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. 2018 Jun 8;25(1):59–69. doi: 10.1007/s12298-018-0564-x

Evaluation of tea (Camellia sinensis L.) biochemical traits in normal and drought stress conditions to identify drought tolerant clones

Mehdi Rahimi 1,✉, Mojtaba Kordrostami 2, Mojtaba Mortezavi 1
PMCID: PMC6352540  PMID: 30804630

Abstract

Abiotic stresses, such as drought, can induce different morphological, physiological and molecular responses in the tea plants. Since there have not been any experiments on the screening of tea genotypes in terms of drought tolerance, this study was conducted to screen the drought resistance of 14 tea clones of Iran germplasm in a randomized complete block design with three replications, separately in two stressed and non-stressed conditions at Fashalam tea station. The results of grouping the clones under normal and stress conditions and comparing their results with the results of mean comparison of the agronomic and biochemical traits showed that in all cases, clones 100, Bazri and 399 were in the group that can be identified as the drought-tolerant group. Also, the results showed that in the most cases, clones 278, 276 and 285 were placed in a group that had low values for all of the traits and could be considered as a group that are susceptible to drought stress.

Keywords: Biochemical traits, Drought stress, Mean comparison, Tea

Introduction

Tea (Camellia sinensis L.) is one of the tropical and subtropical plants that has a special importance due to its nutritional value and its effects on the human body. It is one of the most popular soft drinks due to its quantitative and qualitative values in the human nutrition (Gonbad et al. 2015). Currently, the tea growing areas are located between 42°N and 20°S, with the minimum rainfall of 1040 mm. Temperature, light, location, soil and water are the most important environmental factors that play an important role in the growth and development of the tea plants (Al-Achi 2008). Although, some of the latest reports revealed that Chinese tea plants such as Shuchazao and Yinghong can survive below 15 °C, the minimum temperature for survival of the Chinese type of the tea is 15 °C (Ban et al. 2017; Wang et al. 2013; Zhang et al. 2014). The minimum temperature for the photosynthesis of this plant is 10 °C, and at lower temperatures, the plant growth decreases and the growth stops completely as the temperature continues to decrease (Chen et al. 2013). The proper soil temperature for the root activation and nutrient absorption is between 20 and 25 °C. In Iran, the tea gardens have been widespread in the north of the country in a range of 203 km and a width of 90 km between the Caspian Sea and the mountains of Alborz in two provinces of Gilan and Mazandaran. On the margins of the Caspian Sea, due to its relative humidity and constant cloud cover and low altitude, the air temperature is moderate and the heat range is limited. This area has warm, humid, sultry summers and mild winters, and long frost rarely happen (Gonbad et al. 2015).

The increasing population growth and the need for agricultural products and water resources has to be seriously addressed in the world, including Iran. In the future, the main challenge of developing countries, including Iran, will be water, and food production will depend on the availability of water and the sustainability of water resources (Mondal 2014). Due to the climate restrictions for the development of cultivated gardens, this quantity should be obtained from existing land. For this purpose, it is necessary to develop a program for the production of superior tea clones that can be both functional and sufficient in underwater conditions (Carr 2017). The area under cultivation of tea in Iran is about 32,000 ha, which is located in 2 northern provinces of Gilan and Mazandaran. About 40% of the Earth land is located in arid and semi-arid regions. Iran with an average rainfall of 240 mm/year is classified as arid and semi-arid regions (Basu Majumder et al. 2010). Drought or water deficiency is one of the most important stress-inducing factors in the crop plants. Unfortunately, water scarcity is not restricted to these areas, and sometimes irregular rain distribution in other places creates difficult conditions for the plant growth. Such a stress affects the yield and often causes a loss of yield in the plant (Farooq et al. 2009). Drought in terms of time, length and severity of the stress period causes a decrease in yield through the effect on each of its components. Therefore, leaf yield of tea and its components as one of the most important indices for selection of compatible or tolerant plants should be considered in the breeding programs (Anjum et al. 2011). Thus, drought resistance or tolerance is considered as important physiological and breeding aspect. Water stress in some stages of growth, in comparison with other stages, causes more damage to the plant crop (Anjum et al. 2011).

Abiotic stresses such as drought can induce different morphological, physiological and molecular responses in the tea plants (Jiménez et al. 2013; Shinozaki and Yamaguchi-Shinozaki 2007; Valliyodan and Nguyen 2006). Drought stress significantly reduce the stem, root ratio, leaf area, stem diameter, stomatal conductance, chlorophyll content, photosynthesis rate, transpiration rate, dry matter allocation to the root, and ultimately led to a 14.0–14.3% reduction in the yield of tea plants (Carr and Stephens 1992; De Costa et al. 2007). Drought stress is one of the main causes of yield loss in the plants. In today's world, due to the development of arid and semi-arid regions and the limited availability of water resources, identification and selection of water deficit resistant plants is necessary to minimize the future problems of the world in terms of food supplies. To achieve this goal, it is necessary to determine the degree of resistance of the plant species to drought stress and also to identify the mechanisms involved in the survival of plants under arid and semi-arid conditions (Rahnama and Ebrahimzadeh 2005). The variation in root and stem biomass under the stress conditions among plant varieties, has shown that plant variety is an important factor in determining the resistance to the stress conditions (Boldaji et al. 2012).

Plants have developed various strategies for the stress tolerance. These strategies include changes in metabolic processes, structural changes in the membrane, the expression of specific genes, and production of secondary metabolites (Jiménez et al. 2013). Under the drought stress, tea provides different mechanisms at physiological, biochemical and molecular levels and provides avoidance and tolerance to the drought stress. Drought avoidance in tea is due to the morphological changes such as decreasing the stomatal conduction, leaf area and developing the root system (De Costa et al. 2007; Smith et al. 1994, 1993). On the other hand, drought tolerance is induced through some physiological and molecular mechanisms including photosynthesis regulation, osmotic regulation, antioxidant production, and inhibition of active oxygen species (Gupta et al. 2012, 2013). Various studies have suggested that reducing the moisture content of the tea plants induces the antioxidant activity such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) along with the antioxidants such as polyphenols and proline and the amount of these compounds in tolerant clones is more than the sensitive ones, which may act as a defense mechanism against the water stress (Jeyaramraja et al. 2002; Upadhyaya and Panda 2004; Upadhyaya et al. 2008).

A number of genes have been identified for drought tolerance in tea (Dong et al. 2017; Koech et al. 2018). A study on tea in drought stress condition has shown that the malondialdehyde concentration and electrical conductivity of leaves of stressed plants increased when chlorophyll concentration decreased under severe dry stress (Guo et al. 2017). Therefore, this research was conducted to identify drought-tolerant tea clones based on biochemical and agronomic trait for use in tea breeding programs.

Materials and methods

Geolocation, plant material and the experimental design

This experiment was conducted at Farsalam Tea Station, Lahijan Tea Research Center, Lahijan, Iran, with a latitude of 49° 45′ Eand a latitude of 37° 23′ N with a height of 7 m above the sea level in the 2017 crop year. Since there have not been any experiments on the screening of tea genotypes in terms of drought resistance or tolerance, in this study, 14 tea clones (Table 1), were evaluated in a replicated design. 14 tea clones (Table 1) were studied using a randomized complete block design with three separate replications (biological replicate), separately in two stressed and non-stressed conditions at Fashalam tea station. All the agronomic operations such as fertilization, pest, diseases and weeds management were carried out according to the custom of the area. Irrigation in the both designs was carried out routinely until the end of March and then irrigation was cut off in the drought stress treatment until the end of August (when tea leaves were harvested). Due to the highest damage of drought stress in Guilan tea gardens in July and August, the drought stress was carried out in August. After applying the stress treatment and collecting leaves of plants, agronomic and biochemical traits such as chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, reducing sugars, total protein and proline concentrations were measured in the laboratory.

Table 1.

The name and characteristics of tea clones studied at Fashalam station

Row Clone name Type Origin Yield Quality
1 100 Chinese West Guilan High Good quality
2 277 Chinese West Guilan Medium Medium quality
3 270 Chinese West Guilan Medium Medium quality
4 74 Chinese West Guilan Medium Medium quality
5 269 Chinese West Guilan Medium Medium quality
6 276 Chinese West Guilan High Good quality
7 399 Chinese West Guilan Medium Medium quality
8 272 Chinese West Guilan Medium Medium quality
9 285 Chinese West Guilan High Good quality
10 278 Chinese West Guilan Medium Medium quality
11 252 Chinese West Guilan Medium Medium quality
12 261 Chinese West Guilan Medium Medium quality
13 280 Chinese West Guilan Medium Medium quality
14 Bazri Chinese West Guilan Medium Medium quality
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Regional weather features

The average, standard deviation and variability of monthly, seasonal and annual rainfall were calculated for the time period under investigation. In addition, the proportional contribution of seasonal to annual rainfall was analyzed in order to understand rainfall distribution pattern. The annual rainfall was estimated 1441 mm and the average annual temperature was 16.8 °C, based on an average of 10 years data. Based on the meteorological divisions, this region is the part of warm Mediterranean regions with warm summers and mild winters. Although the average annual precipitation of the test area was very high, the distribution of the rainfall throughout the year was generally so low that during the period of tea growth, from June to September, rainfall wasn’t very and it was about zero. Irrigation was suspended for 1 month in drought stress conditions and evaluation of drought stress was done with gypsum block. Soil moisture content was measured at the field capacity and wilting point in the laboratory and based on block resistance information against volumetric soil moisture, the graph was plotted. Based on block resistance data in different days after stress, it was observed that after 7 days of irrigation interruption, drought stress was gradually applied and a severe stress was observed in the field at the end of irrigation broken off.

Measurement of agronomic and biochemical traits

After applying drought stress treatments for tea clones in the field, photosynthetic pigmentation was measured in leaf of tea clones under drought stress. Chlorophyll a, b and total chlorophyll and carotenoid were quantified in samples by reading the optical density at 663, 645 and 470 nm (Sudhakar et al. 2016). Reducing sugars was measured by the Somogyi method (Somogyi 1952). To measure the protein concentration, 100 μl of protein extract and 5 ml of biuret reagent were added and rapidly strained. After 25 min, the absorbance was read by a spectrophotometer at 595 nm. Protein concentrations were calculated using a standard curve (Bradford 1976).

To measure the amount of proline, 2 ml of supernatant obtained from the centrifugation of the extract was mixed with 2 ml of reagent ninhydrin and 2 ml of pure acetic acid and placed in an air bath for 1 h at 100 °C. Then, the tubes containing the mixture was immediately placed in the ice bath. Next, 4 ml of toluene was added to the mixture and the tubes were well vortexed. By stacking the tubes for 15–20 min, two separate layers were formed. The upper color phase, which included toluene and proline, was used to measure proline concentration. The absorbance of this dye was determined at 518 nm and the amount of proline in each sample was calculated using a standard curve. The results obtained from measuring proline content in grams of dry weight were calculated and presented (Bates et al. 1973). The yield of leaf was harvested in each plot and converted to yield per hectare. The number of shoots and number of bushes per clone were counted in a plot of 25 × 25 cm. Simple analysis of variance for each environment and combined analysis was performed on a randomized complete block design with SAS software and the mean comparison was achieved by Duncan test. Also, cluster analysis was done with the ward method with the PAST software.

Results and discussion

Simple and combined analysis of variance of the traits under normal and drought stress conditions

In Fig. 1, some clones are shown under normal (1) and drought stress (0) conditions. The results of simple analysis of variance showed that there is a significant difference between the clones in normal conditions (data not shown) for the most traits (except for number of shoots). The coefficient of variation varied from 0.9 to 28.73% for the studied traits. The differences between the clones indicate that there are variations among the clones for these traits and the selection can be performed among them. Also, the results of analysis of variance showed that there was a significant difference between the clones in the drought stress conditions for the most studied traits (except for number of shoots and number of plants). The coefficient of variation varied from 1.31 to 45.20% for the studied traits. Differences between the clones indicate that for these traits, there is a variation among the clones and one can choose between them to select a suitable clone for stress conditions.

Fig. 1.

Fig. 1

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Measuring the traits in clones 100 and 285 in normal and drought stress conditions

Descriptive statistics showed that the highest amount of phenotypic variation in normal conditions for chlorophyll b and yield was 71.664 and 45.436, respectively (Table 2). In drought stress condition, proline and chlorophyll a and b (84.865, 62.537 and 62.061, respectively) traits showed the highest phenotypic variation (Table 2). Due to the amount of observed phenotypic variation in the traits, we can use the observed variation for breeding traits in tea clones.

Table 2.

Descriptive statistics for studied traits in normal and drought stress conditions

Traits Mean Standard deviation Phenotypic CV (%) Range
Normal Stress Normal Stress Normal Stress Normal Stress
Protein (mg/gfw) 0.007 0.007 0.0001 0.0002 2.097 2.878 0.0005 0.0006
Proline (mg/gfw) 0.004 0.004 0.0017 0.0037 41.805 84.865 0.0047 0.013
Sugars (mg/gfw) 0.053 0.050 0.0036 0.0031 6.852 6.341 0.01 0.0104
Chl. A (mg/gfw) 6.829 4.196 2.9424 2.6243 43.085 62.537 11.32 9.58
Chl. B (mg/gfw) 2.931 2.412 2.1007 1.4966 71.664 62.061 7.28 5.43
Total Chl. (mg/gfw) 9.618 6.517 4.2500 3.8167 44.189 58.567 16.60 15.12
Carotenoids (mg/gfw) 2.817 1.684 0.9873 0.7805 35.050 46.356 3.00 2.73
Number of shoots 13.357 10.964 2.4529 2.3490 18.364 21.424 9.00 7.5
Number of plants 4.929 4.464 1.4525 1.1843 29.472 26.527 5.50 3.5
Leaf yield (kg/m2) 328.542 168.190 149.2747 81.5994 45.436 48.516 450.00 275
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In order to assess significant difference among the clones more accurately which would not be attributed to the genotype × environment interaction, combined analysis of the variance for two experiments was carried out in the form of pooled analysis. Prior to the combined analysis, to test the uniformity of the variance of experimental errors in the two studied environments, F test was used and the larger variance: smaller variance ratio was calculated for each of the studied traits and compared with the F value of the table. Since the calculated F value was non-significant for all the traits, the combined analysis of variance was carried out. In this analysis, the genotype was considered as a constant factor and environment was considered as an experimental randomized factor. The results of combined ANOVA for the studied traits showed that the effect of the environment for all the studied traits (except for leaf yield) wasn’t significant at 1 or 5% probability level (Table 3). In the sense that the natural environment and drought stress did have the same effect on the mentioned traits, or, in the other words, the change in the amount of these traits in two conditions of moisture was significant. One of the reasons is that most of these traits are influenced by environmental factors due to their occurrence and other factors. Also, there was a significant difference between genotypes for all traits (Table 3). Genotype × environment interaction was significant for all traits (except for number of shoots and number of plants), implying that the reaction of different genotypes under different moisture conditions was not the same.

Table 3.

Combined analysis of variance of the traits in tea clones in two non-stressed and drought stress environments

Source of variation Degree of freedom Mean square of traits
Protein (mg/gfw) Proline (mg/gfw) Sugars (mg/gfw) Chl. a (mg/gfw) Chl. b (mg/gfw)
Stress 1 0.00000011ns 0.0000004ns 0.00017ns 97.04ns 3.78ns
E1 or Rep (stress) 2 0.00000001 0.000003 0.000001 1.42 0.48
Clone 13 0.00000005** 0.00002** 0.00004** 21.62** 8.35**
Clone × stress 13 0.00000009** 0.00002** 0.00001** 9.47** 4.95**
Error 26 0.00000001 0.000002 0.000001 1.23 0.84
CV% 1.33 31.46 2.29 20.10 34.40
Source of variation Degree of freedom Mean square of traits
Total Chl. (mg/gfw) Carotenoids (mg/gfw) Number of shoots Number of plants Leaf yield (kg/m2)
Stress 1 134.62ns 17.98ns 80.16ns 3.02ns 359,980.94**
E1 or Rep (stress) 2 3.31 1.88 9.73 2.45 302.92
Clone 13 43.12** 1.95** 20.37ns 5.58** 47,217.19**
Clone × stress 13 22.14** 1.22** 2.70ns 1.44ns 10,665.64**
Error 26 2.72 0.42 13.92 1.72 793.68
CV% 20.44 28.83 30.69 27.89 11.34
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ns and ** are not-significant and significant at 1% probability level respectively

Mean comparison of the studied traits in normal and drought stress conditions

Total protein

The mean comparison for this trait is shown in Table 3. The results showed that the interaction between stress x clone for total protein was significant at 0.01 probability level. The amount of protein was different for clones under normal and stress conditions. The clone 100 had the highest protein content under stress conditions, followed by clone 252 under normal conditions and clone 277 under stress conditions. Clones 252, Bazri and 276 had the lowest protein in the stress conditions. The clone 100 can tolerate drought stress better because of high protein content. These data are consistent with the results of some studies which reported that with increasing irrigation time, the protein content of the leaves is significantly reduced (Kafi and Mahdavi Damghani 2003).

Proline

The results of combined ANOVA for proline in control and drought stress conditions (Table 3) showed that the clonal effect and the interaction between stress and clones showed a significant difference at 1% probability level. The results of mean comparison for this trait (Table 4) showed that among the tea clones, clones 100 and 399 showed the highest proline content under drought stress conditions. Also, the results of the mean comparison for the yield of these two clones showed that they had high yield under drought stress conditions and the yield loss was low. Thus, the increase of proline in these two clones have been able to increase tolerance to drought stress. The results of this study are consistent with other results which found that increased proline was consistent with increasing drought stress (Johari-Pireivatlou 2010; Maralian et al. 2010). Proline protects plants against environmental stresses through various mechanisms including osmotic regulation, decontamination of active oxygen species and stability of enzymes or proteins. In some plants, it has been proven that proline changes are associated with their ability to tolerate or adapt to stress conditions, and can be used as a parameter for selecting the resistant plants (Niknam et al. 2006). Increase in proline begins with decrease of leaf water potential, which contributes to maintaining inflation and reducing membrane damage in plants. In this way, dehydration stress tolerance is increased by osmotic adjustment (Rahdari and Hoseini 2012).

Table 4.

Mean comparison of the studied traits in normal and drought stress conditions

Treatmens Average of traits
Clone Stress condition Protein (mg/gfw) Proline (mg/gfw) Sugars (mg/gfw) Chl. a (mg/gfw)
74 Normal 0.0068abcde 0.0056bc 0.0566abc 7.2154bcde
74 Drought 0.0066cdef 0.00595bc 0.05005efgh 3.04575defghij
100 Normal 0.0065def 0.0023c 0.0506efgh 9.92545ab
100 Drought 0.00705a 0.0156a 0.0494fgh 3.95675cdefghij
252 Normal 0.007ab 0.0027c 0.0502efgh 7.48715bcd
252 Drought 0.0064f 0.0032bc 0.04815fgh 4.2767cdefghij
261 Normal 0.00685abcd 0.0034bc 0.05785ab 13.4701a
261 Drought 0.0066cdef 0.0032bc 0.04785fgh 9.8618ab
269 Normal 0.0066cdef 0.0025c 0.0485fgh 4.7799cdefghij
269 Drought 0.0066cdef 0.0044bc 0.0473gh 0.9658ij
270 Normal 0.0068abcde 0.0046bc 0.0594a 8.115bc
270 Drought 0.00655cdef 0.0022c 0.056abcd 1.3417hij
272 Normal 0.00665bcdef 0.007bc 0.05445bcde 2.6808efghij
272 Drought 0.0065def 0.0032bc 0.04975efgh 6.8595bcdef
276 Normal 0.0067abcdef 0.00225c 0.0525cdef 7.9415bc
276 Drought 0.00645ef 0.00235c 0.04575 h 5.60595bcdefgh
277 Normal 0.00655cdef 0.00515bc 0.0503efgh 4.1826cdefghij
277 Drought 0.007ab 0.0023c 0.04905fgh 6.4497bcdefg
278 Normal 0.0068abcde 0.0023c 0.04855fgh 7.25815bcde
278 Drought 0.00675abcdef 0.00235c 0.04815fgh 2.6206fghij
280 Normal 0.00655cdef 0.00295bc 0.04965efgh 8.1741bc
280 Drought 0.0065def 0.00285c 0.0489fgh 5.43105bcdefg
285 Normal 0.0069abc 0.00565bc 0.0525cdef 7.1306bcdef
285 Drought 0.0066cdef 0.00235c 0.04605 h 5.42975bcdefghi
399 Normal 0.0068abcde 0.007bc 0.0572abc 2.1501ghij
399 Drought 0.00675abcdef 0.0084b 0.0561abcd 2.6186fghij
Bazri Normal 0.0065def 0.005bc 0.0545bcde 5.0982cdefghi
Bazri Drought 0.0064f 0.0023c 0.0517defg 0.28665j
Treatmens Average of traits
Clone Stress condition Chl. b (mg/gfw) Tot Chl (mg/gfw) Carotenoids (mg/gfw) Leaf yield (kg/m2)
74 Normal 2.33395 cdef 9.5493bcdef 4.1555ab 293.335cdef
74 Drought 2.25445cdef 5.30025efgh 1.43705cdef 191.67fghi
100 Normal 3.1003bcdef 13.02565abcd 3.01335abcde 350c
100 Drought 1.2136def 5.1704efgh 2.12195abcdef 250cdefgh
252 Normal 2.02835cdef 9.5155bcdef 3.5471abc 150hijk
252 Drought 4.4325abcd 8.7092cdefg 1.66715bcdef 65jk
261 Normal 7.70785a 19.17795a 4.40875a 580ab
261 Drought 5.52355abc 15.88535ab 1.95915abcdef 325cd
269 Normal 2.0417cdef 6.8217defgh 3.0223abcde 225defgh
269 Drought 3.6437bcdef 4.6095efgh 2.9908abcde 191.665fghi
270 Normal 2.81985cdef 10.9348bcde 2.8912abcdef 315cde
270 Drought 1.2408def 2.5355gh 0.5241def 200efghi
272 Normal 1.05015def 3.73095fgh 4.0881abc 173.75ghij
272 Drought 2.72875cdef 9.5882bcdef 2.0465abcdef 91.665ijk
276 Normal 1.11845def 9.0599bcdefg 3.16495abcd 195.835fghi
276 Drought 2.70725cdef 8.31325cdefg 2.20825abcdef 181.25fghij
277 Normal 2.538cdef 6.7206defgh 2.1184abcdef 229.165defgh
277 Drought 3.1459bcdef 9.5956bcdef 0.4567ef 50 k
278 Normal 6.82405ab 14.08215abc 1.9225abcdef 250cdefgh
278 Drought 1.33185def 3.95245fgh 1.8689abcdef 91.665ijk
280 Normal 4.1279abcde 12.302bcd 1.75185abcdef 475b
280 Drought 2.5109cdef 5.44195efgh 2.40485abcdef 166.665ghij
285 Normal 1.3939def 8.52445cdefg 1.4132cdef 272.5cdefg
285 Drought 2.45385cdef 7.8836cdefg 1.69725bcdef 91.5ijk
399 Normal 0.42805ef 2.57815gh 2.25445abcdef 600a
399 Drought 0.09575f 3.48395fgh 1.9234abcdef 270cdefg
Bazri Normal 3.52615bcdef 8.62435cdefg 1.68505bcdef 490ab
Bazri Drought 0.4791ef 0.76575 h 0.26515f 188.57fghi
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Treatments with similar alphabets do not differ statistically

Reducing sugars

The results of this study showed that the amount of reducing sugars was significant under different levels of stress, so that the highest amount was obtained in the normal conditions for clones 270, 261 and 399. Also, the lowest values were obtained for clones 276, 285 and 269 under drought stress conditions. It has been shown that the plants have different protective strategies in counteracting drought stress, including the accumulation of osmolytes such as proline, reducing sugars, enzymatic and non-enzymatic strategies against drought-induced oxidative stress (Lotfi et al. 2009, 2010; Nasibi et al. 2011). The accumulation of intracellular soluble sugars plays an important role in the osmotic regulation, helping to reduce the cellular water potential and maintain turgor under drought stress inside the cell (Sato et al. 2004). According to previous studies (Devi and Sujatha 2014; Mohammadkhani and Heidari 2008), proline, and the amount of sugars in the plant increases with increasing the stress time. This indicates the need for more energy to absorb water and, most importantly, to protect the membrane, which results in a glassy state.

Chl. a, Chl. b and total Chl.

The chlorophyll a content in all 14 clones of the tea plants under drought stress was significantly reduced compared to tea clones in normal conditions (Table 4). Tea clones were in good condition under normal conditions and had very dark green leaves. After measuring and analyzing this index, it was determined that the effect of clones and the interaction of stress and clones were significant for this parameter. The highest amount of chlorophyll a was observed in clones 261 and 100 in normal conditions and the least, in clones Bazri and 269 under stress conditions. Water stress reduces photosynthetic pigments, chlorophyll content and degrades photosynthesis aparatus (Anjum et al. 2011). Table 3 also shows the results of composite analysis of variance for chlorophyll b index in 14 tea clones under drought stress. In this index, the effect of clones and the interaction of stress and clone had a very significant difference at 1% level (Table 3). The highest levels of chlorophyll b were obtained from clones 261 and 278 in normal conditions and the lowest one was obtained in clone 399 in normal and stress conditions. Therefore, chlorophyll b is also a trait that is influenced by genotype in addition to environmental factors. The results shows that the total chlorophyll content varied in the tea clones under drought stress and normal conditions. This parameter is the result of the two preceding indicators, chlorophyll a and chlorophyll b. In this index, the effect of clone and the interaction of stress and clone had a very significant difference at 1% level (Table 3). Plants that were under normal conditions had the highest total chlorophyll content than the stressed plants. Chlorophyll catabolism increases under drought stress conditions, which can be the result of premature aging of leaves due to the hormonal imbalance of the water stress (Kafi and Mahdavi Damghani 2003). Under drought stress, photochemical activity of the plant is inhibited, the chlorophyll content of the leaves varies, and the activity of the calvin cycle enzymes decreases in the process of photosynthesis (Monakhova and Chernyad’ev 2002). In many plant species, reduction in crop production under drought stress is often associated with a decrease in photosynthetic capacity (Bacelar et al. 2007). Allen (1995) has reported that the highest damage to plants applied through various stresses is related to the oxidative damage at various levels of the cell. Aerobic photosynthetic organisms are exposed to a different amount of toxins during their lifetime, which is more severe in higher plants under drought stress (Sairam and Srivastava 2001). The results of the studies show that under the stress conditions, and even under high concentrations of CO2 in the environment, photosynthesis is reduced further, which suggests that the photosynthesis apparatus is affected, regardless of the closure of the stomata. With the failure of the photosynthetic apparatus due to the drought stress, plants are faced with oxidative stress and eventually the yield decreases (Sairam and Saxena 2000; Türkan et al. 2005).

Carotenoids

Like other herbal pigments under stress conditions, the amount of carotenoids decreases, the lowest of which is related to clones Bazri and 277 under stress conditions. Also, the highest values were for clones 261 and 74 in normal conditions. Carotenoids act as auxiliary pigment in photosynthetic tissues but also play an antioxidant role and accumulate free oxygen radicals (Egert and Tevini 2002). It is likely that the reduction of carotenoids in severe stress is due to lack of plant resistance to this stress level. Also in another study by Falakroo et al. (2014), the effects of two irrigation cuttings treatments (10 and 20 days) on the process of phenolic and flavonoid compounds, malondialdehyde content, chlorophyll a, total chlorophyll and carotenoids in three clones of tea, DN, 100 and 258, were evaluated. The results showed that phenol content in DN and 100 clones was highest in 20 and 10 days irrigation cuttings, respectively, but in 258 clones, no significant changes were observed in any of the treatments. The amount of flavonoids and antioxidant capacity in the 20-day irrigation cuttings treatment increased in the DN clone, while its amount in the 258 clone decreased and remained constant in the clone 100. The highest amount of proline for all clones was observed only in 20-day irrigation cuttings treatment. Malondialdehyde values increased in clones 100 and 258, but the DN clone did not change. Reduction of chlorophyll a and total chlorophyll was observed in 20-day irrigation cuttings treatment in DN and 100 clones. However, these values did not show significant changes in clone 258. Carotenoid values remained constant in all clones and treatments. Based on the observed changes, DN clone seems to be more tolerant to drought stress and is more stimulated due to 20 days treatment of its defense mechanism.

Yield

The results of mean comparison showed that the yield of the clones was decreased under drought stress conditions. As shown in Table 3, the performance of some clones in stress conditions was better than other clones in normal conditions. Bases on leaf yield, some clones showed a decreased yield under stress conditions than normal. The clones for which leaf yield was close in both conditions can be considered tolerant clones. Although the selection of tolerant clones based on all traits and multivariate methods can have better results. The highest yields belonged to clones 399, 261, Bazri, 280 and 100 in normal condition, and the least belonged to 277, 252, 285 and 278 under drought stress conditions. Recently, research have shown that, in addition to physiological changes caused by water shortages in the plant, oxidative damage is one of the important factors limiting growth and plant production that results from the absence of suitable conditions. In previous studies, it has also been reported that drought as the most important factor controlling the yield of crops that affects almost all plant growth processes (Fahad et al. 2017; Hatfield and Prueger 2015; Kang et al. 2009).

Cluster analysis of clones under normal and drought stress conditions

To classify the clones in terms of traits under normal and stress conditions and identify the group with the best traits as a tolerant group, cluster analysis was used. Cluster analysis divided the clones into two groups and group2 was divided into two subgroups under normal conditions (Fig. 2a). The cluster of 100, 399, 280 and Bazri clones was better for most traits and can be introduced as superior clones for normal conditions. Also, the subgroup2 of clones 269, 277, 278, 252, 272 and 276 with least values of traits can be identified as low-yield clones in normal conditions. The other group was also considered as an intermediate-yield group. The results of this method were in line with the mean comparison results. In this analysis, the clones of the tolerant group were better than other clones in terms of yield, as well as the amount of proline and total protein (Table 3). Under drought stress conditions, the clones were also divided into three groups (Fig. 2b). The cluster with 100, 399 and 261 clones were better for most traits and could be considered as the drought-tolerant group. Also, the group3 with clones 278, 276, 272, 252 and 285 had the least amount for most of the traits that could be considered as sensitive group and the other group was considered as an intermediate group.

Fig. 2.

Fig. 2

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a Grouping tea clones in normal conditions and b grouping tea clones in drought stress conditions

Conclusion

The results of grouping the clones under normal and stress conditions and comparing their results with the results of mean comparison of the agronomic and biochemical traits showed that in all cases, clones 100 and 399 were in the group that can be identified as the drought-tolerant group. Also, the results showed that in most cases, clones 278, 276 and 285 were placed in a group that had low values for all of the traits and could be considered as a group that are susceptible to drought stress.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Contributor Information

Mehdi Rahimi, Phone: 00989177015872, Email: mehdi83ra@yahoo.com.

Mojtaba Kordrostami, Email: kordrostami009@gmail.com.

Mojtaba Mortezavi, Email: mortezavimm@gmail.com.

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