1 Introduction

Plants are often subjected to periods of soil and atmospheric water deficits during their life cycle. Water deficit is one of the most common environmental stresses that affect growth and development of plants (Aslam et al., 2006). According to Passioura (2007) , drought is the most important limiting factor for crop production and it is becoming an increasingly severe problem in many regions of the world.

According to Jajarmi (2009) , current estimates indicate that 25% of the world's agricultural land is now affected by high levels of water stress. Drought is connected with almost all aspects of biology (Bayoumi et al., 2008), and is one of the major causes of crop loss worldwide. Drought commonly reduces average yield for many crop plants by more than 50% (Wang et al., 2003).

For the purpose of crop production and yield improvement, developing of drought tolerant varieties is the best option (Siddique et al., 2000). Water availability mostly affects growth of leaves and roots, photosynthesis and dry mater accumulation (Blum, 1996). Generally, plants accumulate some kind of organic and inorganic solutes in the cytosol to raise osmotic pressure and thereby maintain both turgor and the driving gradient for water uptake (Rhodes and Samaras, 1994). Among these solutes, proline is the most widely studied (Delauney and Verma, 1993). It has been widely reported that plant cells achieve their osmotic adjustment by the accumulation of some kind of compatible solutes such as proline, betaine, sugars and polyols to protect membranes and proteins (Delauney and Verma, 1993).

According to Sanusi and Ayinde (2013) , pepper is the world's second important vegetable, ranking after tomatoes and it is the most produced type of spice flavouring and colouring for food while providing essential vitamins and minerals. Peppers (hot and sweet)belong to the Solanaceae family, genus Capsicum (Greenleaf, 1986). This genus originated from Central and South America (Grubben and El Tahir, 2004). Cobley and Steele (1976) reported that apparently between 5, 200 and 3, 400 B.C., native of Americas were growing Capsicum, which places it among the oldest cultivated crops in the Americas. Nigeria is known to be one of the major producers of pepper in the world accounting for about 50% of the African production and the major area of production is Northern Nigeria. Most of the peppers grown belong to Capsicum frutescens. Present world production is about 19 million tons fresh fruit from 1.5 million ha (FAOSTAT, 2001).

Water stress is considered as one of the abiotic stress factors that restrict growth and plant production (Grand et al., 2014). Plants accumulate different types of organic and inorganic solutes in the cytosol to lower osmotic potential thereby maintaining cell turgor (Rhodes and Samaras, 1994). Under drought, the maintenance of leaf turgor may also be achieved by the way of osmotic adjustment in response to the accumulation of proline, sucrose, soluble carbohydrates, glycinebetaine, and other solutes in the cytoplasm improving water uptake from drying soil. The accumulation process of such solutes under drought stress is known as osmotic adjustment which strongly depends on the rate of plant water stress. Proline accumulation is the first response of plants exposed to water-deficit stress in order to reduce cell injury.

To improve crop productivity, it is necessary to understand the responses of plants to drought condition with the ultimate goal of improving crop performance in the vast areas of the world where rainfall is limiting or unreliable (Passioura, 2007). One mechanism utilized by the plants to overcome water stress is via accumulation of compatible osmolytes, such as proline (Cattivelli et al., 2008)and soluble sugars (Izanloo et al., 2008). A lot of information is available in the literature on the responses of field crops like maize, rice and sorghum to water stress. However, relatively little attention is being paid to vegetable crops despite its much importance.

This study was carried out to determine how the growth stages (vegetative, flowering and fruiting)affect the levels of proline and soluble sugar accumulation in the three pepper species. This is with a view of determining the growth stages at which each of the three pepper species are tolerant to water stress.

2 Materials and methods

Seeds of Capsicum chinense (Variety Rodo), C. annuum (Variety Tatase)and C. frutescens (Variety Wewe)were raised in a nursery inside a screen house in three plastic buckets. The buckets were filled with sterilized top soil collected on the campus of Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. Preliminary soil testing was carried out at the Analytical Laboratory of the Department of Soil Science, Obafemi Awolowo University, Ile Ife, Nigeria. The analyzed soil was air-dried and transferred into seventy-two plastic buckets containing bored holes at the bottom to allow for drainage during the course of the experiment. The buckets each of which was 21 cm in diameter and 24 cm in height was filled near brim with the soil. The experiment was carried out under screen house conditions to minimize extraneous factors such as pests and rodents, supply of water other than the amount specifically applied. The mean daily temperature under the screen house was taken with the aid of a thermometer. Light intensity was also determined using a digital luxmeter LX 1000. Seeds of the three pepper species were sown in the nursery and 25 days after sowing, they were transplanted into the 72 plastic pots at the rate of five seedlings per pot. Treatments were applied at three stages of growth and development: vegetative stage, S₁= 25-40 days, flowering stage, S₂= 45-60 days, and fruiting stage, S₃= 65-75 days. The treatments were: W₁= watering with 200 mL of water once every day (this served as control), W₂ = watering with 200 mL of water at three days interval, W₃ = watering with 200 mL of water at five days interval, W₀ = no watering throughout the particular stage of growth (vegetative, flowering or fruiting).

At the end of each of the growth stages, leaves were randomly harvested from each of the three pepper species. Proline accumulation in the fresh leaves was determined according to the method of Bates et al. (1973) . Free proline was extracted from the plant leaves using aqueous sulfosalicylic acid. The filtrate (1 mL)was mixed with equal volumes of glacial acetic acid and ninhydrin reagent (1.25 g ninhydrin, 30 mL of glacial acetic acid, 20 mL of H₃PO₄)and incubated for 1 hour at 100 ℃. The reaction was stopped by placing the test tubes in cold water. The reaction mixtures were rigorously mixed with 3 mL toluene. The absorbance of toluene phase was estimated at 520 nm using a spectrophotometer. The proline concentration was determined using a standard curve. Free proline content was expressed as μmol/L of plant parts.

Soluble sugars were determined based on the phenol-sulphuric acid method (Dubois et al., 1956). 0.1 g of dry leaves was homogenized with deionized water, filtered and the extract was treated with 2% (w/v)phenol and 98% sulphuric acid. The mixture was incubated at room temperature for 1 hour and then absorbance at 490 nm was read on a spectrophotometer. Contents of soluble sugar were determined by using glucose as a standard and expressed as mg/g. A statistical analysis was performed using SAS version 9.1 quantitative analytical software package and post hoc testing was carried out using Duncan multiple range test to separate the means at 0.05 probability level.

3 Results

In Capsicum chinense subjected to water stress at vegetative stage, there was a significant difference (p<0.05) in proline content of W₁ and W₀ plants at the end of the vegetative, flowering and fruiting stages (Table 1). Plants in the W₀ treatment recorded the highest proline content at the end of each of these stages while those in the W₁ treatment had the lowest. For the plants subjected to water stress at flowering stage, there was a significant difference (p<0.05) in W₁ and W₀ plants in proline content at the end of the flowering and fruiting stages (Table 2). When the plants were subjected to water stress at fruiting stage, W₀ plants were significantly higher (p<0.05) in proline content than W₁ plants at the end of the fruiting stage (Table 3).

In Capsicum annuum subjected to water stress at vegetative stage, W₀ plants were significantly higher (p<0.05) in proline content than plants in all other water treatments at the end of the vegetative stage (Table 1). Plants in the W₀ treatment were also significantly higher (p<0.05) in proline content at the end of the flowering stage, but not significantly different (p>0.05) from those in W₁ treatment at the end of the fruiting stage (Table 1). For the plants subjected to water stress at flowering stage, plants in the W₀ treatment were significantly higher (p<0.05) in proline content than those in the W₁ treatment at the end of the flowering and fruiting stages (Table 2). In C. annuum plants subjected to water stress at fruiting stage, plants in the W₀ treatment were significantly higher (p<0.05) in proline content than those in the W₁ treatment at the end of the fruiting stage (Table 3).

In Capsicum frutescens subjected to water stress at vegetative stage, W₀ plants were significantly higher in proline content than those in the W₁ and W₂ treatments at the end of the vegetative, flowering and fruiting stages (Table 1). In the plants subjected to water stress at flowering stage, plants in the W₀ treatment were significantly higher (p<0.05) in proline content than those in W₁ and W₂ treatments at the end of flowering and fruiting stages (Table 2). In the plants under water stress at fruiting stage, W₀ plants were significantly higher (p<0.05) in proline content than those in W₁ and W₂ treatments at the end of the fruiting stage (Table 3).

In Capsicum chinense subjected to water stress at vegetative stage, W₀ plants were significantly higher (p<0.05) in total soluble sugar than plants in other water treatments at the end of the vegetative stage (Table 4). At the end of the flowering stage, W₀ plants were significantly higher (p<0.05) in total soluble sugar than those in the W₁ and W₂ treatments (Table 4). There was however no significant difference (p>0.05) in total soluble sugar in the different water treatments at the end of the fruiting stage (Table 4). Plants in the W₀ treatment were significantly higher (p<0.05) in total soluble sugar content than those in the W₁ treatment at the end of the flowering stage in plants subjected to water stress at flowering stage (Table 5). There was however no significant difference (p>0.05) in total soluble sugar at the end of the fruiting stage (Table 5). For plants subjected to water stress at fruiting stage, W₀ plants were significantly higher (p<0.05) in total soluble sugar content than those in the W₁ and W₂ treatments at the end of the fruiting stage (Table 6).

In Capsicum annuum subjected to water stress at vegetative stage, W₀ was significantly higher (p<0.05) in total soluble sugar content than in other water treatments at vegetative stage (Table 4). At flowering stage, plants in W₀ treatment were also significantly higher (p<0.05) than those in the W₁ treatment. There was however no significant difference (p>0.05) in total soluble sugar content at the end of the fruiting stage (Table 4). In C. annuum subjected to water stress at flowering stage, plants in the W₃ and W₀ were significantly higher (p>0.05) in total soluble sugar content than those in the W₁ and W₂ plants at the end of flowering and fruiting stages (Table 5). In plants subjected to water stress at fruiting stage, there was no significant difference (p<0.05) in the total soluble content at the end of the fruiting stage (Table 6)

In Capsicum frutescens subjected to water stress at vegetative stage, W₁ plants were significantly different (p<0.05) from other water treatments in total soluble sugar at the end of the vegetative stage (Table 4). There was however no significant difference (p>0.05) in total soluble sugar at the end of the flowering and fruiting stages (Table 4). Plants in the W₀ treatment were also significantly higher (p<0.05) in total soluble sugar content than those in the W₁ and W₂ treatments at the end of flowering stage in the plants subjected to water stress at flowering stage (Table 5). There was however no significant difference (p>0.05) in total soluble content at the end of the fruiting stage (Table 5). For plants that were water stressed at fruiting stage, plants in W₀ treatment were significantly higher (p<0.05) in total soluble sugar content than those in W₁ and W₂ treatments at the end of the fruiting stage (Table 6).

4 Discussions

The concentration of proline and soluble sugar in leaves of the three pepper species were found to be remarkable at the different growth stages in stressed plants. These results suggest that the production of these osmotic adjustments is a common response of plants under drought conditions irrespective of the growth stage of the plant. According to Umezawa et al. (2006) , plants have the ability to accumulate non-toxic compounds such as proline which protects cell damage due to low water potential of cells, which is a way of plant adaptation to drought stress tolerance. When plants are faced by drought stress, the osmotic pressure of the plant cell regulates many processes through the accumulation of non-toxic solutes inside the cell (Lipiec et al., 2013). This osmotic accumulation occurs because the cell water potential decrease thereby increasing the concentration of dissolved material to maintain turgidity of the cell.

The role of proline in adaptation and survival of plants had been observed by Watanabe et al. (2000) and Saruhan et al. (2006) . These results suggest that the production of these osmotic adjustments is a common response of plants under drought conditions. Osmotic adjustment through the accumulation of cellular solutes, such as proline, has been suggested as one of the possible means for overcoming osmotic stress caused by water loss (Caballero et al., 2005). The significant reduction in the proline content in the three pepper species after the plants were re-watered corroborated the findings of Singh et al. (2000) . Proline is a non-protein amino acid that forms in most tissues subjected to water stress and together with sugar, it is readily metabolized upon recovery from drought. In addition to acting as an osmo-protectant, proline also serves as an energy sink to regulate redox potentials, as a hydroxyl radical scavenger (Sharma et al., 2006), as a solute that protects macromolecules against denaturation and as a means of reducing acidity in the cell (Kishor et al., 2005). It has been shown that, the concentration of soluble sugars increased under drought stress in the three pepper species at the different growth stages. Capsicum chinense and C. annuum subjected to water stress at vegetative stage was unable to recuperate until the end of the fruiting stage. Capsicum frutescens plants that were subjected to water stress at vegetative stage were able to recuperate at flowering stage. Capsicum chinense and C.frutescens subjected to water stress at flowering stage was unable to recuperate at the end of the fruiting stage. The accumulation of sugars in response to drought stress is also quite well documented (Watanabe et al., 2000 ). A complex essential role of soluble sugars in plant metabolism is well known as products of hydrolytic processes, substrates in biosynthesis processes, energy production but also in sugar sensing and signaling systems.

Osmotic Potential can be adjusted by increasing the concentration of total soluble sugar which can decrease water potential of cells without inhibiting enzyme function and does not reduce turgidity of the cell. Sugar accumulation in drought stress conditions helps to maintain membrane stability, prevent and protect membrane fusion and; keep protein so as to remain functional (Xonostle-Cazares et al., 2011; Arabzadeh, 2012; Lipiec et al., 2013).

Recently it has-been claimed that, under drought stress condition, even sugar flux may be a signal for metabolic regulation (Kishor et al., 2005). Soluble sugars may also function as a typical osmoprotectant, stabilizing cellular membranes and maintaining turgor pressure. The presence of genes functionally associated with other abiotic stresses among the drought-up-regulated genes suggested that different stresses share some common signaling pathways. Gene ontology attributes such as proline and soluble sugar accumulations were highly enriched in the drought-up-regulated genes, suggesting that those metabolic pathways are important in responses to drought stress. Indeed the importance of many of these pathways to drought tolerance has been empirically supported by transgenic experiments (Umezawa et al., 2006). The accumulation of osmolyte compounds in the cells, as a result of water stress is often associated with a possible mechanism to tolerate the harmful effect of water shortage. The contribution of sugars as an osmotic solute in expanded and partly expanded sunflower leaves was studied by Jones and Turner (1980) . They found that contents of sugars did not change in fully expanded leaves. In addition to sugars, some plants also accumulate other low molecular mass compounds, such as proline (Gzik, 1996; Bajji et al., 2001). One of the most studied solutes is the amino acid proline and high proline content in plants under water stress is frequently observed in several species (Clifford et al., 1998; Bajji et al., 2001)and may act as a regulatory or signaling molecule to activate multiple responses that are part of the adaptation process (Maggio et al., 2002; Claussen, 2005).