Effects of watering regime on the morphological, physiological and functional traits of seedlings of cacao provenances under screen house conditions

In the present study, morphological and physiological responses of cocoa provenances to watering regimes under screen house conditions and the implications of the measured variables as drought tolerance strategy in Theobroma was discussed. A 4 by 3 factorial scheme involving four cacao provenances and watering regimes (well watering at full field capacity, 60 and 40% field capacity: 1.5, 0.9 and 0.6 L/plant at each watering event) the cocoa genotypes evaluated are PA 150 Series (the elite varieties), F3 Amazon and Amelonado. Observations were made on the morphological and physiological traits of seedlings of the cacao genotypes affected by watering regimes. The measured variables were deployed to rank the drought performance of cacao genotypes following nursery desiccation studies. Data on root and shoot biomass, water use, stomatal conductance, proline, water soluble carbohydrate and leaf chlorophyll concentrations of cacao seedlings were collected. The results showed that root zone moisture status affected the morphological and physiological characteristics of cacao provenances. Differences were obtained in root and shoot biomass, water use, the densities of stomatal and its conductance of gases, and the concentrations of leaf chlorophyll, and shoot and leaf proline and water soluble carbohydrates among the watering regimes imposed. Cacao provenances evaluated also differed in their responses to watering regimes and in morphological and physiological characters. The imposed root zone moisture scenarios elicited differences in the responses of cacao provenances evaluated. Most of the measured morphological and physiological variables were driven by root zone moisture status among cacao provenances, the measured traits appeared to have played important roles as root zone moisture deficit stress tolerance mechanisms in cacao. Seedlings of cocoa provenances had better vigour of growth when grown under 100 and 60% field capacity watering compared with 40% FC. Adequacy of soil moisture promotes growth and physiological functions in the seedlings of cacao provenances tested. The measured morpho-physiological variables were statistically superior under well watered situations (100% FC) compared with the 40% FC. The results confirmed that cocoa seedlings cannot withstand soil moisture deficit stress as was obtained for seedlings that were watered with 40% FC. It is recommended that watering cacao seedlings at full field capacity (FC) and at 70% FC (mild root zone moisture stress) will ensure the production of vigorous seedlings of cacao in the nursery.


INTRODUCTION
Cocoa seedlings are raised in nurseries by smallholder farmers for planting out on the field to meet the increasing demand for vigorous seedlings for the establishment of new plantations or rehabilitation of old/moribund fields. Therefore it is necessary to develop sustainable nursery management practices for the production of vigorous cocoa seedlings. Cocoa seeds are sown in pots in the nursery to raise seedlings which are subjected to variable watering regimes (soil moisture status and wet-dry cycles) while in the nursery. The watering regimes under which seedlings are raised in the nursery affect their vigour of growth (Famuwagun et al., 2017;Agele et al., 2018).
There is an increasing need to establish new cocoa fields, and or rehabilitate old and moribund plantations using more productive cocoa stock for increased productivity. The success of the establishment and rehabilitation of cocoa farms, aimed at replacing ageing and non-productive cocoa stocks in the field may be limited by the inadequacy of healthy cocoa seedlings (CRIN, 2010;Agele et al., 2016;Famuwagun et al., 2017). The vigour of seedlings plays an important role in the establishment, survival percentage and growth following transplanting on the field. Daymond and Hadley (2008) and Tezara et al. (2016) reported that varietal improvement for tolerance to abiotic and biotic stress factors has been identified as research priority programs for cocoa producing countries. Thus, cocoa varieties that are high yielding, and tolerant to pests and diseases and environmental stresses have been developed (CRIN, 2010).
Cocoa is highly sensitive to changes in climate from hours of sunshine to rainfall and application of water, soil condition and temperature (Almeida et al., 2002;Acheampong et al., 2013, Tezara et al., 2020. In nature, water is usually the most limiting factor for plant growth. If plants do not receive adequate rainfall or irrigation, the resulting drought stress can reduce growth more than all other environmental stresses combined. Inadequate root zone moisture status halts plant growth and development resulting in reduced vigour and yield (Daymond & Hadley, 2008;Agele et al., 2018). As water loss progresses, leaves of some species may change color, wilt and, if the plant is not irrigated, leaves will fall off followed by death (mortality) Almeida et al., 2018).
In plants, changes in the morphological and physiological traits of annual and perennial species under drought or soil moisture deficit have been evaluated severally (Glenn et al., 2014;Tezara et al., 2016). These reports affirmed that plants exhibit adaptive strategies to cope with root zone moisture deficit stress and recovery following stress alleviation via rewatering (Tyree et al., 2003;Glenn et al., 2014;Haeberle et al., 2016). These strategies may involve complex, interacting mechanisms (e.g. desiccation tolerance and drought performance) (Tyree et al., 2003;Li & Liu, 2016;Tombesia et al., 2018).
Root-zone moisture deficits impinge on stomatal density, a parameter that is closely and inversely correlated with a starch concentration in roots and trunks of plants (Putra et al., 2012). It has been suggested that the carbohydrate reserve status of plants may be an important endogenous determinant of stomatal density. Putra et al. (2012) found that the stomatal density of newly emerging leaves plants grown in dry and warm soil increased after the treatment had been removed. The differences in stomatal densities have consequences on stomatal gas exchange (conductance of the stomatal to gases, g) (Putra et al., 2012;Glenn et al., 2014). In addition, it has been reported that the low stomatal conductance characteristic of a dry root-zone environment may not be due to low stomatal density alone but also to the stomatal aperture (Putra et al., 2012;Glenn et al., 2014).
Physiological variables such as water relations and stomatal gas exchange are important to plant survival under unfavourable environmental conditions Haeberle et al., 2016;Tezara et al., 2020). Limitation of the water supply has an impact on photosynthesis, plant growth, and yield production in plants, and drought is an important environmental stress which affects various levels of plant metabolism (Sheffield et al., 2012;Witt et al., 2012;Glenn et al., 2014;Li & Liu, 2016;Zhang et al., 2017). The effects of limitation in soil moisture status on phytochemistry, especially parameters such as total soluble solids, soluble sugar, organic acids and vitamin C has been reported .
The accumulation of osmolytes exerts protection against drought stress (Yancy et al., 1982;Herbinger et al., 2002;Verbruggen & Hermans, 2008). For example, proline accumulation in plant tissue serves as a marker and an important part of the stress signal influencing adaptive responses to environmental stress, particularly in plants under drought stress (Routley, 1966;Sanchez et al., 1998;Maggio et al., 2002;Mafakheri et al., 2010). Osmolytes accumulation offers protective functions to plants under environmental stress (Tokihiko et al., 2003). Studies have shown that proline content increased under drought stress in peas (Sanchez et al., 1998;Verbruggen & Hermans, 2008) while proline accumulation has also been obtained for plants under high temperature and poor soil fertility status. Severe wilting under soil drought has been reported to stimulate proline synthesis and accumulation of carbohydrates (Routley, 1966;Mafakheri et al., 2010).
In addition to ecophysiology, various fields of omics including molecular biology, transcriptomics and metabolomics have been deployed to clarify mechanisms of abiotic stress tolerance in plants (Cramer et al., 2007;Kantar et al., 2011;Wang et al., 2015). Results affirmed the potential of metabolic pathways and their manipulation for ameliorating adverse effects of root zone moisture deficits on plant performance (Budak et al., 2015;Soni et al., 2015;Zhang et al., 2017). Drought-induced accumulated solutes offer plant protective functions in response to environmental stresses, as was reported in studies with Arabidopsis (Tokihiko et al., 2003) and grapevine (Kantar et al., 2011;Soni et al., 2015;Chmielewska et al., 2016).
Changes in chlorophyll and carotenoid contents have served as an index for the evaluation of plant response to drought or soil moisture deficit stress (Pastori & Trippi, 1992;Kpyoarissis et al., 1995;Zobayed et al., 2005;Khayatnezha & Gholamin, 2012). Decreases in chlorophyll concentration are referred to as a nonstomatal limiting factor for plants under drought stress (Pastori & Trippi, 1992;Kpyoarissis et al., 1995). Ommen et al. (1999) and Agele et al. (2018) reported that drought stress caused a large decline in leaf chlorophyll a, b and total chlorophyll content in cacao. The decrease in chlorophyll under drought stress has been ascribed to damage to chloroplasts caused by active oxygen species (Smirnoff, 1995;Khayatnezha & Gholamin, 2012).
The implications of soil moisture deficit stress on stomatal gas exchange in plants have been variously reported (Berninger et al., 1996;Daymond & Hadley, 2008;Miranda et al., 2013;Martin-StPaul et al., 2017). Reports of gas exchange variable measurements (net CO 2 assimilation rate (A), stomatal conductance (g s ) and transpiration (E) of leaves) spanning daytime hours (early morning, midday and late afternoon periods) have confirmed the effects of various root zone moisture status (Cuevas et al., 2006;Glenn et al., 2014;Agele et al., 2016). Stomatal conductance increases with sunlight intensity from sunrise, attaining a maximum at mid-morning followed by midday depression (Tyree et al., 2003;Agele et al., 2016;Haeberle et al., 2016). The decline in stomatal conductance (gs) just after sunrise toward midday had been attributed to increases in leaf temperature and vapor pressure deficit and incident photon flux density (PPFD) which increased after sunrise and reached a maximum around noon (Cuevas et al., 2006;Agele et al., 2016;Haeberle et al., 2016). Changes in stomatal conductance under root-zone moisture deficits appear as a regulatory signal for transpirational water loss (Tyree et al., 2003;Agele et al., 2016;Li & Liu, 2016). Changes in the daytine course of gas exchange have been reported for plant species including forest trees (Cuevas et al., 2006;Haeberle et al., 2016). The response of gs and leaf transpiration characterized by midday depression is known to increase daily water-use efficiency (Cuevas et al., 2006;Agele et al., 2016) while the midday depression of plant water status (water potential), stomatal conductance and photosynthesis have important consequences for ecosystem water and carbon exchange Haeberle et al., 2016). Several studies have reported the midday and afternoon reductions in stomatal conductance as a common phenomenon across species, and that this phenomenon be incorporated leaf-level stomatal regulation into process-based models of ecosystem gas exchanges of forest trees (Tyree et al., 2003;Glenn et al., 2014;Trenberth et al., 2014;Li & Liu, 2016).
Drought affects the establishment, growth and yield of cocoa , especially during the juvenile stage (Almeida et al., 2016;Famuwagun et al., 2017). Inadequate (sub-optimal) water application may profoundly affect the vigour of cacao seedlings (growth, development and survival rate) Improved insight is required for the adjustment of physiological processes for enhanced tolerance of drought including photochemical compounds of cacao (Daymond et al., 2011;Tezara et al., 2020).
Can the expression of physiological attributes of cacao provenances, varieties and clones to drought suggest differences in drought tolerance? Are the morphological (growth, development) and physiological traits (water use, leaf proline, chlorophyll and water soluble carbohydrate contents) measured indicators of the sensitivity of cacao provenances to variables of soil moisture status (adequacy and deficits)? It is hypothesized that during episodes of drought (root zone moisture deficit stress), changes in physiology and functional attributes would cause physiological plasticity with implications for drought tolerance in cacao provenances.
This study aims to investigate morphological and physiological responses of cocoa provenances to watering regimes under screen house conditions and the implications of the measured variables as drought tolerance strategy in Theobroma.

MATERIALS AND METHODS
Experiments were conducted at the Nursery and Experiment Station of the Department of Crop, Soil and Pest Management, Federal University of Technology, Akure. Two months old potted cacao seedlings were subjected to three watering regimes in the screen house. The cacao provenances are Amelonado and F3 Amazon and two elite lines (PA 150/34 and PA 150/36).

Planting Materials, Experimental Design and Treatments
Seedlings of the cacao genotypes were obtained from the Cocoa Research Institute of Nigeria (CRIN) in Ibadan, Nigeria. The seeds of the cacao genotypes were planted in polythene pots (10cm diameter x 30cm length) filled with topsoil obtained from fallow vegetation. After 16 weeks at the nursery, the seedlings were later transplanted into standard plastic pots (25 x 15cm) with drainage holes at the bottom and transferred to the screen house. The seedlings of cacao genotypes were subjected to wet-dry cycles. The seedlings were well watered before treatments were imposed.
Treatments consisting of 4 x 3 factorial combinations of 4 cacao genotypes by 3 watering regimes were assigned using a complete randomized design with five replications. Cacao seedlings were subjected to three (3) field capacity percentages; 100, 60 and 40%. The full field capacity (100% FC: 1.5 L/plant), 60% field capacity (0.9 L/plant) and 40% field capacity (0.6 L/plant) were applied once a week throughout the experiment. To maintain the preferred percentage FC in the soil, water was added whenever the water level is lower than the required field capacity conditions.

Data Collection
Measurement of agronomic variables started 4 weeks after transplanting (WAT) and lasted for 10 months before they were transplanted to field plots.

Water use characters of cacao
Seedling water use was by weighing method, the weights of the potted seedlings were measured using a weighing balance before watering and a day after watering. The differences between measurement periods were carefully recorded. Plant water use (kg) was determined using a weighing balance before and 24 hours after watering.

Growth parameters
Cacao height, number of leaves, leaf area and number of branches were measured weekly. Plant height was measured from the base to the crown of the plant using a meter tape graduated in millimetres. The total number of leaves was obtained by counting. Leaf area was measured on a Leaf area Meter (Delta T, UK). The average leaf area measured was multiplied by the total number of leaves on the plant to obtain the total leaf area available for photosynthesis. The total number of branches was also determined by counting. For root and shoot biomass determination, cacao seedlings were gently uprooted and separated into roots and shoots, then oven-dried separately at 60 °C for 48 hours and re-weighed to obtain the dry weight of samples. The mass of each fraction was then averaged to obtain the corresponding final dry mass per plant. The final biomass was a combination of root and shoot dry masses: where DM = dry mass.

Stomatal densities
The Impression Method uses clear nail polish to make an impression or cast of the leaf surface placed on a microscope slide. Leaf samples were harvested from each treatment replication and were placed into plastic envelopes and transported immediately to the laboratory, where leaf surface impressions were taken using clear fingernail polish. The upper and lower surfaces of leaves were identified as they are under normal conditions. Clear nail polish was spread as a thin layer on each surface, upper side and lower side, the leaf surface and left to dry. The casts of fully expanded mature leaves were made by pressing leaf sections onto a microscope. The number of stomata of the sampled leaves was counted using a light microscope, and stomatal density was averaged per ring and viewed under the microscope (100 or 400x magnification). The number of stomata of the sampled leaves was counted using a light microscope, and stomatal density was averaged per ring.

Stomatal conductance
Stomatal conductance was measured using a steady state porometer device (Delta T, UK). Seedling water use was estimated by a weighing method in which the weights of the potted seedlings were measured using a weighing balance before watering and 24 hours day after watering. The differences between measurements were recorded. Soil moisture content of each watering treatment was determined using soil moisture sensor (Decagon Device, USA), at a three weeks interval. The ambient air temperature during the period of analysis was measured using ordinary mercury in a glass thermometer, suspended at 1.5 m above ground level.

Proline content
The proline content was determined following the free proline accumulation method. Plant samples were harvested, and approximately 100 mg of fresh samples were deployed for analysis. Plant samples can be snap frozen in liquid nitrogen and when necessary stored below 80 • C. The plant materials are ground and kept in tubes and stored on ice. The samples are centrifuged for 5 min at room temperature using a benchtop centrifuge with maximum speed. Afterwards, exactly 100 μL of 3% sulfosalicylic acid and 200 μLglacial acetic acid is added in addition to 200 μL acidic ninhydrin to 100 μl to the supernatant of the plant extract and properly mixed while the tubes are incubated at 96 • C for 60 mins. Plant samples are extracted with toluene (1 mL toluene which was added to the mixture in the tube). Samples are taken and vortexed for 20 seconds, and left on the bench for 5 min to allow the separation of the organic and water phases. The chromophore containing toluene is removed into a fresh tube while the absorbance of the extract is measured using a Spectrophotometer at 520 nm using toluene as a reference. The proline concentration was determined using a standard concentration curve and calculated on a fresh weight basis (usually expressed as microgram per gram FW or micromole per gram FW: μmoles g -1 ).

Relative water content
After harvesting, the samples were immediately weighed (Wf). Plant samples were then oven dried at 70°C for 2 days and dry weight was calculated (Wd). Then their average was calculated.
Relative Water Content = (Wf -Wd)/Wt ×100 2 Where Wf is fresh weight and wd is dry weight of samples.

Determination of chlorophyll
The leaf chlorophyll concentration was determined using the acetone method. Leaf samples were collected from intact leaves still attached, and placed in vials with 10 mL 95% ethanol in the cold room for 24 hours for chlorophyll extraction and chlorophyll determination. Leaf chlorophyll was extracted and determined using leaf samples from the uppermost leaves of the cacao genotypes from each treatment. One gram of the fresh plant samples was cut into pieces and smashed in a mortar. The samples were put in a test tube and their chlorophyll content was repeatedly extracted with successive volumes of 100 mL acetone/ water (80:20 v/v) until no traces of green colour were noticed (residue became white). While adding the solvent (acetone), the test tubes containing the samples were kept boiling in a hot water bath. The total volume of the extract was also recorded at the end of the extraction. Three millimeters (3 mL) of the extract was taken and the absorbance of chlorophyll was determined with a spectrophotometer at two wavelengths of 663 nm and 645 nm that corresponds to the maximum absorption of chlorophyll "a" and "b" respectively. The total chlorophyll content was calculated as follows: Total chlorophyll content (mg/100 g tissue) = (20.2A 645 + 8.02A 663 ) (V/10 w) 3 Where, A645 = absorbance at 645 nm wavelength; A663 = absorbance at 663 nm wavelength; A = absorbance, C a = chlorophyll a, C b = chlorophyll b, C a+b = total chlorophyll, V is the final volume (cm 3 ) of chlorophyll extract in 80% acetone and W is fresh weight (g) of tissue extracted.

Water soluble carbohydrate
Water soluble carbohydrate was determined using the Anthrone extraction method. About 1.0 g of plant samples were ground and transferred into a 250 mL test tube and 220 mL of water was added. The bottles were capped and shaken on a shaker for about an hour and filtered. The first few ml were ejected and the filtrate was retained for the determination of soluble carbohydrates using Anthrone reagents. 770 mL of concentrated H 2 SO 4 was added to 330 mL of distilled water, in addition to 1 g of thiourea, and 1 g of anthrone, stirred until dissolved and stored in a refrigerator. Glucose stock solution, 1.0 g of anhydrous D (+) glucose in water and diluted to one litre prepared immediately before use. From the glucose working standard solutions, 10 mL of stock to 100 mL was diluted to produce 100 ppm. From these, 0, 5, 10, 20, 40, and 80 mL were pipetted and made up to 100 ml and these produced 0, 5, 10, 20, 40, and 80 ppm. Samples of 2 mL of each glucose working standard solution were pipetted into the glass test tube and rapidly, 10 mL of anthrone reagent was added and mixed by shaking. The test tube was loosely covered with a glass bulb stopper and placed immediately in boiling water for 20 minutes. The absorbance was measured using a spectrophotometer device in a 10 mm optical cell at 620 mm. The graph of absorbance was plotted against glucose concentration in ppm and prepared a standard graph with each batch of extracts examined. The glucose standard becomes 0, 0.8, 1.7, 3.3, 6.7, and 13.3 ppm respectively.
Data analysis was carried out using the Minitab Version 17 statistical package (Minitab Inc., PA, USA) and where necessary, significance was determined at the 95% level (α = 0.05) using Tukey HSD test at α =.05, after tests for normality and homogeneity of variance

RESULTS
The watering regimes and cocoa provenances evaluated affected root zone moisture. The 40% Fc had consistently lower root zone moisture followed by 60% Fc and highest under full field capacity watering. The trends in root zone moisture were similar among the provenances. While root zone moisture for 40 and 560 5 Fc among provenances, differences were found for root zone moisture among the provenances under field capacity conditions ( Figure 1).
There was a gradual decline in root and shoot biomass as the quantity of water applied increased (40, 60 and 100% FC) ( were profound on the root characteristics of cocoa. Root biomass was highest at 60% FC (15.3 g) and lowest at 40% FC (12.8 g). There were increases in the tap root length and the number of root hairs as the quantity of water applied increased. Taproot length was longest at 100% FC (33.7 cm), and the number of root hairs was significantly higher at 100% FC compared to other watering regimes. The number of lateral roots was significantly higher at 60% FC (71.1) and least at FC (47.1).
Cacao seedlings that were watered at 40% field capacity consistently consumed the highest amount of water while the least was obtained for full field capacity watering while the provenances differed in their water use ( The effects of differential watering were significant on the relative water content and stomatal density of cacao ( There were significant (P<0.05) effects of watering regime and variety on the proline content of cocoa seedlings (Table 4). F3 Amazon had the highest leaf, stem and root proline contents. While F3 Amazon produced a significantly (P<0.05) higher proline content on the leaves and stem respectively at 40% FC and on the root at 60% FC, Amelonado on the other hand produced the least proline content on the leaves, stem and roots at 60% FC. The watering regime affected the proline content on the leaf, stem and root of cocoa seedlings. Proline content was significantly (P<0.05) higher in the leaves and stem at 40% FC and for root at 60% FC. Proline concentrations increased with decreases in water application (40<60<100 FC) and in plant tissue relative water content (moisture status of cell sap). Proline content was 4.4 μmoles/g of fresh weight in well watered treatment compared with 5.8 μmoles/g fresh weight under soil moisture deficit (40% FC watering). Decreases in proline contents were observed as tissue relative water content (water content of cell sap), decreased. The proline content was 4.4 μmoles/g for well watered treatment compared with 5.8 μmoles/g fresh weight under soil moisture deficit (40% FC watering).    The trend among cocoa provenances and watering regimes of the time (900 to 1600 h) course of stomatal conductance (gs) is presented in Figures 2, 3 & 4). Root zone moisture affected stomatal conductance among cocoa provenances (Figure 2). Stomatal conductance was highest for F3 Amazon it was lowest for PA 150/36 series, however, at 40% FC, F3 Amazon had the lowest gs while the highest value was obtained for PA 150/34 and Amelonado (Figure 3). Among the varieties and across the daytime course of stomatal conductance, the highest stomatal conductance was observed for F3 Amazon and lowest for Amelonado for both morning and afternoon hours. The daytime pattern of stomatal conductance was similar between the PA 150 Series and F3 Amazon at 60% and 100 FC while differences were found for Amelonado for stomatal conductance under the various watering regimes. The responses of stomatal conductance to watering regimes (40 and 60% field capacity (FC) and full (100% FC) is presented in Figure 4. Increases in root zone moisture deficit consistently reduced stomatal conductance during the morning and afternoon hours of the day. Across the varieties and watering regimes, stomatal conductance was significantly higher during the morning hours (900 to 1100) and lowest in the late afternoon (1300 to 1600 hours) after which recovery of conductance occurred. The highest stomatal conductance values were obtained for the 60 and 100% field capacity moisture (0.9 and 1.5 L/plant) compared to 40% FC (0.6 L/plant). At the various measurement points during the day, 40% FC had the lowest stomatal conductance followed by 60% field capacity watering. Thus, the increasing trends in stomatal conductance were 40 < 60 < 100% field capacity (FC).

DISCUSSION
The responses of cacao provenances to watering regimes were assessed through the measurement of morphological and physiological traits in screen house condition. The imposed root zone moisture scenarios elicited different responses in the evaluated provenances.
The status of root zone moisture and water use was affected by the watering regime and cocoa provenances. Across watering regimes, the highest soil moisture values were found for well watered plants while non-significant differences were found between 0.6 and 0.9 litres/plant (70 and 40% FC watering). Differences which were found among the provenances for status of soil moisture and cacao water use can be explained by genotypic differences among provenances. These observations, which were consistent with reports of Agele et al. (2018), imply that cocoa seedlings require a consistent moist root zone environment for optimum growth (Haeberle et al., 2016;Agele et al., 2018;Tezara et al., 2020). The enhancement of crop water use (evapotranspiration) under adequacy of soil moisture would have promoted seedling growth compared with root zone moisture deficits.
Cacao biomass (root and shoot): Watering regime and provenances affected root and shoot biomass production in cocoa. The provenances exhibited a gradual decline in shoot root biomass as the quantity of water applied decreased (100 < 60 < 40% FC). The effects of differential watering were profound for other root parameters, tap root length was longest at 100% FC, and the number of root hairs was significantly higher at 100% FC compared to other watering regimes. There were increases in the tap root length and the number of root hairs as the quantity of water applied increased. The number of lateral roots was significantly higher at 60% FC and the least at FC. F3 Amazon had the highest dry leaf biomass at 100% FC, F3 Amazon and Amelonado had similar leaf biomass at 60% FC while PA150/36 and PA150/34 had similar leaf biomass at 40% FC. However, PA150/36 had the least leaf biomass at 100% FC (1g). Amelonado consistently had the heaviest root biomass at 40, 60 and 100% FC. Haeberle et al. (2016) stated that water stress reduces plant growth through inhibition of physiological and biochemical processes, including nutrient uptake and metabolism. Water stress reduces vigour and biomass (Agele et al., 2018;Tezara et al., 2020). Cocoa provenances subjected to mild and severe root zone moisture deficits had decreased root and stem biomass which had been described as survival (tolerance) strategic among cacao progenies under drought (Tezara et al., 2020).
Differential watering affected relative water content and stomatal densities for both the upper (adaxial) and lower (abaxial) leaf surfaces. Stomatal densities also differed among the provenances which were highest at 100 FC for the PA 150 series (the elite varieties) and least for F3 Amazon at this level of watering level. PA150/36 had the highest abaxial stomatal density at 60% FC and the least values for Amelonado at 40% FC. Variable root-zone moisture impinged on stomatal density, this parameter has been closely and inversely correlated with a starch concentration in roots and trunks of plants. It has been suggested that the carbohydrate reserve status of plants may be an important endogenous determinant of stomatal density. Depleted starch reserves, elicited by long periods (several weeks) of high metabolism in dry root zones, would require replenishment. The stomatal density of newly emerging leaves plants grown in dry and warm soil increased after the treatment had been removed. The high stomatal densities obtained for well-watered treatment led to improved stomatal gas exchange (conductance of the stomatal to gases, g). Low stomatal conductance is characteristic of dry root-zone environments and may not be due to low stomatal density and stomatal aperture.
There were significant (P<0.05) effects of watering regime and variety on the proline content of cocoa seedlings. F3 Amazon had the highest leaf, stem and root proline contents compared with other provenances, especially for leaf and stem at 40% FC and on the root at 60% FC. Amelonado on the other hand produced the least proline content on the leaves, stem and roots across the watering regimes evaluated. Plants can protect themselves against mild drought stress by accumulating osmolytes (Yancy et al., 1982;Herbinger et al., 2002;Verbruggen & Hermans, 2008). Proline has been identified as an important compatible osmolyte in drought stressed plants (Sanchez et al., 1998;Verbruggen & Hermans, 2008;Mafakheri et al., 2010). For example, the proline content increased under drought stress in peas (Sanchez et al., 1998). Proline accumulation in plant tissues has been described as a marker for environmental stress, and as an important part of the stress signal influencing adaptive responses (Routley, 1966;Yancy et al., 1982;Herbinger et al., 2002;Maggio et al., 2002;Tokihiko et al., 2003;Verbruggen & Hermans, 2008, Mafakheri et al., 2010. Water soluble carbohydrates (WSC) and chlorophyll concentration were affected by the watering regime and cacao provenance. PA150/36 at 100% FC and F3 Amazon at 40% FC had the highest leaf and stem water soluble carbohydrate contents (WSC), and PA150/34 has the least leaf WSC and stem WSC for F3 Amazon both at 100% FC. The results showed that root zone moisture status affected the quantities of total soluble sugars in plant parts with increases in the intensity of soil moisture deficit stress. Increased soil moisture deficits brought about increased accumulation of soluble sugars and proline. These observations confirmed other reports that soil moisture deficit stress increases the content of soluble sugars in plant tissues with increases in proportion to intensity of moisture deficits (LiXin et al., 2009).
Soluble carbohydrates (sugars) are among metabolites and osmolytes (Bray, 1997) which increased with increasing drought stress (reduced soil water content) (Hoekstra et al., 1994;Garcia-Sanchez et al., 2007;LiXin et al., 2009). Considerable changes in the accumulation of soluble sugars in response to drought stress have been observed both at intra-and inter-species levels in plants subjected to drought (dryness) (Ashraf & Harris, 2004;Verbruggen & Hermans, 2008;Mafakheri et al., 2010). The increase and accumulation of soluble sugars maintain leaf turgidity under soil moisture deficits and they prevent dehydration of proteins and cell membranes (Sawhney & Singh, 2002). Under drought stress or reduction of soil water content, soluble carbohydrates accumulate and would have served to activate protective enzymes in cacao.
Soil moisture status has been found to affect stomatal gas exchange and regulate stomatal conductance (g), transpiration (E) and carbon dioxide fixation (photosynthesis: A) in arables and perennial crops. The main effect of water stress is the reduction in carbon fixation associated with stomatal closure, reduction of photosynthesis and resultant decreases in carbohydrate synthesis and plant growth and yield. Cuevas et al. (2006) reported that approximately 60% of the variation in stomatal conductance was attributable to changes in soil water content (θ v ), and obtained a close correlation between g s and θ v ) while net CO 2 assimilation rates were significantly correlated with g s . Change in the daytime course of stomatal conductance (gs) has been reported in plants when measured between 09.00 h and 15:00 h with increases approximately by 20% from morning to afternoon and generally decreasing trends with decreasing soil water status (Cuevas et al., 2006;Agele et al., 2016;Haeberle et al., 2016).
Chlorophyll a and b contents of cocoa leaves differed among varieties and watering levels. Chlorophyll contents increased on average, from well watered (FC) to deficit watering (40 and 60% FC) showing decreases in total chlorophyll concentrations in leaves with increasing soil moisture deficit Among the provenances, the highest total chlorophyll contents were recorded for PA 150/34 and PA150/36 at 100 and 60% FC while the least was for Amelonado at 40%. Changes in chlorophyll and carotenoids have been associated with drought stress tolerance in plants (Pastori & Trippi, 1992). Zobayed et al. (2005) also reported that chlorophyll concentration is an important plant response to drought or soil moisture deficit stress. In crop species, changes in chlorophyll contents during drought stress have been reported depending on the duration and severity of drought (Kpyoarissis et al., 1995;Ommen et al., 1999;Mafakheri et al., 2010). Drought stress significantly decreased chlorophyll a, chlorophyll b and total chlorophyll and increase in proline content due to drought stress in Chickpeas (Verbruggen & Hermans, 2008;Mafakheri et al., 2010). The results were in agreement with earlier reports (Nyachiro et al., 2001;Agele et al., 2018) where a significant decrease of chlorophyll a and b was obtained under water deficits. It has been reported that the production of reactive oxygen species is mainly driven by excess energy absorption in the photosynthetic apparatus, and this might be avoided by degrading the absorbing pigments (Yancy et al., 1982;Smirnoff, 1995;Herbinger et al., 2002). Therefore, a decrease in total chlorophyll with drought stress implies a lowered capacity for light harvesting and thus photosynthesis.
Daytime changes in magnitudes of stomatal conductance among cocoa provenances and watering regimes were obtained. Stomatal conductance (gs) was higher during the morning hours (900 to 1100) and decreased at midday under the various watering regimes. Gs was highest at FC and 60% field capacity moisture (1.5 and 0.9 L/plant/day). The depression of gs at noonday was followed by recovery of conductance late afternoon (1500 and 1600 h) hours. Across the varieties and watering regimes, values of gs were significantly higher during the morning hours (900 to 1100) and lowest in the late afternoon (1300 to 16 00 hours) during which recovery of conductance occurred. Among the varieties, the daytime course of stomatal conductance (gs) showed that higher values were observed at mid-morning hours (1000 and 1100 hours) and lowest in the afternoon. Environmental factors drive stomatal closure and result in leaf transpiration (Cuevas et al., 2006;Agele et al., 2016;Haeberle et al., 2016). The changes in the daytime course of stomatal conductance (gs) from after sunrise toward midday were attributed to increases in leaf temperature and vapor pressure deficit and incident photon flux density (PPFD) which increased after sunrise and reached a maximum around noon (Cuevas et al., 2006;Agele et al., 2016). It has been reported that midday depression can be considered to result from the combination of the effects of light intensity of stomatal opening. The daytime carbon and water exchanges of plant leaves reflect a balance between stimulation from high light exposure and depression from a high vapor pressure deficit. High light exposure during the day stimulates stomata opening, thereby driving gas exchange. Stomatal opening (aperture) negatively affects transpiration rate and stomata often close during the day when the humidity deficit is high (Cuevas et al., 2006;Agele et al., 2016;Haeberle et al., 2016).
Previous studies of forest trees had demonstrated a reduction of gs from sunrise followed by apparent midday depression (Cuevas et al., 2006;Agele et al., 2016;Haeberle et al., 2016). Cacao provenances differed in the daytime course of stomatal gas exchange, the highest gs was observed for PA 150/34 and the lowest for Amelonado for both morning and afternoon hours.
Across the watering regimes, the highest stomatal conductance was consistently obtained under field capacity watering closely followed by 60% FC (0.9 L/plant) and lowest for 40%FC (0.6 L/ plant). The decrease in a daytime hour for stomatal conductance was substantially less in the water stressed treatment compared with the well watered. Reports of measurement net CO 2 assimilation rate (A), stomatal conductance (g s ) and transpiration (E) of leaves spanning daytime hours (early morning, midday and late afternoon periods) have shown these variables differed under various root zone moisture scenarios Haeberle et al., 2016). Stomatal conductance was responsive to root zone moisture and appeared as a regulatory signal that is operative during the day for regulation of transpiration. Changes in the daytime course of gas exchange have been reported for plant species including forest trees (Cuevas et al., 2006;Haeberle et al., 2016). Cuevas et al. (2006) reported that approximately 60% of the variation in stomatal conductance was attributable to changes in soil water content (θ v ), and obtained a close correlation between g s and θ v ) while net CO 2 assimilation rates were significantly correlated with g s . The main effect of water stress is the reduction in carbon fixation associated with stomatal closure, reduction of photosynthesis and resultant decreases in carbohydrate synthesis and plant growth and yield (Tyree et al., 2003;Agele et al., 2016;Haeberle et al., 2016). The consequences of leaf gas exchange and midday depression of gs on sunny days have been reported not only in dry regions but also in wet temperate and humid regions with implications for ecosystemlevel carbon uptake (Tyree et al., 2003;Agele et al., 2016;Li & Liu, 2016). The stomatal gas exchange appears as an informative indicator of drought tolerance in cacao due to its high sensitivity to root zone moisture status (Daymond et al., 2011;Almeida et al., 2018;Tezara et al., 2020). In this study, stomatal gas exchange was highly responsive to the root zone moisture environment. The stomatal gas exchange has been reported as an informative indicator of drought tolerance in plants because of its high sensitivity to environmental stress factors (Massacci et al., 2008;Tezara et al., 2020). The effect of water stress on plants to the reduction in carbon fixation associated with stomatal closure and concluded that the reduction of photosynthesis and resultant decreases in carbohydrate synthesis reduces plant growth and, therefore, impacts crop yield.
In this study, the growth and vigour of cocoa provenances tested were statistically superior under the full FC regimes compared with the 40% FC watering. This observation supported the findings of Agele et al. (2011) on the effects of soil moisture deficit on the of Shea butter seedlings. Our findings were also substantiated by the reports of DaMatta, Carr and Burkhardt on the drought stress responses of coffee varieties and cacao (Tezara et al., 2020). Moisture deficit stress reduces leaf area and biomass accumulation which affirmed that plants which grow under water stress will end up smaller and poor in vigour. The results of this study showed that the measured morphological and physiological variables on the seedlings of cacao genotypes responded to watering regimes. This implies that cocoa seedlings require a consistently moist root zone environment for optimum growth and development (Haeberle et al., 2016;Agele et al., 2018).

CONCLUSIONS
The study investigated morphological and physiological responses of cocoa provenances to watering regimes under screen house conditions and the implications of the measured variables as drought tolerance strategy in Theobroma was discussed. The results showed that root zone moisture status affected the morphological and physiological characteristics of cacao provenances. The imposed root zone moisture scenarios elicited differences in the responses of cacao provenances evaluated. Differences were obtained in root and shoot biomass, water use, the densities of stomatal and its conductance of gases, and the concentrations of leaf chlorophyll, and shoot and leaf proline and water soluble carbohydrates among the watering regimes imposed. Cacao provenances evaluated also differed in their responses to watering regimes and in morphological and physiological characters. Most of the measured morphological and physiological variables were driven by root zone moisture status among cacao provenances, the measured traits appeared to have played important roles as root zone moisture deficit stress tolerance mechanisms in cacao. The measured morphophysiological variables were statistically superior in well-watered situations (100% FC) compared with the 40% FC. Root zone moisture deficit stress reduced relative water content, chlorophyll and water soluble carbohydrate concentrations but reduced proline contents, stomatal densities and the conductance of the stomatal to gases. The best performance during drought was shown by the PA series while F3Amazon and Amelonado seemed to be less tolerant of drought. The latter provenance exhibited less change in chlorophyll, proline and total soluble carbohydrates concentration, and stomatal gas exchange with increasing soil moisture deficits. Adequacy of soil moisture promotes growth and physiological functions in the seedlings of cacao provenances tested. The results confirmed that cocoa seedlings cannot withstand soil moisture deficit stress as was obtained for seedlings that were watered with 40% FC. The responses of measured photochemical compounds under variable root zone moisture environments suggest inhibition of physiological functions for which the loss of phytochemical constituents can be implicated. It is concluded that root zone moisture stress-induced loss of physiological integrity is mediated by changes in phytochemistry. Seedlings of cocoa provenances had better vigour of growth when grown under 100 and 60% field capacity watering compared with 40% FC. Seedlings had greater growth when grown under 100 and 70% field capacity watering compared with the 40% FC in the nursery. The measured physiological variables varied among the provenances and root zone moisture appeared as important to cacao growth and survival, and thus, expression of cacao acclimation potential under current and future climate change scenarios of variable drought stress conditions. It is recommended that watering cacao seedlings at full field capacity (adequacy of watering/root zone moisture conditions) and at 70% FC (mild root zone moisture stress) will ensure the production of vigorous seedlings of cacao in the nursery.