the light (6:00pm -12:00am) introduction from 16 th week to 46 th week according to treatment groups. Three birds were randomly selected out of each treatment weekly and with a digital stethoscope and thermometer the heart rates, respiratory rate and rectal temperature were determined according to Sturkie, (1976). Three birds per treatment was randomly selected and blood samples were collected and analysed according to Coles, (1986) and Cheesbrough, (2000).
Data collected from the experiment were subjected to statistical analysis using Mini-tab version 18 software analysis of variance (ANOVA). Treatment means were separated using Fisher pairwise comparisons

Effect of Melatonin on Physiological Responses
Results of the physiological response is presented in Table  1.1. The rectal temperature and respiratory rate of layers administered different dosages of melatonin differed significantly (p<0.05) among the treatment groups. The rectal temperature was significantly (p<0.05) higher on control group ISM 0 mg (43.56  C) compared to those on levels of melatonin which recorded lower rectal temperatures of 40.54  C and 41.52  C for ISM 5 and ISM 10 respectively.
The reduced rectal temperature among the treatments on levels of melatonin indicated that melatonin alleviated the negative impact of heat stress. The high rectal temperature in the ISM 0 mg group showed that the layers were already in a stressful condition as the temperature already increased from 41  C to 43  C. This finding is in agreement with the report of Nalini et al. (2008) who stated during heat stress physiological adjustment can occur when the temperature is above 41  C. Exposure of poultry to ambient temperature outside the thermo-neutral zone during the course of production may affect production and feed conversion efficiency due to decreased feed intake, immune response and mortality (Howlinder and Rose, 1989). Results from the study showed that inclusion of melatonin decreased the rectal temperature of the Isa Brown laying birds. This report is in agreement with the report of Zeman et al. (2001), who noted that the supplementation of melatonin at a dose of 150mg/kg of feed resulted in a highly significant decline in heat production of 2-week and 3-week-old chickens (8.38% and 13.05%, respectively). Report from the experiment is consistent with report of Apeldoorn et al. (1999) who reported that melatonin supplementation in a broiler diet reduces heat production, and more significantly, the physical activity related to heat production. Siegel (1995) also stated that melatonin can be used in controlling temperature stress, since epiphyseal gland and its metabolites play an important role in the circadian thermoregulation in many animal species (Reiter, 1995). Saarela and Reiter (1994) also observed that melatonin aids in circadian thermoregulatory adj ustments of body temperature. The decline in heat production could be attributed to the hypothermic effect of melatonin (George, 1999). A dose-dependent reduction in basal temperature after a single melatonin injection was recorded in chickens by Rozenboim et al., (1998). This is also in line with the report of Minka and Ayo, (2012) who observed that the mean colonic temperature value of 40.8 ± 0.2  C was recorded in melatonin-treated quails during transportation and the value was significantly (p<0.05) lower than that of 42.4 ± 0.7  C recorded in the control group. The respiratory rates of the laying birds were significantly influenced (p<0.05) by the

Effect of Melatonin and Lighting Regime on Physiological Responses and Haematological Profile of Isa Brown laying Birds
treatment. There were drastic reductions in the respiratory rate of the layers on the 5mg and 10mg of melatonin compared to those on 0mg. This indicates that melatonin maintained the thermo-neutral or comfort zone of the thermally stressed layers. This finding is in agreement with the report of Sinkalu et al. (2010) who noted that broilers presented a significant increase of respiratory rate under high temperatures with values up to 165 breaths per minute.
Result from this finding is also in agreement with the report of Rozenboim et al. (1998) who recorded a dose dependent reduction in basal temperature after a single melatonin injection was recorded in chickens. The decreased basal temperature subsequently resulted in a lower dissipation of heat to environment. The finding of the present study has, thus, demonstrated that the increases in internal heat energy generation from feed metabolism, associated with rearing layers under continuous photoperiods may be ameliorated by melatonin.

Effect of Lighting on Physiological Responses
The result of the effect of lighting on physiological responses are presented in Table1.2. Significant difference was observed in the rectal temperature, respiratory rate and heart rate of the birds. There were significant differences (p<0.05) on the mean of respiratory rate recorded. Significant difference (p<0.05) was also obtained on the mean of heart rate among the treatment groups. Isa Brown on ISL 18 (350bpm) had the highest mean values compared to ISL 15 (328.11 bpm) and ISL 12 (320.00bpm) groups. Results from this study showed that longer duration of lighting increased the environmental temperature of the poultry house which in turn elevated the rectal temperature with increased respiratory rate of the layers, the birds were also deprived of sleep due to continues lighting which led to increased activity. These report are in agreement with the findings of Aengwanich, (2008) who stated that sleep deprivation increased body temperature and activity due to lack of rest. The increased respiratory rate is as a result of high heat production. This finding is in agreement with the report of Zhou and Yamamoto (1997) who stated that higher environmental temperature is responsible for high heat production thereby increasing respiratory rate (209.25bpm) after 3 hours of heat exposure than normal condition of (48.50/minute). Bottje et al. (1990) also reported similar findings as respiratory rate of (140-170 breaths/minute) increased with increasing body temperature when chicken was exposed to 40.5  C.

Effect of Melatonin on Haematological Parameters
The result of the effect of melatonin on the haematological parameters are presented in Table 2.1. Layers on 5 mg ISM 5 (28.00%) of melatonin had the highest mean value of PCV followed by group ISM 10 (28.11%) while the control ISM 0 (25.66%). RBC significantly increased with increased melatonin, layers in melatonin administration had the highest mean values compared to the control. The mean values of MCV, MCH and MCHC did not differ significantly (p>0.05) among the treatment groups. MCH and MCHC mean values decreased with increased melatonin administration compared to the control. There was significant difference (p<0.03) among the treatment means of W BC. Heterophils also differed among the treatment means. The layers in the control had increased number of heterophils compared to those on melatonin treatment. Lymphocyte differed significantly with increased mean values on those with melatonin treatment compared to the control. Melatonin increased the circulating monocytes but decreased eosinophils concentration while basophils mean values were not significantly (p>.005) influenced by melatonin administration. The monocytes mean values were numerically high on melatonin treated group compared to the control. The beneficial effects of melatonin are not confined to direct modulation of the immune system it diminished the parasite load in blood and tissue (Erbaş et al., 2012). Indeed, melatonin increases total antioxidant capacity and/or reduces the production of reactive oxygen species (Espino et al., 2012). The antioxidant activity of melatonin and its metabolites may also account for its antiapoptotic effects on immune cells (Maestroni, 2001). The   results from this study indicate that melatonin improved the health status of Isa Brown laying birds. Melatonin level with immune processes and the immune-hematopoietic system has been shown to improve immune response (Pacini and Borziani, 2016). Results of this study affirms the findings of Ben et al. (1995) who stated that administration of daily melatonin starting three days before stress increased PCV, significantly reduced heterophils circulation, mortality, and postponed the onset of disease and death. This also affirms the report of Rodriguez et al. (2001) observation that melatonin is also able to enhance the phagocytic capacity of heterophils. Melatonin administration upon reperfusion decreased the migration of circulatory neutrophils and macrophages/monocytes into stressed rats and inhibited microglial activation following transient focal cerebral ischemia in rats (Ben et al., 1995). Table 2.2 There were significant differences on the PCV, heterophils and monocytes mean values. The PCV mean values were ISL 12 (28.55%), ISL 15 (25.44%) and ISL 18 (22.77%). This shows that the control group on 12 hours lighting ISL 12 had the highest PCV mean values compared to those on ISL 15 AND ISL 18 respectively. This suggests that lighting decreased the PCV mean values across the treatment means. Heterophil values were significantly (p<0.05) higher in the treatment groups with the longest duration of lighting compared to the others. The mean values were ISL 12 (51.00%), ISL 15 (51.80%) and ISL 18 (55.66%). Lighting also increased the monocytes mean values compared to the control group. Those on group ISL 15 and ISL 18 had higher number of monocytes compared to ISL12. The increased heterophils and monocytes values indicate possible presence of pathogenic organisms which results to increased body temperature which ultimately made the body unfavourable for further pathogenic growth.

CONCLUSION
The results from this study have shown that 5mg of melatonin reduced the rectal temperature, respiratory rate and heart rate of Isa Brown laying birds during thermal stress. Increased lighting had a deleterious effect on the physiological responses of the birds especially with 18hrs of lighting which increased the heart rate and rectal temperature leading to distress n the birds. The results of the haematological profiles of the birds indicate that melatonin improved the overall health status of the Isa Brown laying birds especially those in 5mg of melatonin and 15hrs of lighting. Lighting alone did not affect the haematological indices.
It is therefore recommended that 5mg of melatonin be used to reduce thermal stress in Isa Brown laying birds during thermal stress to improve the wellbeing of the birds which in turn enhance productivity of the birds.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Stocks and Distribution of Soil Carbon, Nitrogen, Phosphorus and Sulfur in an Integrated Crop-Livestock System Treated with Phosphates

Conservation agriculture practices can contribute to changes in soil nutrient dynamics over time. This experiment evaluated the changes in total stocks and distribution of carbon, nitrogen, phosphorus and sulfur concentrations in soil, during 60 months, in an integrated crop-livestock system (ICLS) due to anticipated fertilization of sources and doses phosphates applied in soil surface. The experiment was conducted over a period of five years, under Typic Dystrudept, using a randomized block design, in an incomplete factorial scheme (3×3+1), with four replications. Treatments consisted of three sources of P [triple superphosphate (TSP), rock phosphate – Arad (RP) and magnesium thermophosphate (MTP)], along with four doses of P (0, 60, 120 and 180 kg ha P2O5 total). Samples of soil were collected in 0-5, 5-10, 10-15, 15-20 and 20-30 cm layers at 24, 36, 48 and 60 months after beggining of experiment where the following chemical attributes were evaluated: (i) total organic carbon (TOC); (ii) total nitrogen Kjeldahl (TNK); (iii) available P by ion exchange resin method (P-IER); and (iv) available S-SO4. The ICLS conditions provided increased total stocks and concentrations of TOC, TNK, P-IER and S-SO4 over time. The applications of different phosphates had no influence on soil TOC concentrations during the five years of experimentation. HIGHLIGHTS  Conservation agriculture practices under Typic Dystrudept increase stocks of C, N, P and S over time of five years.  The annual applications of soluble phosphates to soil surface changes concentrations of phosphate and sulfate in the soil under no-tillage, in an integrated crop-livestock system.  In situation of phosphorus-fixing soils, anticipated fertilization of sources and doses phosphates applied in the soil surface supply this nutrient for forage and grain crop production systems over time. 2 Guera, K.C.S; et al. Brazilian Archives of Biology and Technology. Vol.63 no.spe: e20190520, 2020 www.scielo.br/babt The concentrations of TNK, P-IER and S-SO4 showed an increase in different layers of soil, with the application of sources and doses of P. The P fertilization practice that was anticipated can consist of an efficient management of soil fertility, using properly managed conservation systems.

INTRODUCTION
Conservation agriculture practices can effectively contribute to enrichment of soil fertility, as well as ensuring the sustainability of the agroecosystem. Thus, an integrated crop-livestock system (ICLS) is a practice of soil conservationist management that, owing to its benefits (e.g., deposition of residues in the soil (animals + plants) [1]; high nutrient cycling rate [2]; and the increase in the soil organic matter [3]), promotes biotic and abiotic modifications in the soil-plant-atmosphere system, which alters the biogeochemical process of soil nutrients [1,4].
The ICLS was widely evaluated in relation to carbon (C) gains in the soil [3][4][5][6][7][8] over several decades. On the other hand, little is studied considering levels of nitrogen (N) and phosphorus (P) [7], and rare are those that consider sulfur (S) [8], in ICLS. In these systems, cattle grazing alters direction, magnitude and composition of nutrient fluxes, affecting residues decomposition and nutrient release rates. Thus, deposition of organic residues in the soil, accumulation of carbon in the soil and maximization of above-and belowground plant development will promote higher nitrogen, phosphorus and sulphur levels in the soil. Moreover, increase in the soil organic matter resulting from this production system, helps to reduce adsorption of anions on the surfaces of colloids, such as phosphates and sulfates, over time, and can maximize efficient use of phosphorus [9].
Among the most commonly used phosphate sources in Brazil are phosphates water-soluble, citrate soluble and with low solubility in both water and citrate. The P sources that provide higher solubility, such as triple superphosphate (TSP) and magnesium thermophosphate (MTP), present better performance in relation to sources of lower solubility, such as rock phosphate (RP), since the latter phosphate guarantees release of P [10]. In addition, the definition of phosphate fertilizer dose to be added to soil depends on the fertilization practice adopted. Corrective fertilization followed by maintenance and the gradual construction of soil fertility is basically the practices in use in Brazil. However, according to soil content, the dose should be adjusted to recompose export by crop and achieve or maintain optimal soil content [11].
Anticipated fertilization is a practice that provides for total or partial application of recommended doses of fertilizer to the summer crop at the time of sowing of the previous crop, in an incorporated form or at soil surface [12]. Thus the application of soil surface P to winter crops is an interesting practice in production systems such as ICLS, because it would improve P recycling, especially in the medium and/or long-term and ensure a better time and operational efficiency in sowing summer crop [13]. However, scientific information on practice of anticipated fertilization with different sources and doses of phosphates is still scarce, especially in the medium and/or long-term in ICLS.
The aim was to evaluate the changes in total stocks and distribution of carbon, nitrogen, phosphorus and sulfur concentrations in the soil, during 60 months, in an integrated crop-livestock system (ICLS) due to anticipated fertilization of sources and doses phosphates applied in soil surface.
The experimental design was a randomized complete block design in an incomplete factorial scheme (3×3+1), with four replications. Treatments consisted of different sources [triple superphosphate (TSP), rock phosphate -Arad (RP) and magnesium thermophosphate (MTP)] and doses [absolute control (0 kg ha -1 of total P2O5), 60, 120 and 180 kg ha -1 of total P2O5] of P. Quantities utilized from each source were calculated based on total P2O5 content of fertilizers and applied annually at soil surface at the time of winter forage sowing (anticipated fertilization). Chemical composition of different phosphate sources used is shown in Table  1.
Crops rotation that was followed during the experimental period of 60 months in ICLS were as follows:   The soil acidity control was carried out in 2008. After the beginning of the experiment (April 2009), no liming was applied. However, except for P, all other nutrients were being applied at recommended doses, according to results of soil chemical analysis and crop requirements, during the experimental period. Potassium chloride (KCl) and urea [CO(NH2)2] fertilizers were used for the plants as sources of K and N, respectively, and distributed at soil surface. Soybean seeds were inoculated with strains selected from Bradyrhizobium japonicum and treated with cobalt and molybdenum at doses commonly used in that region [15]. When necessary, other agronomic practices (e.g., seed treatment, weed control, pests and diseases) were carried out in order to facilitate adequate growth and development of crops.

Soil sampling and chemical determination
In April, at 24, 36, 48 and 60 months after the beggining of the experiment, soil samples were collected at the following depths: 0-5, 5-10, 10-15, 15-20 and 20-30 cm. Samples for analysis were composed of these 12 simple randomized samples to form a composite sample. After sampling, the soil was taken to the laboratory in plastic bags, air dried at 40 °C, ground and sieved in a 2.0 mm mesh sieve for later analysis [16]. Samples were analyzed to determine: (i) total organic carbon (TOC) by the Walkley-Black method and factor-corrected for no-till managed soils [17]; (ii) total nitrogen by the semi-micro-Kjeldahl method (TNK); (iii) available P by the IER method (Amberlite IRA-410 and Amberlite IRA-120) (P-IER) and; (iv) available S-SO4 2extract by ammonium acetate (0.5 mol L -1 ) in acetic acid (0.25 mol L -1 ) solution, and measure by turbidimetric method. Total stocks were estimated to be 0-30 cm based on weighted average of soil layers analyzed.

Statistical analysis
The results of stock and distribution of TOC, TNK, P-IER and S-SO4 2in the soil were submitted to univariate statistical analysis according to the experimental model in a randomized complete block design. In cases where significant F (p<0.05), Tukey test (α = 0.05) was applied. Equation (1) shows the statistical model used for analysis of variance (ANOVA) for each soil layer evaluated (for stocks, 0-30 cm and, for Brazilian Archives of Biology and Technology. Vol.63 no.spe: e20190520, 2020 www.scielo.br/babt distribuition in the soil, 0-5, 5-10, 10-15, 15-20 e 20-30 cm). All statistical analyses were performed using SAS Version 9.2 program (SAS Institute Inc. 9.1.2).

Stocks of soil carbon, nitrogen, phosphorus and sulfur in integrated crop-livestock system
The total stocks of TOC, TNK, P-IER and S-SO4 2were strongly influenced by the experimentation period ( Table 2). The greater deposition of residues (vegetable + animal) in ICLS may have led to an increase in stocks over time. There was a progressive annual addition in TOC stock (on average, 4.3 Mg ha -1 year -1 ) in the 0-30 cm layer ( Figure 1A). For TNK, a small annual increase (approximately 0.81 Mg ha -1 year -1 ) was observed ( Figure 1B). However, the sources and doses of total P2O5, did not change the total stocks for these elements ( Table 2).
The P-IER and S-SO4 2stocks increased at 36 and 48 months ( Figure 1C and 1D), where doses of total P2O5 were applied. The P-IER stock in the soil was superior when applying 120 kg ha -1 of total P2O5. Higher stocks of S-SO4 2were observed in the absence of phosphate application (0 kg ha -1 of total P2O5). In this case, plants showed a reduction in growth as a result of the absence of P, and consequently, they uptake smaller amounts of nutrients, particularly S-SO4 2-.
Brazilian Archives of Biology and Technology. Vol.63 no.spe: e20190520, 2020 www.scielo.br/babt Table 2. F values for stocks and distribution of total organic carbon (TOC), total nitrogen Kjeldahl (TNK), available P (P-RTI) and S-SO4 2in the different soil layers.

Distribution of carbon, nitrogen, phosphorus and sulfur in the soil
The experimentation period explained (Table 2), on average, 33%, 34%, 15% and 20% of total data variability, in different soil layers, for TOC, TNK, P-IER and S-SO4 2-, respectively. This shows that, over time, conservation agriculture practices in an production system, such as ICLS, can influence on changes in the distribution of TOC, TNK, P and S-SO4 2in the soil. Higher concentrations of TOC in the soil were observed at 48 and 60 months of experimentation, except for the 20-30 cm soil layer that presented higher concentrations at 24 months (Figure 2).
There was an increase of 83% (1.75 to 3.22 g dm -3 ) in TNK concentrations in the soil 10-15 cm layer, with application of 180 kg ha -1 of total P2O5 as RP at 36 months of treatment as compared to other treatments during the same period ( Figure 3). For other layers of soil, TNK concentrations did not undergo any change due to P fertilizers; however, the greatest values were observed at 48 and 60 months of experimentation.
The concentrations of P-IER and S-SO4 2-, in the 0-5 cm soil layer, were not influenced due to P fertilizers, during five years of experimentation (Table 2). However, higher concentrations of P-IER ( Figure 4) and S-SO4 2- (Figure 5), in the 0-5 cm soil layer, was observed at 36 months. In the same conditions was also observed the increase of 34% of P-IER (35.1 to 46.9 mg dm -3 ) as a result from the application of 120 kg ha -1 of total P2O5. For the 5-10, 10-15, 15-20 and 20-30 cm soil layers, higher concentrations of P-IER at 48 months due to the application of 180 kg ha -1 of total P2O5 as TSP.
Only the experimentation period changed the concentrations of S-SO4 2in the 5-10 (48 and 60 months) and 15-20 cm layer (36 months). An increase in concentrations was observed in the 10-15 cm layer of soil with annual application of 180 kg ha -1 of total P2O5 in the form of TSP at 48 and 60 months of experimentation. Also, in the 20-30 cm soil layer, the use of the same dose of total P2O5 increased the concentrations of S-SO4 2-, but without any influence of different phosphate sources ( Figure 5).

Impact of soil management system on carbon, nitrogen, phosphorus and sulfur stocks over time
Conservation agriculture (CA) practices, such as ICLS, results in higher deposition and quality of organic residues, and an increase in the C, N, P and S stocks [1,3,18,19]. It has been observed when using global meta-analysis, that CA practices can be increased to 3.3 Mg ha -1 of organic C in 0-30 cm layer of soil [8]. Increases in N and P stocks were also related in different regions of Brazil under ICLS. For these systems, the 0-30 cm soil layer presented, on average, 3.94 Mg ha -1 of N and 49.5 kg ha -1 of P [7]. In our work, N total LSD stocks were 6.81 Mg ha -1 of N, on average, during the five years of the experiment, and P stocks (43.7 kg ha -1 of P) had similar results, compared to data found in other regions [7]. The fluxes between the soil-plant-atmosphere compartments are modified by introduction of animals into production systems, through ingestion, consumption and return of nutrients to the soil (nutrient cycling) [20,21]. Thus, the cycle of plant and animal residues becomes an important source of maintaining the equilibrium of the agroecosystem, as it contributes to the supply of nutrients to the crops [22], and C sequestration in the soil management systems [23]. In addition, defoliation process imposed by animals allows greater activity in the roots and, consequently, increases release of organic compounds in the soil. In this way, it favors storage of C in the soil, and ameliorates physical, chemical and biological soil attributes, maximizing the release of nutrients through rhizodeposites [1,23].
Small C inputs from release of exudates and root renewal, boost P and S cycling [24]. Soil P stocks increase considerably in ICLS. Compared to native vegetation (21.74 kg ha -1 of P), in ICLS, P stocks in the 0-30 cm soil layer more than doubled (49.50 kg ha -1 P) with the conversion to these systems [7]. This can be explained by the addition of phosphate fertilizers to cultivated areas [9] and/or deposition of organic residues in the soil [1,9].
However, the dynamics of P, after addition of a source in the soil, vary considerably depending on how it is added (organic waste and/or fertilizer) [24], and this fact may interfere in the dynamics of other anions present in the soil. In our work, phosphate sources did not interfere with changes in P and S stocks in the soil. Nevertheless, the total P2O5 doses created a response gradient, where the presence of P in the soil led to the displacement of S. Anions, such as phosphates, have a high affinity for oxides and hydroxides of iron and aluminum. In this case, it has been observed that a decrease of inorganic anions adsorption with equal or lesser affinity, such as sulfate, takes place over a wide pH range. The orthophosphate being present in the soil solution in greater quantity can displace sulfate, due to higher adsorption affinity on colloid surface and increased P in the presence of available S in the soil [25].
Studies on soil P and S stocks and their interactions are scarce, especially in ICLS [8]. P nutrition is highly important, with global crop production restricted by phosphorus-fixing soils. In fact, it is estimated that by the year 2050, a total of 169 to 365 million hectares of land will contain low levels of available P in world [26]. In fact, agriculture places a high demand for phosphate fertilizers. Of the 14 essential nutrients to plants, global reserves of P are the smallest [26][27][28]. Finally, nutrition S is of increasing importance due to reduction of atmospheric inputs and decrease of additions of S into fertilizers or coated [8]. Thus, studies aimed at elucidating storage and availability processes of P and S for plants are extremely important from the point of view of the sustainability of cropping systems.

Carbon, nitrogen, phosphorus and sulfur soil distribution over time
In general, distribution of TOC, TNK, P-IER and S-SO4 2in the soil was higher in the upper soil layer (0-15 cm of soil) compared to deep soil layers. Studies have shown that soil C, N and P content decreases with increasing soil depth [4,7]. However, CA practices, such as ICLS, concentrations of C and nutrients increase over time [18,19]. Higher root yield of plants under grazing conditions [1,29] allows a greater accumulation of C in the soil, thus becoming an important pool of C, and also promotes formation of biopores, leading to migration of nutrients to deeper layers of the soil [9]. However, information about distribution of S in the soil under ICLS, are still inconsistent. The applications of different phosphate sources had no influence on soil TOC concentrations (Figure 2), and TNK, P-IER and S-SO4 2concentrations in the soil surface layer of 0-5 cm. This is due to microbial activity, fed by high levels of C in a superficial layer, which has its mineralization potentiated, releasing organic and inorganic forms in the soil solution [30,31]. Thus, this contributes significantly to maintenance of high concentrations of elements in these layers of soil, not being changed by use of different sources of P applied to the soil. In addition, a higher concentration of P is observed at the soil surface due to low mobility, especially in no-tillage systems [32,33].
In the soil, in the 10-15 cm layer, TNK concentrations increased 83% due to RP treatment up to a maximum dose (180 kg ha -1 of total P2O5) (Figure 3), at 36 months of experimentation. Under these conditions, there was an increase in production of 1781 kg ha -1 of total dry matter in the soybean crop (crop grown in the referred period) compared to other treatments in the same period. Nitrogen is one of the most important ecosystemic nutrients, and its availability is directly linked to primary net production, as well as stabilization Brazilian Archives of Biology and Technology. Vol.63 no.spe: e20190520, 2020 www.scielo.br/babt of soil organic matter. The long-term storage of nitrogen-containing organic matter in the soil is attributed to chemical complexity of nitrogen-rich plant residues and soil microbial activity [34].
The distribution of P-IER (below 5 cm soil layer) and S-SO4 2-(10-15 cm soil layer) changed due to application of TSP at a dose of 180 kg ha -1 of total P2O5. The major water soluble fertilizers include simple superphosphate (SSP), monoammonium phosphates (MAP), diammonium phosphates (DAP) and TSP. All these compounds are readily soluble in the soil and readily release phosphates in the soil solution for plant uptake [10]. Thus, P concentration increases in the soil, favored by migration of nutrients by formation of bioporosity in ICLS.
For concentrations of S-SO4 2-, there are many factors that control sulfate adsorption and leaching. Some of these factors are: (i) soil pH [35]; (ii) the presence of other anions, such as phosphate [35]; and (iii) organic matter content [8]. The superficial layer of no-tillage soil presents unfavorable conditions to sulfate adsorption due to additions of phosphate fertilizers, superficial application of limestone and practices that increase organic matter content of soil. This conditions can lead to displacement of S from superficial layers, for deeper layers of soil, over the long term [36].
In the 0-20 cm layer, P (on average, 17.8 mg dm -3 ) and S (on average, 5.1 mg dm -3 ) concentrations were above critical levels, in addition to a high level of organic C (on average 29 g dm -3 ) [15,37] after 60 months of experimentation. This shows that anticipated fertilization of P is an efficient practice using properly managed conservation systems with no detriment to productivity [4762.8 and 10559.8 kg ha -1 in soybean (2012/2013) and maize (2013/2014), respectively]. In addition, this practice provides greater efficiency in sowing operation, resulting in a reduction of operational and total costs, when compared to the traditional practice (sedding furrow application) [12,38].
During the 60 months of experimentation, experimental conditions favored high concentrations of carbon, nitrogen, phosphorus and sulphur due to: (i) high clay content [11]; (ii) high organic matter content [3]; (iii) high nutrient recycling [2]; (iv) no-tillage and no erosion [39]; (v) correct pasture management [1]; (vi) doses used in response intervals of regional indications [15] resulting in maintenance of soil fertility, productivity and sustainability of the production system.

CONCLUSIONS
Conservation agriculture practices, in an integrated crop-livestock system under Typic Dystrudept, resulted in an increase in total stocks of organic carbon, nitrogen, phosphorus and sulfur available, over five years, regardless of the phosphate source used. The concentrations of phosphorus and sulfur available in the soil were changed with the annual applications of water-soluble phosphate on the soil surface, under notillage in an integrated grain crops and forage system. The anticipated fertilization of sources and doses of phosphates applied annually to the soil surface proved to be an efficient practice, guaranteeing supply of P to crops over time and increasing soil fertility, without losses in productivity.


extfieldsofstudy: Physics Chemistry

text:
Growth and production of purple waxy corn (Zea mays ceratina Kulesh) on the application of NPK fertilizers and humic acid

The research aims to study the effect of various doses of NPK fertilizer and humic acid on the growth and production of purple waxy corn. The study was conducted in Bilonga Village, Bontonompo District, Gowa Regency, from September to December 2018. The method used was a two-factor factorial experiment and a randomized block design as the environmental design. The first factor was NPK fertilization consisting of four levels, namely Control, 250, 300, and 350 kg ha−1. Meanwhile, the second factor was humic acid which consists of three levels, namely 0, 15, and 20 kg ha−1 resulted in total of 12 treatment combinations that are repeated three times. The results show that NPK fertilization of 350 kg ha−1 resulted in the highest production of the purple waxy corn of 2.95 ton ha−1. The application of humic acid had no significant effect on the growth and production of the purple waxy corn, but the humic acid affected the increase in soil pH and cation exchanges capacity.

Introduction
Indonesia is an agrarian country whose agricultural land area reaches around 107 hectares from the land area of Indonesia which reaches 192 million hectares. From the area of agricultural land, the area of corn is around 5.73 million hectares in 2018. Corn as a food crop in Indonesia ranks second after rice, but corn has an equally important role compared to rice. Its position as the main source of carbohydrates and protein after rice makes corn has an economic value and has a high enough opportunity to be developed as a raw material for the food processing industry [1]. Corn has a great influence on economic stability. This is because of the increasing needs in the manufacture of food ingredients, as well as animal feed and industrial raw materials. Stems, leaves and husks of the plant are used as organic mulch or compost fertilizer. Along with the increasing science and technology, corn plants are currently being developed as a producer of energy because corn produces bioethanol in large quantities [2].
Corn production in Indonesia in 2018 amounted to 30.05 million tons, or increased by 3.91 million tons compared to 2017. Maize productivity increased by 0.27%, ie 52.27 ku / ha in 2017, rising to 52.41 ku / ha in 2018. Corn production in South Sulawesi Province in 2018 was 2.34 million tons or increased 0.01 million tons compared to 2017. Corn demand in 2018 was 16.14 million tons with consumption level 1.64 kg / capita / year [3].  [4], South Sulawesi is the best waxy corn producing region in Indonesia, due to the high amylopectin content (> 90%) so that when compared to waxy corn from other regions, the taste is more savoury, fluffier and soft. In the outer layer of corn seeds there is lycopene, an antioxidant that can prevent various types of cancer. According to Iriani [5], waxy corn is a local corn that has a low yield potential, which is less than 2 tons / ha, small cob with a diameter of 10-11 mm and very sensitive to downy mildew. The purple waxy corn is one of the food commodities that is still less well known because it has not been widely cultivated in Indonesia. One of the reasons for the lack of recognition of the purple waxy corn because it has not received serious attention. Purple corn seeds are difficult to obtain because they are rarely cultivated by farmers. Nutrient composition of corn is not much different from the yellow sweet corn and white waxy corn. The constraints of purple waxy corn production are not much different from the constraints on the yellow or white waxy corn, which are less optimal cultivation techniques, inappropriate dosage fertilization, and planting of local varieties continuously.
The initial stage of developing the purple waxy corn can be started by utilizing available seeds and fertilizing them with the right dosage. Fertilization is one of the important activities in cultivation to increase crop productivity. Cultivated plants generally require macro or micro nutrients [6]. The use of fertilizers for unfertile land is one way to increase plant growth and production. According to Novizan [7], fertilizer is defined as material added to the soil or to the canopy of plants with the aim of completing the availability of nutrients. Fertilizers used in the form of organic and inorganic fertilizers. To achieve optimum results, corn plants require adequate nutrient input. Nutrient is an important factor that influences plant growth and development. The availability of nutrients in the soil decreases due to intensive crop cultivation, especially macro nutrients such as nitrogen, phosphorus, and potassium due to transported by crops.
NPK fertilizer is a compound fertilizer that contains elements of N, P and K in a balanced proportion and can be used as a basic fertilizer and supplement in the growth and production of a plant. In addition to providing NPK elements at once, the NPK fertilizer types usually also contained other elements, both macro and micro elements. Inorganic fertilizers have a large role to fulfil the nutrients for plants, because organic fertilizers provide more limited nutrients. The elements N, P, and K are essential nutrients that must be available for plant metabolism. The function of N for plants is as a constituent component of amino acids, proteins, enzymes, vitamin B complex, hormones and chlorophyll [8]. The phosphate functions in energy transfer, cell membrane formation, metabolism, carbohydrates and protein. On the other hand, potassium functions as an enzyme activator, triggering the translocation of carbohydrates from leaves to other organs, an important component in the mechanism of osmotic regulation in cells [9].
One alternative that can be used to help the absorption of nutrients in plants is humic acid. Humic acid can affect directly and indirectly. Indirectly, namely improving soil fertility status both in physical, chemical, and biological soil properties [10]. Increasing the fertility status of the soil will result in increased nutrient uptake in plants, hence plant growth and production will be more optimal. The effect of humic acid directly is to improve metabolic processes in plants, such as increasing the rate of plant photosynthesis. Humic acid affecting corn plants is seen by increasing the growth of the plant's upper parts and roots.
Humic acid is an organic soil conditioner. Testing the effect of a combination of NPK fertilizer and humic acid has been carried out on corn plants in Gowa Regency. The results showed the addition of 0.15% humic acid reduced the use of NPK fertilizer by 25%. The dosage of NPK fertilizer 350 kg / ha only can produce 10.14 tons / ha while the use of NPK fertilizer 257.5 kg / ha and 0.15% humic acid can produce higher yield (10.21 tons / ha) [11]. Based on the description that has been stated, then research on growth and production of purple waxy corn on NPK fertilizer and humic acid is carried out.

Experimental design
The study was conducted in Bilonga Village, Bontonompo District, Gowa Regency. The study was carried out from September to December 2018. The study employed a two-factor factorial design using a randomized block design as the experimental design. The first factor was NPK fertilizer consisted of four levels namely 0, 250, 300 and 350 kg ha -1 equivalent to 150, 180 and 210 g per plot, respectively. The second factor was the application of humic acid consisted of three levels, namely 0, 15 and 20 kg ha -1 . Based on the number of treatments tested, 12 combination trials were repeated three times to produce 36 experimental plots.

Planting
Prior to planting, soil tillage was performed using a hand tractor to obtain loose soil texture that will improve soil aeration. Subsequently, a 3 m × 2 m with a height of ± 30cm were made and the distance between the beds was set to 100 cm. Seeds of local purple waxy corn were soaked in warm water for ± 30 minutes. Two seeds were planted in planting holes with plant spacing of 60 cm × 20 cm.

NPK application
The NPK fertilizer treatment was carried out twice according to the dosage treatment by making a hole with planting stick between planting holes. The first fertilization was conducted at 10 days after planting (DAP) with NPK fertilizer dose of 150 kg ha -1 (p1), 200 kg ha -1 (p2) and 250 kg ha -1 (p3). The second fertilization was carried out at 30 DAP with NPK fertilizer dose of 100 kg ha -1 for each NPK dose treatment (p1, p2 and p3).

Humic acid application
The application of humic acid was conducted by split application at planting and at 15 DAP. Humic acid was spread on the surface of the ground. The first application was 10 kg ha -1 (h1) and 15 kg ha -1 (h2) and the second application was 5 kg ha -1 for each humic acid treatment dosage (h1 and h2).

Data analysis
Data were analysed using two way analysis of variance based on randomized block design. A further test of least significant difference (LSD) at level of 5% was conducted if there was a significant effect of the treatments.

Effect of NPK fertilizer and humic acid on the growth of purple waxy corn
Variance analysis results show that NPK fertilization treatment significantly affected plant height, number of leaves, and leaf area but did not significantly affect the stem diameter of the purple waxy corn plants. Humic acid treatment and its interactions with the NPK treatments did not have significant effect on the growth parameter of the purple waxy corn. The average values of the purple waxy corn growth parameters on different application of NPK fertilizer and humic acid are presented in table 1. Table 1 shows that the growth of purple waxy corn increased with the dosage of NPK given. As general, the application of NPK fertilizer at higher dose increase the plant height, number of leaves and leaf area of the purple waxy corn. Plant treated with the 350 kg ha -1 NPK fertilizer, resulted in the highest plant, widest leaf area and plant with biggest number of leaves. However, the values of these parameters seems to be not different significantly with the other NPK fertilizer dosages (250 and 300 kg ha -1 ). No significant differences were found between the humic acid treatments on the growth parameters of the purple waxy corn. In the recent study, application of this compound did not necessarily increase the plan height, number of leaves, and leaf area of the waxy corn variety.
Similarly to the response of the purple waxy corn on the humic acid application, none of the treatment applied had significant effect on the stem diameter of the waxy corn. Nevertheless, biggest stem diameter was observed in the application of NPK fertilizer of 350 kg ha -1 , and 15 kg ha -1 of

Effect of NPK fertilizer and humic acid on the production parameters
Application of NPK fertilizer significantly increased production of purple waxy corn such as number of row per cob, number of kernels per row (figure 1), weight of 1000 kernels, rendement and productivity (table 2). Figure 1 shows that application of NPK fertilizer increase the quality of the purple waxy corn kernels indicated by higher number of row per cob and kernels per row. Even though there was no significant effect of the NPK fertilizer on the ear diameter, the average values of the parameter increased with the dose of NPK fertilizer. This caused higher number of row and kernels per cob at higher dose of NPK fertilizer. Table 2 reveals that application of NPK fertilizer increased the productivity of the purple waxy corn. The higher the dose of NPK fertilizers applied, the higher the size of the kernels, the rendemen and the productivity of the purple waxy corn. Although no significant differences between the humic ICROEST 2020 IOP Conf. Series: Earth and Environmental Science 575 (2020) 012118 IOP Publishing doi:10.1088/1755-1315/575/1/012118 5 acid doses applied, the weight of 1000 kernels, rendemen and productivity of the purple waxy corn increased with the humic acid dose. Highest productivity was shown by the application of NPK fertilizer of 350 kg per hectare and 25 kg per hectare humic acid (3.05 ton per hectare).

Discussion
The results of the experiment show that NPK fertilization treatment had a very significant effect on the observed growth and production parameters of the purple waxy corn. The application of NPK fertilizer produced higher plants compared to the control treatment or without application of NPK fertilizer. This is due to increase in the availability of N nutrient which is needed for vegetative growth of plants. This is in accordance with the opinion of Law-Ogbomo & Law-Ogbomo [12] which states that the administration of NPK compound fertilizer significantly increases plant height, plant dry weight and yield of corn kernels.
In addition to plant height, the number of leaves is one important aspect for plants and the growth and yield of plants is influenced by the number of leaves as a place for photosynthesis to produce the energy needed for plant growth [13]. The number of leaves is influenced by the availability of Nitrogen (N), N nutrients absorbed by plants then play an important role in increasing chlorophyll in the leaves, with increasing chlorophyll also increases the rate of photosynthesis which affects the formation of the number of leaves in the corn plant [14].
Some balanced fertilization research can be achieved if we pay attention to soil nutrient status, soil nutrient dynamics, and plant requirements to achieve optimum production. According to Hardjowigeno [1], NPK fertilizer application has the advantage that a one-time application of fertilizer can include several elements so that it is more efficient than using a single fertilizer. The application of 350 kg per hectare of NPK fertilizer resulted in higher plant height and leaf area, highest weight of 1000 kernels and produced the highest production per hectare. Thus, this dose is the best potential fertilizer dose to be used based on the level of production and other parameters.
Humic acid treatment did not significantly affect all growth and production parameters observed. Several studies have shown that the application of humic acid had an effect on increasing growth, nutrient uptake, and production in various plants [16]. Despite this, the application of humic acid showed an increase in soil cation exchanges capacity (CEC) values in the plot with of the 25 kg per hectare humic acid (figure 2). This is consistent with the opinion of Stevenson [17] which states that the addition of humic acid can increase CEC and improve the chemical properties of the soil. The non-significant effect of the humic acid in the recent study might due to the event of high rainfall following the application of humic acid, which is applied on the surface of the soil. Rainfall in Gowa Regency was 205.25 mm with a temperature of 27.1 ℃, the highest rainfall monitored by


extfieldsofstudy: Biology Medicine

text:
Integrative Transcriptomic and Metabolic Analyses Provide Insights into the Role of Trichomes in Tea Plant (Camellia Sinensis)

Trichomes, which develop from epidermal cells, are regarded as one of the key features that are involved in the evaluation of tea quality and tea germplasm resources. The metabolites from trichomes have been well characterized in tea products. However, little is known regarding the metabolites in fresh tea trichomes and the molecular differences in trichomes and tea leaves per se. In this study, we developed a method to collect trichomes from tea plant tender shoots, and their main secondary metabolites, including catechins, caffeine, amino acids, and aroma compounds, were determined. We found that the majority of these compounds were significantly less abundant in trichomes than in tea leaves. RNA-Seq was used to investigate the differences in the molecular regulatory mechanism between trichomes and leaves to gain further insight into the differences in trichomes and tea leaves. In total, 52.96 Gb of clean data were generated, and 6560 differentially expressed genes (DEGs), including 4471 upregulated and 2089 downregulated genes, were identified in the trichomes vs. leaves comparison. Notably, the structural genes of the major metabolite biosynthesis pathways, transcription factors, and other key DEGs were identified and comparatively analyzed between trichomes and leaves, while trichome-specific genes were also identified. Our results provide new insights into the differences between tea trichomes and leaves at the metabolic and transcriptomic levels, and open up new doors to further recognize and re-evaluate the role of trichomes in tea quality formation and tea plant growth and development.

Introduction
Trichomes are developed from epidermal cells and they can be uni-or multicellular, glandular or non-glandular, branched or unbranched, and extensively distributed on plant tissues, playing critical roles in plant growth and the development and stress responses [1][2][3][4][5][6]. In tea plants (Camellia sinensis), trichomes are one of the main characteristics that are critical for tea plant breeding and quality formation of tea products. Trichomes are mainly present on the abaxial surface and stems of tender leaves and abscise as the shoots mature [7]; therefore, teas with an abundance of trichomes are usually processed as tender and fresh tea shoots, and an abundance of trichomes seems to be a key feature indicating high quality. Previously, we observed that tea plants primarily have a single type of unicellular, unbranched, and non-glandular trichome [7]. It has been well established that the most notable difference between glandular and non-glandular trichomes is that glandular trichomes have the capability to synthesize, store, and secrete a wide variety of metabolites, including polysaccharides, organic acids, proteins, terpenoids, alkaloids, and polyphenols [1,5,8,9], suggesting that tea trichomes may not have the capability to synthesize, store, or secrete these metabolites. Nonetheless, several previous studies showed that tea trichomes also had a wide variety of metabolites, such as catechins, caffeine, amino acids, and aromatic compounds. Given that almost all tea plant cultivars or germplasm resources have trichomes on the tender shoot surfaces, if these trichomes do not synthesize metabolites, what is their function in the tea plant?
Trichomes have been regarded as a perfect system in which to study cell development and secondary metabolite synthesis in the model plants Arabidopsis [10][11][12][13][14][15][16], tomato [17][18][19][20][21][22][23], and Artemisia annua [24][25][26][27][28][29][30], and many genes that are involved in trichome differentiation have been identified, especially various transcription factors (TFs) encoding genes, such as positive regulators of GLABROUS 3 (GL1, GL2, and GL3) and ENHANCER OF GLABROUS 3 (EGL3), TRANSPARENT TESTA 1 (TTG1 and TTG2) and repressors of TRIPTYCHON (TRY), CAPRICE (CPC), ENHANCER OF TRY, CPC 1 (ETC1, ETC2, and ETC3), and TRICHOMELESS 1 (TCL1 and TCL2) [6,12,13,16]. Moreover, an increasing number of studies have indicated that trichomes seem to be an excellent model in which to study the molecular mechanisms of secondary cell wall deposition and fiber synthesis [31,32], which have been well studied in plants. Recently, we performed RNA-Seq analysis while using hairy and hairless cultivars, and many genes were identified to investigate the genes involved in tea trichome formation [7]. Studies on fresh trichomes, especially comparisons of trichomes and tea leaves per se at the molecular level, are lacking since it is difficult to collect fresh trichomes from tea shoots and all trichomes used in previous studies were isolated from tea products that were subjected to a series of processes.
In the present study, we first developed a method to collect trichomes from fresh tender shoots, which could satisfy the research requirements at the molecular level. The major non-volatile metabolites, including catechins, caffeine, amino acids, and aromatic compounds, in the trichomes were determined while using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) and gas chromatography mass spectrometry (GC-MS). Most importantly, the trichome and tea leaf samples were subjected to RNA-Seq analysis, and the differentially expressed genes (DEGs) were identified and compared. When combined with the metabolite contents and gene expression patterns, we showed that tea trichomes might not have obvious contributions, as previously expected, to tea biochemical quality, but might only contribute to tea appearance quality as well as function as the first barriers against a variety of abiotic and biotic stresses.

Plant Materials and Trichomes Collection
In this study, the hairy tea plant Camellia sinensis var. Fudingdabaicha growing in a natural tea garden at Fujian Agriculture and Forestry University (Fuzhou, China, 26 • 05 N, 119 • 18 E) was used as the plant material. One-bud-and-one-leaf tissue samples were collected and then frozen quickly using liquid nitrogen, and trichome isolation was performed by shaking the tissues in liquid nitrogen, as previously described by Spyropoulou, et al. [33], with minor modifications, to isolate fresh trichomes from tender tea shoots in April 2019. Briefly, the tender tissues were collected and stored in a 50 mL tube and quickly frozen in liquid nitrogen. After that, the tube was held on a motor to shake the samples for 10 sec. until the trichomes fell off, and the trichomes were collected from the tubes using spoons. To isolate enough trichomes for further analysis, at least 400 shoots of one-bud-and-one-leaf were sampled from over 40 tea individuals for each replicate, and three biological replicates were used to investigate. The isolated trichomes and processed leaves were used for total RNA extraction and metabolite detection.

Determination of Non-Volatile and Volatile Compounds in Tea Trichomes and Leaves
Tissues of trichomes and processed leaves were dried by freezing and ground to fine powders while using mortars and pestles. 30 mg of each sample was extracted using 1.2 mL of 70% (v/v) Biomolecules 2020, 10, 311 3 of 20 methanol and then vortexed and sonicated at 25 • C for 20 min. to extract the main non-volatile metabolites in the tissues. After centrifugation (10 min., 12,000×), the supernatants were transferred and filtered through a 0.22 µm PVDF filter (Millipore) and stored at −20 • C until analysis. The contents of amino acids, catechins and caffeine were determined while using UPLC-QqQ MS-based targeted quantifications based on authentic standard, as previously described by Chen, et al. [34].
The volatile compounds of fresh trichomes and processed leaves were extracted and determined, as previously described by Dong, et al. [35] and Zeng, et al. [36], with minor adjustments. Briefly, 250 mg of each finely powdered fresh sample was extracted while using dichloromethane (2 mL), and ethyl n-decanoate (5 nmol) was added as an internal standard. After overnight shaking, the extraction solution was collected, dried using anhydrous sodium sulfate, and concentrated to 100-200 µL under a stream of nitrogen. The extract (1 µL) was then subjected to GC-MS that was carried out on a GC-MS QP2010 SE (Shimadzu Corporation, Kyoto, Japan) that was equipped with GC-MS Solution software (Version 2.72, Shimadzu Corporation, Japan). Samples were injected into the GC injection port and held at 230 • C for 1 min., with all injections made in splitless mode. The aroma compounds were separated on a SUPELCOWAX 10 column (30 m × 0.25 mm × 0.25 µm, Supelco Inc., Bellefonte, PA, USA). Helium was used as the carrier gas, with a flow rate of 1 mL/min. The initial GC oven temperature was 60 • C for 3 min., which was ramped up to 240 • C at a rate of 4 • C/min, and then held at 240 • C for 20 min. The mass spectrometer was operated in full scan mode (mass range, m/z 40-200). Some volatile compounds were identified by direct comparison with authentic standards and then quantified according to their calibration curves. Some aromatic compounds without authentic standards were tentatively identified by comparison with mass spectra, and the relative content of each compound was calculated by comparison with the peak area of the internal standard. Three biological replicates were used for each tissue.

RNA Isolation, Library Preparation, and Illumina Sequencing
Total RNA was extracted from tea leaves and trichomes using the TIANGEN RNAprep Pure kit (TIANGEN, Beijing, China), and genomic DNA elimination by treated with DNase I (Invitrogen, Carlsbad, CA, USA) and RNA quality and quantity determination were performed, as previously described by Yue, et al. [7]. Only RNA samples with an A260/A280 > 1.8, A260/A230 > 1.8, and RNA integrity number (RIN) > 8 were used for library construction. A total amount of 3 µg of RNA per sample was used to generate sequencing libraries while using the NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, USA), following the manufacturer's recommendations. To preferentially select cDNA fragments 250~300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Subsequently, 3 µL of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 • C for 15 min. followed by 5 min. at 95 • C before PCR. Afterwards, the PCR products were purified (AMPure XP system), and the library quality was assessed on the Agilent Bioanalyzer 2100 system.
Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform (Novogene, Beijing, China).

Transcriptome Data Processing
The quality of raw data was processed through in-house Perl scripts. Clean reads were obtained by removing reads containing adaptors, reads containing poly-N and low-quality reads from raw data. The Q20, Q30, and GC contents of the clean data were calculated. All the downstream analyses were based on clean data with high quality.
The paired-end clean reads of each sample were aligned to the tea plant genome while using Hisat2 v2.0.5 with the default parameters [37]. The feature Counts v1.5.0-p3 was used to count the read numbers mapped to each gene [38]. Subsequently, the fragments per kilobase of transcript per Biomolecules 2020, 10, 311 4 of 20 million mapped reads (FPKM) value of each gene was calculated based on the length of the gene and read count mapped to that gene. The DESeq2 R package (1.16.1) was used to identify DEGs [39]. The resulting P-values were adjusted while using Benjamini and Hochberg's approach for controlling the false discovery rate, and the unigenes with a |log 2 Fold Change| > 1 and adjusted p-value < 0.05 were considered to be differentially expressed.
The DEGs were subjected to Gene Ontology (GO) enrichment analysis while using the clusterProfiler R package, and the GO terms with corrected p-values < 0.05 were considered significantly enriched [40]. The clusterProfiler R package was used to test the statistically significant enrichment of the DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to understand the high-level biological functions and pathways of DEGs [41]. An FDR ≤ 0.05 was set as a threshold value to identify enrichment pathways.

Quantitative Real-Time PCR Validation
In total, 21 DEGs were selected for real-time qPCR analysis to validate the transcriptome data. qRT-PCR was carried out while using SYBR Premix Ex Taq™ II (TaKaRa, Dalian, China) and run on a CFX96 Touch Real-Time PCR System (BIO-RAD, California, CA, USA), according to the manufacturer's protocol. The Camellia sinensis GADPH reference gene was used as an internal control, as described in previous studies [7]. Table S1 shows the primers for RT-qPCR. Each sample was repeated in triplicate, and the relative gene expression levels were determined while using the 2 −∆∆CT method. The results are presented as the mean ± standard deviation (SD).

Statistical Analysis
Statistical analyses were performed while using SPSS (SPSS Inc., Chicago, IL, USA) and Prism7 (GraphPad Software Inc., La Jolla, CA, USA). The differences among the means were analyzed using the two-tailed Student's t test at a 0.01 probability level.

Comparison of the Key Secondary Metabolites Between Tea Trichomes and Leaves
Nineteen amino acids were determined from tea trichomes and leaves while using UPLC-QqQ-MS. However, threonine was not detected in trichomes, and serine was not detected in leaves. The total contents of amino acids in the leaves was more than 10 times higher than that in the trichomes ( Table 1).
The most abundant amino acids in both the trichomes and leaves were theanine, glutamic acid, glutamine, aspartic acid, and arginine, which accounted for 93.1% and 94.4% of the amino acids, respectively. Theanine was the most abundant amino acid in both the leaves and trichomes, containing 4.49 mg/g and 0.76 mg/g of this amino acid, respectively.
The catechin detection results showed that the content of catechins was significantly higher (more than 61 times) in the leaves than in trichomes. Epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) were the most abundant catechins. In leaves, the contents of EGCG, ECG, EGC, and EC were 82.7 mg/g, 28.8 mg/g, 17.7 mg/g, and 4.2 mg/g, respectively, whereas they were 1.1 mg/g, 0.54 mg/g, 0.085 mg/g, and 0.037 mg/g in trichomes ( Table 2).
The rutin content in tea leaves (0.39 mg/g) was more than six-fold higher than that in trichomes (0.058 mg/g). The caffeine content in tea leaves (31.2 mg/g) was more than 30-fold higher than that in trichomes (1.01 mg/g). Values shown are means ± SD (n = 3); n.d., non-detectable; ** indicates significant difference at the 0.01 level; Std., quantitative analysis based on authentic standard Values shown are means ± SD (n = 3) ** indicates significant difference at the 0.01 level; Std., quantitative analysis based on authentic standard.

Comparison of Aromatic Compounds Between Tea Trichomes and Leaves
34 of the main aromatic compounds were identified and quantified while using GC-MS to investigate the aromatic compounds in fresh trichomes and processed tea leaves. Among the tested compounds, the content of phytol was the highest in both trichomes and leaves, followed by 3,5-dimethylbenzaldehyde, 2-phenylethanol, benzyl alcohol, 2-hexenal, (Z)-3-hexenol, and linalool, as shown in Table 3.

RNA Sequencing, Reference Genome Alignment, and New Gene Annotation
Six cDNA libraries were constructed while using trichomes and leaves to investigate the differences in genes between tea trichomes and leaves (each sample was repeated three times). In total, 353.0 million high-quality clean reads were generated from six libraries, and the sequence data were deposited in NCBI (SRA accession: PRJNA560722). The average clean bases, Q20 values, and Q30 values of each sample were 8.83 Gb, 97.88%, and 93.95%, respectively (Table S2). The read mapping ratio per sample to the reference genome was 86.55-90.50% (Table S2). A total of 11460 transcripts were identified as novel genes and annotated to the Pfam, SUPERFAMILY, GO, and KEGG databases.

Differential Expression Profiling between Tea Trichomes and Leaves
A volcano plot was constructed to determine the genes that were significantly changed between trichomes and leaves. In total, 6560 DEGs were identified from the comparison of trichomes and leaves according to the parameters (p-value < 0.01 and |log 2 Fold Change|>1). Among these DEGs, there were more upregulated DEGs (4471) than downregulated DEGs (2089), while the fold change values of the upregulated DEGs were also larger than that of downregulated DEGs ( Figure S1). GO term and KEGG pathway enrichment analyses were performed on the upregulated and downregulated DEGs, respectively, to analyze the functional enrichment of the DEGs in trichomes in comparison with leaves. For upregulated DEGs, 62 GO terms, including 29 biological process (BP) terms, 27 molecular function (MF) terms, and five cellular component (CC) terms, were significantly enriched (p ≤ 0.05). Among these terms, the most enriched components were categorized as nucleic acid binding transcription factor activity (GO:0001071), transcription factor activity, sequence-specific DNA binding (GO:0003700), iron ion binding (GO:0005506), oxidoreductase activity, acting on paired donors, with the incorporation or reduction in molecular oxygen (GO:0016705), response to stress (GO:0006950), hydrolase activity, acting on glycosyl bonds (GO:0016798), hydrolase activity, hydrolyzing O-glycosyl compounds (GO:0004553), sequence-specific DNA binding (GO:0043565), and multiorganism process (GO:0051704) ( Figure 1A).

Differential Expression Profiling between Tea Trichomes and Leaves
A volcano plot was constructed to determine the genes that were significantly changed between trichomes and leaves. In total, 6560 DEGs were identified from the comparison of trichomes and leaves according to the parameters (p-value < 0.01 and |log2 Fold Change|>1). Among these DEGs, there were more upregulated DEGs (4471) than downregulated DEGs (2089), while the fold change values of the upregulated DEGs were also larger than that of downregulated DEGs ( Figure S1).

Differential Expression Profiling of Genes Involved in the Characteristic Metabolite Biosynthesis Pathways Between Tea Trichomes and Leaves
The expression patterns of the target genes that are involved in the biosynthetic pathways of theanine, catechins, and caffeine were screened and analyzed. 32 genes that were involved in the caffeine biosynthesis pathway were identified, and the expression profiles showed that 20 genes were downregulated and 12 genes were upregulated in the trichomes, as shown in Figure 3A. However, among these genes, only the expression levels of caffeine synthase (TEA010054) and theobromine synthase (TEA028049) were downregulated at a significant level (log2 FoldChange > 1.0). Additionally, 27 genes involved in the theanine biosynthesis pathway were identified ( Figure 3B).

Differential Expression Profiling of Genes Involved in the Characteristic Metabolite Biosynthesis Pathways Between Tea Trichomes and Leaves
The expression patterns of the target genes that are involved in the biosynthetic pathways of theanine, catechins, and caffeine were screened and analyzed. 32 genes that were involved in the caffeine biosynthesis pathway were identified, and the expression profiles showed that 20 genes were downregulated and 12 genes were upregulated in the trichomes, as shown in Figure 3A. However, among these genes, only the expression levels of caffeine synthase (TEA010054) and theobromine synthase (TEA028049) were downregulated at a significant level (log 2 FoldChange > 1.0). Additionally, 27 genes involved in the theanine biosynthesis pathway were identified ( Figure 3B).
Among these genes, two glutamine synthetase (GS)-encoding genes (TEA032125 and TEA028194) were significantly downregulated (log2 FoldChange >1.0), and arginine decarboxylase (ADC, TEA032991) was upregulated in tea trichomes. We found that the theanine synthase gene (TS, TEA015198) was also downregulated, but not at a significant level. Seventy-eight genes, including 41 upregulated and 31 downregulated genes that were involved in regulatory catechin biosynthesis, were isolated from the trichomes vs. leaves dataset ( Figure 3C). Among these genes, 14, including

Differential Expression Profiling of Tfs Between Tea Trichomes and Leaves
TFs, such as MYB, NAC, WRKY, C2H2, bHLH, and homeobox-leucine zipper protein, play pivotal roles in the regulation of secondary metabolism and trichome development. 2369 TFs were identified and classified into 66 types to further analyze the differential regulation between trichomes and leaves ( Figure S2). A total of 117 MYB DEGs were screened, and 95 of those genes were differentially expressed in the trichomes vs. leaves comparison. Among these genes, 74 DEGs were significantly upregulated, and 21 DEGs were notably downregulated in trichomes ( Figure 4A). synthesis of lignin while using 4-coumaroyl-CoA, were considerably upregulated in the trichomes.

Differential Expression Profiling of Tfs Between Tea Trichomes and Leaves
TFs, such as MYB, NAC, WRKY, C2H2, bHLH, and homeobox-leucine zipper protein, play pivotal roles in the regulation of secondary metabolism and trichome development. 2369 TFs were identified and classified into 66 types to further analyze the differential regulation between trichomes and leaves ( Figure S2). A total of 117 MYB DEGs were screened, and 95 of those genes were differentially expressed in the trichomes vs. leaves comparison. Among these genes, 74 DEGs were significantly upregulated, and 21 DEGs were notably downregulated in trichomes ( Figure 4A). We found that several MYB DEGs, such as TEA003067, TEA031473, TEA024116, TEA007100,  TEA014321, TEA017098, TEA023817, TEA018834, TEA001697, TEA011677, TEA025707, TEA023311, and TEA026096, were not expressed in tea leaves, and TEA027578 was not transcribed in trichomes. A total of 108 NAC DEGs were identified, and 33 of those genes were significantly expressed between trichomes and leaves. Thirty of 33 DEGs were upregulated in trichomes, and the DEGs TEA013629, TEA013108, TEA022743, TEA007908, TEA015555, TEA013548, and TEA011210 were specifically expressed in trichomes ( Figure 4B). Additionally, 40 WRKY DEGs were identified as being significantly differentially expressed between trichomes and leaves, including 33 upregulated and six downregulated DEGs in the trichomes vs. leaves dataset. The DEGs TEA027312, TEA012365, TEA027198, and TEA003077 were specifically expressed in trichomes, whereas TEA002329 was not detectable in trichomes ( Figure 4C). Thirty-one C2H2 zinc finger protein DEGs, which include 19 upregulated and 12 downregulated DEGs, were identified to be significantly expressed in the trichomes vs. leaves comparison ( Figure 4D). Similarly, 16 of 24 significantly changed homeobox-leucine zipper protein DEGs were upregulated in the trichomes, and the rest of the DEGs were downregulated in the trichomes ( Figure 4E). On the other hand, 33 bHLH DEGs were identified as being significantly differentially expressed between trichomes and leaves, 19 of 33 DEGs were downregulated, and the rest of the genes were upregulated in trichomes, and TEA011884 was not transcribed in trichomes ( Figure 4F). Thirteen TCP DEGs, including six upregulated and seven downregulated DEGs, were significantly expressed in the trichomes vs. leaves comparison ( Figure 4G). Moreover, 10 WD40 DEGs, including seven upregulated and three downregulated DEGs, were also identified in the trichomes vs. leaves comparison ( Figure 4H).

Validation of Differential Expression Data
21 DEGs regulatory TFs and structural genes that were involved in the trichome development regulation and major secondary metabolic pathways were selected and examined using RT-qPCR to validate the reliability of the RNA-Seq results. The RT-qPCR results exhibited similar expression patterns between trichomes and leaves as the RNA-Seq data, which suggested that our transcriptomic data are reliable and valid ( Figure 6). compared with leaves ( Figure 5E). Moreover, 26 DEGs belonging to GDSL-type lipase/esterase were identified, and all of them showed significantly upregulated expression patterns in trichomes ( Figure  5F). Additionally, 21 DEGs of pectinesterase, including 12 upregulated and nine downregulated DEGs, were identified, and TEA004577 and TEA003984 were not transcribed in leaves, showing that they were trichome specific ( Figure 5G).

Validation of Differential Expression Data
21 DEGs regulatory TFs and structural genes that were involved in the trichome development regulation and major secondary metabolic pathways were selected and examined using RT-qPCR to validate the reliability of the RNA-Seq results. The RT-qPCR results exhibited similar expression patterns between trichomes and leaves as the RNA-Seq data, which suggested that our transcriptomic data are reliable and valid ( Figure 6).  MYB5a, ZAT5, ZAT12, ATHB40, WUSCHEL3, GL2, bHLH162, bHLH83, TTG1, FLS, C4H, CHS1, CHS3, DFR, LAR, TS1, GS2, AMPD, TCS1, and TPS1 were selected for RT-qPCR determination. The GAPDH gene was used as an internal control.
Moreover, we observed that the genes of TCS1 and TPS1 were highly expressed in leaves and related to secondary metabolite synthesis. The TFs ZAT12, ATHB40, and WUSCHEL3 were specifically expressed in trichomes and they might be associated with trichome development in tea plants. Moreover, we observed that the genes of TCS1 and TPS1 were highly expressed in leaves and related to secondary metabolite synthesis. The TFs ZAT12, ATHB40, and WUSCHEL3 were specifically expressed in trichomes and they might be associated with trichome development in tea plants.

Discussion
It has been well established that trichomes (or hairs) are vital characteristics of tea germplasm that are important for tea quality formation in terms of tea appearance and tea infusion. Therefore, the metabolites in tea trichomes, which have been isolated from white tea, green tea, black tea, and yellow tea, have been broadly determined, and the trichomes have been found to contain certain levels of metabolites, such as catechins, amino acids, and caffeine [42]. Moreover, several studies have shown that trichomes also contain aromatic components [43]. However, we found that the trichomes that were used in previous studies were isolated from tea products that have encountered different manufacturing processes, which might impact the accuracy of the results.
In this study, we developed a method to isolate trichomes from fresh tea tissues for the first time to gain a more precise understanding of the metabolites in trichomes. We found that, as compared with tea leaves, the major metabolites of tea, including amino acids, catechins, and caffeine, had low contents in trichomes. In particular, the catechin content in leaves was over 50 times higher than that in trichomes, and the caffeine content in leaves were more than 30-fold higher than that in trichomes ( Table 2); although the amino acids did not exhibit large fold changes between trichomes and leaves, the content of amino acids was 10 times higher in leaves than in trichomes (Table 1), which suggested that the metabolites were mainly synthesized in the mesophyll cells of leaf tissues. Trichomes are developed from epidermal cells. Previously, we showed that the tea trichomes were single-celled and non-glandular [7]. It has been well established that glandular trichomes are the major tissues that are involved in the synthesis and accumulation of secondary metabolites, such as in Artemisia annua and tomato [30,44], whereas non-glandular trichomes have scarcely any ability to synthesize and accumulate secondary metabolites. Hence, we could conclude that tea trichomes had slight or no ability to synthesize the key secondary metabolites of tea, in plants as catechins, caffeine, and amino acids (theanine). Recently, Li, et al. [45] reported that tea trichomes collected in September were rich in free catechins, caffeine, and amino acids, implying that the synthesis and accumulation of metabolites in tea trichomes might be affected by seasonal and cultivar differences.
RNA-Seq technology has been widely used to investigate the differential transcription of genes in glandular trichomes, such as those of tomato [33], Salvia pomifera [46], and Artemisia annua [26], which have been found to play multiple functions in plants. However, the non-glandular trichomes from other plants remain to be examined using this method, except for studies in Arabidopsis [13,15]. Hence, in this study, we compared the differential gene expression between trichomes and leaves in tea plants while using RNA-Seq technology for the first time. A total of 6560 DEGs, including 4471 upregulated and 2089 downregulated DEGs, were identified from the comparison of trichomes and leaves ( Figure  S1). Interestingly, in addition to there being more upregulated DEGs than downregulated DEGs, the fold change values of upregulated DEGs were also larger than those of the downregulated DEGs ( Figure S1). In addition, these upregulated DEGs were significantly enriched in the "phenylpropanoid biosynthesis (ath00940)", "glutathione metabolism (ath00480)", "cutin, suberine and wax biosynthesis (ath00073)", and "cysteine and methionine metabolism (ath00270)" pathways (Figure 2A), and these pathways play fundamental roles in trichome growth by supporting cytoskeleton formation and maintaining the structure of the cell wall. On the other hand, we found that the downregulated DEGs were significantly enriched in MF items, such as terpene synthase activity (GO:0010333), carbon-oxygen lyase activity, acting on phosphates (GO:0016838) ( Figure 1B), and in the pathways of "photosynthesis (ath00195)" and "plant hormone signal transduction enrichment (ath04075)" (Figure 2B), indicating that the elementary functions of trichomes and leaves are different. While considering the structure of tea trichomes, it is impossible that trichomes have photosynthetic ability, and, therefore, they might not be able to synthesize metabolites, including amino acids, catechins, and caffeine. Therefore, we proposed that the structural genes that are involved in the amino acid, catechin, and caffeine synthesis pathways are repressed or not transcribed in trichomes, thus causing the different contents of amino acids, catechins, and caffeine between trichomes and leaves (Tables 1 and 2). Consistently, among 32 DEGs that were involved in the caffeine biosynthesis pathway, the expression levels of caffeine synthase (TEA010054) and theobromine synthase (TEA028049) were significantly downregulated in trichomes, whereas the rest of the DEGs had no significant changes between trichomes and leaves ( Figure 3A). Similarly, in the theanine biosynthesis pathway, two GS DEGs (TEA032125, TEA028194) were notably repressed, and one ADC DEG (TEA032991) was induced in trichomes ( Figure 3B). The TS DEGs (TEA015198) were also downregulated, but not significantly downregulated, in trichomes. GS shares high homology with TS, and both of them have the ability to synthesize theanine [47][48][49], reflecting, to some extent, that the content of theanine in trichomes was lower than that in leaves. In the catechin biosynthesis pathway, 41 of 78 DEGs were significantly upregulated, and only seven DEGs were markedly downregulated in the trichomes ( Figure 3C). Although the structural genes that were involved in theanine, catechins, and caffeine pathways were detected in the trichomes, the key genes generally exhibited downregulated patterns that might influence the biosynthesis of these metabolites. On the other hand, several upregulated genes, such as cinnamate 4-hydroxylase (TEA016772, TEA014864) and phenylalanine ammonia-lyase (TEA003374, TEA014056, TEA003137, TEA014166, and TEA034008) ( Figure 3C), which are critical for the phenylpropanoid pathway, participate in a variety of secondary metabolite biosynthesis pathways, especially lignin synthesis, which might be important for trichome growth [50,51].
It has been demonstrated that the phenylpropanoid and flavonoid biosynthesis pathways are regulated by different TF classes, such as MYB, WRKY, NAC, and bHLH, through modulating the expression of the related structural genes. Among these TFs, MYB-bHLH-WD40 repeat (MBW) ternary complexes have been illustrated to be one of the most important regulatory components that are involved in phenylpropanoid and flavonoid biosynthesis [52][53][54][55][56]. To date, certain MBW component genes that regulate flavonoid biosynthesis have been identified in tea plants. For instance, Sun, et al. [57] showed that R2R3-MYB CsAN1 could interact with bHLH TFs (CsGL3 and CsEGL3) and CsTTG1 (a WD-repeat protein) to form an MBW complex to regulate anthocyanin accumulation; Liu, et al. [58] found that CsWD40 (homologous to AtTTG1 TF) partners with bHLH (CsGL3 and CsTT8) and MYB (CsAN2 and CsMYB5e) TFs to form WBM complexes to control anthocyanin and proanthocyanidin biosynthesis; finally, the overexpression of certain tea plant MYB genes, such as CsMYB5a, CsMYB5b, CsMYB5e, CsMYB5-1, and CsMYB5-2, could alter anthocyanin and proanthocyanidin accumulation in other plants [59][60][61]. We found that most of the DEGs that were involved in the MYB, NAC, and WRKY classes were significantly upregulated in trichomes since tea trichomes usually exhibit glossy characteristics and might not have the capability to synthesize and store flavonoids and phenylpropanoids, especially anthocyanins and proanthocyanidins (Figure 4). The WBM complexes not only regulate flavonoid synthesis, but also modulate trichome development through different TFs competing to form WBM complexes [6,16,62]. Recently, Liu, et al. [58] indicated that the tea plant CsWD40 interacted with bHLH and MYB TFs to form WBM complexes that could alter anthocyanin accumulation and regulate trichome development. Nonetheless, known key TFs, such as GL1, GL3, EGL3, and TTG1, which were involved in trichome development, were not determined to be expressed at a significant level, although TFs, such as TTG1 (TEA000080), were upregulated in trichomes. Consistently, Jakoby, et al. [15] found that the known key genes, such as GL1 and TTG2, which are involved in trichome development, had low or no expression in mature trichomes in Arabidopsis. The reason for this result might be related to the spatiotemporal specificity of gene expression. For example, genes, such as GL3, are highly expressed in initiating trichome cells, but become less detectable or nonexistent in mature trichomes [63], indicating that these genes primarily function in trichome differentiation rather than in trichome growth. Unlike in other plants, trichome initiation is probably combined with tea bud development at an early stage, and the trichomes on the first tea leaf are lengthier than those on the bud. Therefore, these key regulatory genes are slightly or not differentially expressed in the comparisons of trichomes vs. leaves and hairy vs. hairless cultivars [7]. On the other hand, we found that several genes that are involved in the negative regulation of trichome development were downregulated in trichomes, such as MYB82 (TEA023420), whose homologous gene was overexpressed in Arabidopsis, leading to reduced trichome numbers [10]. In this study, we found that some MYB (13), NAC (7), and WRKY (4) DEGs were trichome specific (Figure 4), and most trichome-specific genes were associated with cell wall synthesis, such as TEA013629 (homologues to AtNST1 gene) [64,65] and TEA007243 (homologues to AtMYB86 gene) [66], indicating that these TFs might be mainly involved in trichome development and growth regulation through modulating the cell wall or fiber synthesis. These trichome-specific TFs appear to participate in trichome elongation rather than initiation, and their potential functions in cell expansion and cell wall synthesis deserve further investigation.
In this study, we found that most gene family members, such as cupin superfamily proteins, GDSL-type lipase/esterase, GST, CesA, TPS, peroxidase, and pectinesterase, which are generally involved in cell wall synthesis and abiotic stress response, were significantly upregulated in trichomes ( Figure 5). Similarly, previous transcriptome analysis also showed that the genes that were involved in these pathways were expressed at high levels in Arabidopsis trichomes [13,15]. Genes, such as GST, CesA, and peroxidase, are regarded as critical for cellulose and lignin synthesis, which are important for trichome cell proliferation [67][68][69], and trichomes are rich in cellulose and contain lignin [32]. Consistently, CCR and CAD were both markedly upregulated in the trichomes ( Figure 3C) and have been reported to play a critical role in lignin biosynthesis [50,51]. In particular, these types of genes, including cupin superfamily proteins, GDSL-type lipase/esterase, and peroxidase, also have multiple functions in the plant stress response and cell wall synthesis [70][71][72][73][74], which might be correlated to the roles of trichomes in tea plants. Trichomes are thought to be the first physical barrier for protecting plants against insect and pathogen attack, lessening the heat load of leaves, reducing transpiration rates, and enhancing drought and cold resistance [75][76][77]. Generally, tea plants with high-density or long-length trichomes have a high ability to resist cold stress, drought stress, and biotic stress, including feeding by Acaphylla theae and leafhoppers [78]. Tea plants originate from warm areas in Southwest China, where natural conditions, including temperature and humidity, are suitable for plant growth, and they have been planted in more than 60 countries with a variety of adverse conditions, such as cold and drought [79][80][81][82]. It has been observed that the density and length of trichomes on wild-type tea plants were partially lower than on the cultivated species [83]. Currently, more than 300 tea plant cultivars have been selected in China, and none of them have glabrous tender shoots [84], which indicates that tea plant trichomes may evolve to adapt to environmental changes and mainly result from artificial selection.
Generally, tea products processed using tender hairy shoots are commonly recognized to have the "háoxiang" aroma, a term used to describe a fragrance that has been previously reported to might be secreted by tea trichomes. In this study, we detected the aromatic compounds in trichomes and observed that 26 out of 34 aromatic compounds, especially those regulating tea aromatic quality formation, such as linalool and its oxides, nerol, jasmine lactone, indole, methyl salicylate, and alpha-farnesene, were accumulated in significantly higher levels in tea leaves than in trichomes (Table 3). Interestingly, the contents of (E)-2-hexenol, hexyl butanoate, and trans-2-hexenyl hexanoate in trichomes were considerably higher than those in tea leaves. The contents of major terpene compounds, such as linalool, trans-nerolidol, and nerol, were low in trichomes (Table 3) given that nearly half of the TPS genes (16/33) were downregulated, and some of the genes were markedly upregulated in trichomes ( Figure 5D), indicating that these upregulated TPS genes might be involved in other types of non-volatile terpenoid synthesis and that their functions are worthy of further investigation. Although the trichomes also had a high content of certain aromatic compounds, such as phytol, acetate, 3,5-dimethylbenzaldehyde, benzyl alcohol, and 2-hexenal, their contribution to the tea aroma might not be greater than that from the leaves. Specifically, we showed that tea trichomes do not appear to be capable of synthesizing secondary metabolites, and even if they could synthesize these compounds or other special compounds, the contents of these compounds in trichomes are very low when compared to those in leaves. Moreover, the tea manufacturing processes might not influence aromatic compound synthesis and secretion in tea trichomes since there are not enough substrates stored in the trichomes, and, therefore, the "háoxiang" aroma, which is regarded as a key feature of tender shoots, might not be released from trichomes but is closely related to trichomes. Hence, the function of trichomes in tea products seems to need reappraisal.

Conclusions
In this study, trichomes were first isolated from fresh tea plant shoots while using a novel method, and the difference between trichomes and tea leaves per se was uncovered from metabolite and gene expression patterns using RNA-Seq analysis. Our results indicate that tea trichomes might not have the ability to synthetize metabolites; when compared with tea leaves, the contents of the main catechins, amino acids, caffeine, and volatile aromatic compounds in trichomes were low. Importantly, a total of 6560 DEGs, including 4471 upregulated and 2089 downregulated DEGs, were identified from the comparison of trichomes and leaves, and the molecular regulatory differences between trichomes and leaves were explored. In addition, certain tea trichome-specific genes, especially different types of TFs, were identified, showing that tea trichomes are closely related to the environmental adaptation of tea plants. The findings from the present study can provide fundamental information for understanding the role of trichomes in tea quality formation and in tea plant growth and development.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/2/311/s1, Figure S1: Global distribution of DEGs identified from the comparison of trichomes vs. leaves; Figure S2: Transcription factors identified from RNA-Seq analysis; Table S1: Primers used for qRT-PCR validation; Table S2: Summary of the RNA-Seq data obtained from trichomes and leaves; Table S3: Expression levels of key genes involved in the caffeine biosynthesis pathway, theanine biosynthesis pathway, and catechin biosynthesis pathway.