1

1 Influence of potassium on photosynthetic and nutritional responses of cocoa

2 genotypes under drought stress
3
4
5 Felipe L. Neves*1, Alex-Alan F. Almeida2, Juliane D. Carvalho Neves1, José O. Souza Jú-
6 nior1, Fábio P. Gomes2, Dário Ahnert2, Virupax C. Baligar3
7
1
8 Department of Agricultural and Environmental Sciences, 2 Department of Biological Sciences, State
9 University of Santa Cruz, Campus Soane Nazareth de Andrade, Rod. Jorge Amado, km 16, 45662-900,
10 Ilhéus, Bahia, Brazil, 3 United States Department of Agriculture, Agricultural Research Service, Belts-
11 ville, Maryland, 20705-2350, United States of America
12
13 *Felipe Lopes Neves
14 felipeneves@agronomo.eng.br
15
16 ABSTRACT
17
18 In many crops, those plants well-nourished with K, and other mineral nutrients, are more tol-
19 erant to adverse effects of water deficiency. The objective of this paper was to evaluate the
20 effect of potassium fertilization on the photosynthetic and nutritional response of two drought
21 contrasting cacao (Theobroma cacao) genotypes [PA 13 (tolerant) and CC 40 (intolerant)]. [F1] Comentário: O revisor pede
para trocar a palavra intolerante pela
22 We evaluated three levels of K in the soil [100, 200 and 400 mg dm-3] and four classes of wa- susceptível
23 ter potential (ΨWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19
24 MPa; and IV = - 0.25 to - 0.51 MPa). The PA 13 genotype presented higher net photosynthet-
25 ic rate (A) compared to CC 40, in soil moisture levels of III and IV, associated with K doses
26 of 200 and 400 mg K dm-3 of soil. The K doses of 200 and 400 mg K dm-3 soil contributed to
27 decrease in stomatal conductance (gs) in both genotypes. ΨWsoil classes did not influence sig-
28 nificantly (p<0.05) the reduction of gs for PA 13. On the other hand, there was a reduction of
29 gs from class I to class II for CC 40. There has been a reduction in transpiration (E) with the
30 decrease of ΨWsoil, regardless of genotypes and doses of K in the soil. Inter-genotypic differ-
31 ences were observed (p<0.05) for intrinsic (A/gs) and instantaneous (A/E) water use efficien-
32 cies, and lowest values were recorded by the PA 13, on the lowest level of K in the soil. There
33 was an intra-genotypic difference (p<0.05) observed for N, Ca, and Mg contents in leaves,
34 with the highest levels found for PA 13. The CC 40 genotype had the highest content of K in
35 the leaves. Inter-genotypic difference were found (p<0.05) for leaf Mn contents, and highest
36 Mn content was observed for PA 13. In the roots, inter-genotypic differences (p<0.05) were
37 observed only for K and Ca contents, which were higher for PA 13. Similarly, there was inter-
38 genotypic variation observed for Mn and Zn content in roots mainly for PA 13, which pre-
39 sented the highest values. The PA 13 genotype had higher variability for fluorescence (Fv)
40 where as CC 40 had greater maximum fluorescence (Fm) which was independent of the ΨWsoil
41 classes and levels of K in the soil, while PA 13 showed the highest Fv/Fm ratio at the highest
42 levels of K. The application of K to the soil provided greater tolerance to drought for the
43 evaluated genotypes, mainly for PA 13. There was a positive interaction observed between
44 increasing levels of K in the soil and drought tolerance. The highest drought tolerance of PA
45 13 with respect to CC 40, enhanced by the K fertilization, was related in part to the greater
46 absorption and accumulation of K, Ca, Mn and Zn in roots and N, Ca, Mg and Mn in the
47 leaves, coupled with the ability to keep the stomata partially opened in conditions of low lev-
48 els of ΨWsoil. [F2] Comentário: Faz o seguinte
comentário no trecho destacado: “Ne-
49 cessita de mais coerência e estrutura.
50 Keywords: Theobroma cacao  Drought tolerance  Mineral nutrition  Leaf gas exchange  Chloro- A mensagem final não está clara”
51 phyll fluorescence
 2

52 INTRODUCTION
53
54 The episodes of drought in the world are being exacerbated because of global climate
55 changes. Many agricultural crops are already suffering from deficit of water in the soil (Mori-
56 son et al., 2008). Mortality of trees and forest decline associated with drought and heat has
57 been reported (Allen et al., 2015; Steinkamp and Hickler, 2015). There is a general consensus
58 that reduction of vascular hydraulic efficiency induced by drought is one of the main causes
59 of tree mortality (Nardini et al., 2013; Anderegg et al., 2015; Sperry and Love, 2015).
60 The vulnerability of vascular embolism formation varies greatly between the different
61 plant species. Woody plants apparently operate internal pressures of xylem near the critical
62 threshold of hydraulic failure (Nardini and Salleo, 2000), making the xylem embolism a po-
63 tentially important threat in forest biomes (Choat et al., 2012). The accumulation of embolism
64 in the xylem can lead to dehydration of the plant due to interruption of water flow from the
65 roots to the leaves. Even when it is not lethal, reduction of hydraulic conductance induced by
66 embolism from the root to the leaf can induce the closing of stomata (Salleo et al., 2000).
67 This, in turn, decreases the absorption of CO2 by the plant, reduces the carbon balance to neg-
68 ative values, when the consumption of non-structural carbohydrates through respiration ex-
69 ceeds carbon sequestration by photosynthesis, putting the plants at risk of hunger for carbon
70 (Adams et al., 2013; Hartmann et al., 2013). This risk is compounded when drought is pro-
71 longed and followed by waves of heat, stimulating the plant breathing in tissue level (Zhao et
72 al., 2013).
73 Despite the existence of many studies which evaluated the responses of different plant
74 species to drought, just a few have made comparisons between contrasting genotypes tolerant
75 to drought (Xiao et al., 2009) and, even less have focused on critical level of water deficit for
76 the studied species (Xiao et al., 2009). According to Miller et al. (2010), drought, especially
77 under high light intensity or in combination with other types of stresses affects the net photo-
78 synthesis (A) and increases photorespiration, changing the cell homeostasis causing an in-
79 crease in the reactive oxygen species production. In addition, drought combined with high
80 temperatures causes significant damage to the integrity of cell membranes and promotes liq-
81 uid peroxidation which in combination increases physiological disorders in plants, such as
82 CO2 photosynthetic assimilation and photochemical activity (Silva et al., 2010).
83 Some plant species develop adaptive mechanisms of escape or drought tolerance, re-
84 sulting from a large number of molecular, biochemical, physiological and morphological
85 changes (Ashraf et al., 2011). In leaves of T. cacao, the stomatal aperture is related to relative
86 humidity (RH), because their stomata remain more open at high RH than at low RH (Sena
87 Gomes et al., 1987). On the other hand, the stomatal closure effectively controls the loss of
88 water probably due to high cuticle transpiration (E) (Raja Harun and Hardwick, 1988). Ac-
89 cording to these authors, a leaf of T. cacao does not present high values of stomatal conduct-
90 ance (gs) under water stress and low RH. However, genotypes of T. cacao with an efficient
91 mechanism of stomatal regulation show a reduction in water loss through transpiration under
92 water deficit, considered an important adaptation strategy of the species to drought (Balasim-
93 ha, 1988).
94 The nutritional aspect is important for the maintenance of metabolic functions in ideal
95 conditions. It has been observed for various crops that plants well-nourished with K and other
96 mineral nutrients are more tolerant to the adverse effects of drought (Bosshart and Uexkull,
97 1987; Santos et al., 2014). The accumulation of K and P in leaf tissue of T. cacao assists in
98 osmotic adjustment and leaf turgor maintenance in leaf water potential values of less than -
99 1.5 MPa (Almeida et al., 2002). There are genotypes efficient in absorption and use of soil K
100 in which efficiencies is interconnected (Zhang et al., 2007). Differences between clones of T.
101 cacao in relation to uptake and use efficiency of K are a proven fact (Jones et al., 1972; Li et
 3

102 al., 2015). According to Almeida et al. (2002) there were inter-genotypic differences observed
103 in relation to the accumulation of K and P in leaves for the genotypes of T. cacao SPA 5, SI-
104 AL 70 and TSH 516 when they were exposed to drought. Potassium in soil moves to root
105 zone by mass-flow and diffusion process, under low soil moisture, rate of K flux towards the
106 root is minimized (Barber, 1995). Plant cultivars/species that are efficient in absorption and
107 utilization of low amount of K probably have advantage in overcoming the drought effects.
108 Differential response have been reported for soil K for macro and micronutrient uptake and
109 use efficiency, and growth and physiological traits among cacao genotypes such as Amelona-
110 do, EET-400 and ICS-95 (Li et al., 2013; 2015).
111 The K+ ion acts predominantly as free cation or as adsorbed cation and can easily be
112 moved from the cells or plant tissues (Lindhauer, 1985). This high mobility in plants explains
113 the main functions and features of K+ as the primary cation on neutralization of charges and
114 as the most important and active inorganic osmotic component (Clarkson and Hanson, 1980).
115 Thus, it is reasonable to assume that the genotypic variation in the utilization and efficiency of
116 K+ exists within all the major cultivated plant species. Despite the numerous studies that have
117 identified inter and intraspecific differences for the K+ use efficiency, few have made detailed
118 assessments of the observed differences and, or approached the selection, inheritance or mo-
119 lecular markers associated with the efficiency mechanisms for K+ usage (Rengel and Damon,
120 2008).
121 The species T. cacao is generally cultivated in areas of high annual precipitation as it
122 is considered a species intolerant to drought. In addition, some regions where cacao is grown
123 have shallow soils with low water holding capacity and others are prone to irregular rain pre-
124 cipitation, and these are the main causes of changes in productivity. According to Zuidema et
125 al. (2005) the annual solar radiation and rainfall during the dry season explained 70% of the
126 variations in annual seed yields obtained for 30 sites along the tropical cacao producing areas
127 of the world. Therefore, selection of genotypes tolerant to drought is very important for cacao
128 cultivation (Santos, et al., 2014). The objective of this research was to evaluate the effects of
129 soil potassium fertilization on the photosynthetic and nutritional responses of two cacao geno-
130 types contrasting for drought tolerance PA 13 (tolerant) and CC 40 (intolerant) (Santos et al.,
131 2014), under adequate and drought soil moisture conditions.
132
133 MATERIAL AND METHODS
134
135 Plant material and cultivation conditions
136
137 The experiment was conducted under the greenhouse conditions, at the State University
138 of Santa Cruz (UESC), Ilhéus, Bahia (14°47'00" S, 39°02'00" W), Brazil. Self-pollinated
139 progenies of two clonal accessions of T. cacao, PA 13 and CC 40 contrasting for drought tol-
140 erance were used (Santos et al., 2014). The progenies were obtained from the cacao
141 germplasm bank of the Centro de Pesquisas do Cacau (CEPEC), Bahia Brazil. The PA 13
142 genotype is originally from Peru belongs to the Forastero Amazonic genetic group and is con-
143 sidered drought tolerant. The CC 40 genotype is originally from Costa Rica, a hybrid between
144 Forastero and Criollo, considered intolerant to drought.
145 The self-pollinated progenies were grown in plastic pots with capacity of 12 L contain-
146 ing a mixture of soil + quartz sand, in a 2:1 v/v ratio. Water retention curve (Figure 1) and
147 chemical and textural compositions (Table 1) were determined for the soil. Based on the [F3] Comentário: Faz o seguinte
questionamento no trecho em desta-
148 chemical analysis 21.4 g of dolomite limestone/pot was added to raise the soil base saturation que: “Isso é para o solo unicamente ou
149 to 80%. At planting, the soil was fertilized with the macronutrient phosphorus and micronu- a mistura final?”
150 trients boron, copper, zinc and molybdenum (Table 2). Between 30 and 60 days after planting,
151 biweekly fertilizations were performed with nitrogen coverage at a dose of 25 mg dm-3. From
 4

152 75 days after planting up to 180 days, the fertilizer top dressing was done on biweekly accord-
153 ing to the doses indicated in Table 2. During the initial phase of the experiment, before the
154 application of treatments, soil water potential (ΨWsoil) was kept close to field capacity and had
155 its water content close to 31% which corresponded to a ѰWsoil of - 0.002 MPa.
156 The pots were divided into three groups to accommodate three levels of K treatments of
157 100, 200 and 400 mg K dm-3 which corresponds to sub-optimal, adequate or optimal (control)
158 and higher than the optimal, respectively. The treatments with the potassium doses at any giv- [F4] Comentário: Levanta o seguinte
questionamento no trecho em desta-
159 en time are listed in Table 3 with K source. Different soil water potentials were imposed on que: “Como esses níveis foram
160 45 days after planting, and continued up to 180 days of growth, totaling 10 fertilizations every estabelecidos? como sabemos que 200
é adequado? Como esses níveis foram
161 15 days, which in the end corresponded to 100, 200 and 400 mg K dm-3 soil, which were car- estabelecidos? como sabemos que 200
162 ried out on the same day of the complementary fertilization with N, B, Cu, Zn and Mo (Table é adequado?”
163 2). Additions of macro-micronutrients to soil provided all the essential nutrients to support
164 good and healthy cacao. After applying K to the soil, ten plants from each genotype and from
165 each K-treatment were subjected to four classes of soil water levels. These soil water levels
166 are classified into four ѰWsoil classes (class I = - 0.01 to - 0.03 MPa; Class II = - 0.04 to - 0.07
167 MPa; Class III = - 0.11 to -0.19 MPa; and class IV = - 0.25 to - 0.51 MPa). Total of 120 plants [F5] Comentário: Levanta o seguinte
questionamento no trecho em desta-
168 were involved in this study. The pots were covered with black tarpaulin of plastic to minimize que: “Quais foram as bases para
169 soil water loss by evaporation. Pots were weighed daily and original pot wet was maintained selecionar essas umidades? As plantas
sofrem de déficit hídrico? em qual
170 by adding rain water to compensate daily water loss by transpiration. Soil water content in nível?”
171 each of soil water treatment was calculated in accordance with the water retention curve of
172 soil (Table 4; Fig 1).
173 During the experimental period, we monitored the photosynthetically active radiation
174 (PAR), using light radiation sensors S-LIA-M003 coupled with the climatological Hobo Mi-
175 cro Station Hobo Data Logger (Onset Computer, Massachusetts, USA) and the temperature
176 (T) and air relative humidity (RH) inside the greenhouse, through sensors Hobo H8 Pro (On-
177 set, Computer, Massachusetts, USA). The average values of PAR, T and RH were 774 ± 6
178 µmol photons m-2 s-1, 25 ± 2 °C and 78 ± 3%, respectively.
179
180 Leaf gas exchange
181
182 Leaf gas exchange measurements were carried out using a portable photosynthesis me-
183 ter LI-6400 (Li-Cor Inc., Nebraska, USA), equipped with an artificial light source. Individual
184 measurements were carried out in completely expanded and mature leaves (2nd or 3rd leaf)
185 from the branch apex, between 8:00 AM and 12:00 AM, in five plants. During measurements,
186 irradiance was kept constant at 800 µmol photons m-2 s-1. Net photosynthesis ratio per unit of
187 leaf area (A), stomatal conductance to water vapor (gs), leaf transpiration rate (E) were esti-
188 mated from the values of CO2 changes and from air humidity inside the chamber. The mini-
189 mum pre-established time limit for reading stabilization was 30 s and the maximum to save
190 each reading was 60 s. The maximum coefficient of variation (CV) admitted to save each
191 reading was 0.3%. In addition to the photosynthetically active radiation (PAR) we kept the
192 block temperature constant (26°C) and so the concentration of CO2 (380 µmol mol-1) inside
193 the leaf chamber. In addition, A changes were estimated as a function of gs, the intrinsic water
194 use efficiency (A/gs) and the instant water use efficiency (A/E).
195
196 Fluorescence emission
197
198 Measurements of chlorophyll fluorescence emissions were made on the same leaves
199 used for the measurements of gas exchange, using a portable fluorimeter (Pocket PEA Chlo-
200 rophyll Fluorimeter-v 1.10 - Hansatech Instruments, Norfolk, UK), between 8 AM and 12
201 AM. The selected leaves were adapted to dark for a period of at least 30 min, for reflection of
 5

202 incident solar radiation, leaf temperature reduction and oxidation of the whole photosynthetic
203 electron transport system, using appropriate clips. After adaptation to dark the leaves were
204 exposed to a saturating light (3500 μmol m-2 s-1, wavelength of 650 nm, per 1 s), and the fluo-
205 rescence emission signals recorded in the data acquisition system of Pocket PEA, using a spe-
206 cific software. Among the obtained parameters we evaluated only the variable fluorescence
207 (Fv) and maximum (Fm) and the maximum quantum yield of PS2 (Fv/Fm).
208
209 Mineral macro and micronutrients
210
211 At the end of the experimental period, leaves and roots of the plants from each treat-
212 ment were collected, thoroughly washed and stored separately in paper bags and dried in a
213 forced-air circulation oven at 75 ºC to constant mass. Dry biomass was recorded. Plant mate-
214 rials were ground by mill similar to Willey mill and stored in paper bags. To carry out the
215 mineral macro and micronutrient analysis, 100 mg of each sample was digested in nitric per-
216 chloric digestion for determination of Ca, Mg, Fe, Zn, Mn and Cu contents by atomic absorp-
217 tion spectrophotometry, P by colorimetry, and K and Na by photometrical flame emission.
218 Sulfuric digestion and Kjedahl method was used to determine N (Jones et al., 1991).
219 The soil and sand mixture in the proportion of 2:1 v/v was collected at the time of sub-
220 strate preparation for planting the seeds. Then the substrate was air dried and passed through
221 2 mm sieve and physical and chemical properties were determined by using methodology
222 described by EMBRAPA (2011). The results of the chemical and physical analysis of growth
223 medium are presented in Table 1.
224
225 Statistical analysis
226
227 Completely randomized design was used in a 2x3x4 factorial arrangement with 24
228 treatments, two genotypes of T. cacao (PA 13 and CC 40), three levels of soil K [100, 200
229 and 400 mg dm-3] and four class of ѰWsoil [I (- 0.01 to - 0.03 MPa), II (- 0.04 to - 0.07 MPa),
230 III (- 0.11 to - 0.19 MPa) and IV (- 0.25 to -0.51 MPa)]. Each treatment was replicated five
231 times and one plant per experimental unit was used. Data were subjected to analysis of vari-
232 ance (ANOVA) and the comparison of means performed based on the Scott-Knott test
233 (p<0.05).
234
235 RESULTS [F6] Comentário: LEVANTA O SE-
236 GUINTE QUESTIONAMENTO NO
TRECHO EM DESTAQUE: “que
237 Leaf gas exchanges variação significa em linguagem de
238 pesquisa?”
239 The stomatal conductance (gs) showed significant interactions (p<0.01) between geno- [F7] Comentário: LEVANTA O SE-
GUINTE QUESTIONAMENTO NO
240 types and ѰWsoil classes. The intrinsic water use efficiency (A/gs) was significant (p<0.05) for TRECHO EM DESTAQUE: É muito
241 the interaction genotype and doses of K. On the other hand, the instantaneous water use effi- difícil acompanhar o resultado relacio-
nado às Bolsas de Gases Leaf, no final
242 ciency (A/E) showed strong variation between genotypes (p<0.01), whereas E ranged only for o objetivo principal foi testar se há
243 the ѰWsoil classes. When A was plotted as function of gs for the four classes of Ѱ Wsoil, geno- interação entre os teores de K, teor de
umidade e cultivares. Os autores fize-
244 type PA 13 presented a quadratic mathematical model, whereas for genotype CC 40 the mod- ram uma análise fatorial?
245 el was linear (Figure 2). [F8] Comentário: LEVANTA O SE-
246 ѰWsoil classes were very different for the CC 40 genotype, mainly for the variable gs in GUINTE QUESTIONAMENTO NO
247 function of ѰWsoil, where gs values decreased with the decrease of ѰWsoil. The smallest gs val- TRECHO EM DESTAQUE: Eles eram
muito diferentes? Não foram os
248 ue for this genotype was 0.02 mol H2O m-2 s-1 in class III and the largest value was 0.11 mol mesmos quatro níveis para todas as
249 H2O m-2 s-1 in class I. Highlighting the differences between the classes I and IV (Figure 3 A). plantas? OBS: não entendi muito
bem o comentário
250 PA 13 genotype did not show any significant distinctions among the first three classes of soil
[F9] Comentário: Pode ser algum
251 moisture. The dispersion of the gs values were maintained between classes I, II and III, and erro na tradução que deixou o revisor
confuso.
 6

252 for the class IV there was only a slight decrease of gs when compared with class I (Figure 3 [F10] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
253 B). The CC 40 genotype presented a sharp drop in the A values in ѰWsoil function in class IV TRECHO EM DESTAQUE: Os autores
254 (Figure 3 C). The greatest A value for the CC 40 genotype was 8.7 µmol CO 2 m-2 s-1 in class I devem usar linguagem científica, este
comentário se aplica em todo o MS.
255 and the lowest was 0.07 µmol CO2 m-2 s-1 in class IV. However, for the drought tolerant PA
256 13 genotype observed A values between classes I and II rather similar (Figure 3 D). The PA [F11] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
257 13 genotype showed the highest A value (7.8 µmol CO2 m-2 s-1) in class I, while the lowest A TRECHO EM DESTAQUE: Realmente
258 value was 1.2 µmol CO2 m-2 s-1 in class III. é significante¿¿

259 The average gs did not differ significantly (p<0.05) in doses of 200 mg dm-3 (control) [F12] Comentário: CORRIGIR
260 and 400 mg dm-3 of K in the soil. However, if compared to the lowest dose of K applied (100
261 mg dm-3 soil), there was a reduction in these values (Figure 4 A). In relation to ѰWsoil classes,
262 there was inter-genotypic difference for class I (Figure 4 B), in which the CC 40 genotype
263 achieved the highest gs value (0.07 mol H2O m-2 s-1) in relation to the PA 13 (0.05 mol H2O
264 m-2 s-1). When we compared the same genotype between classes, we observed that the PA 13
265 remained with a relatively constant gs value for all ѰWsoil classes, while the CC 40 had its gs
266 values reduced from class I to class II. On the other hand, from the class II, the gs values did
267 not differ significantly (p<0.05) (Figure 4 B). Besides, it was found that there was no signifi-
268 cant difference (p<0.05) observed for classes II and III, on E values. However, there was a
269 decrease of E as ѰWsoil classes from I to IV (Figure 5). E values ranged from 0.59 ± 0.08
270 mmol m-2 s-1 for class I to 0.31 ± 0.02 mmol m-2 s-1 for class IV.
271 For the intrinsic water use efficiency (A/gs) inter-genotypic significant differences were
272 found (p<0.05) only at equivalent dose to 100 mg K dm-3 soil, with emphasis on the CC 40
273 genotype that presented A/gs value of 108 µmol CO2 mol H2O-1, whereas for the PA 13 the
274 A/gs was 88 µmol CO2 mol H2O-1, a difference of 19%. However, this important difference
275 disappeared as doses of K applied to the soil increased. There was also a significant difference
276 (p<0.05) observed between the doses of K to a single genotype (Figure 6 A). On the other
277 hand, the instantaneous water use efficiency (A/E) value for CC 40 genotype was higher when
278 compared with PA 13 respective of soil K dose (Figure 6 B).
279
280 Fluorescence emission
281
282 The Fv value showed a subtle increase of 3.3% for PA 13 genotype in relation to CC 40 [F13] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
283 genotype (Figure 7 A). On the other hand, CC 40 genotype presented a higher F m value than TRECHO EM DESTAQUE: Ele pergun-
284 PA 13 genotype (Figure 7 B). A significant interaction was observed (p<0.05) between geno- ta se a aumento SUTIL de 3,3% é
significativo¿¿ e Pede novamente para
285 type and dose of K applied in soil for the maximum quantum yield of PS2 (Fv/Fm). In addi- rever a linguem do MS.
286 tion, there was an inter-genotypic significant difference (p<0.05) only for the corresponding
287 dose to 40 mg K dm-3 soil (Figure 8). The CC 40 genotype presented a Fv/Fm ratio value 2.7%
288 higher than PA 13.
289
290 Mineral macro and micronutrients
291
292 Significant inter-genotypic differences (p<0.05) were found for N contents in leaf dry
293 biomass (DB) (Figure 9 A). PA 13 genotype, considered drought tolerant, had N content of [F14] Comentário: Ele exclui a pala-
vra contente e sugere colocar concen-
294 26.3 g kg-1 DB, whereas for the genotype CC 40, considered intolerant to drought, the leaf N tração.
295 content was 24.2 g kg-1 DB. On the other hand, the CC 40 genotype contained the highest leaf
296 K content (10.3 g kg-1 DB), in relation to PA 13 (8.4 g kg-1 DB) (Figure 9 B). Besides that, for
297 Ca and Mg contents (Figure 9 C and D), the highest values were observed for PA 13 genotype
298 (11.9 and 8.7 g kg-1 DB, respectively), while for the CC 40 genotype leaf contents of Ca and
299 Mg were 10.7 and 7.4 g kg-1 DB, respectively.
300 There was also an increase in Ca and leaf Mg contents as ѰWsoil classes were more nega-
301 tive. This increase was significant (p<0.05) for Ca, in classes III and IV, when compared to
 7

302 classes I and II (Figure 10 A). For Mg content, this increase was observed only in the class [F15] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
303 with most negative ѰWsoil (class IV) (Figure 10 B). In addition, it was observed that PA 13 TRECHO EM DESTAQUE: Isso foi
304 genotype presented a higher content of Mn in leaf tissue, when compared with its contrasting observado para ambas as cultivares?
Em caso afirmativo, como as cultivares
305 CC 40, in which variation was 37.8% higher (Figure 11). eram distintas na resistência ao estres-
306 There were significant intergenotypic differences (p <0.05) for the levels of k in the root se hídrico?
307 (Figure 12 A). Contrary to leaf K content, genotype CC 40 presented lower values of K at
308 root, when compared to genotype PA 13 (Figure 12 A). For CC 40 genotype the root K con-
309 tent was 7.5 g kg-1 DB whereas for PA 13 the content was 8.9 g kg-1 DB. The increase of K
310 doses applied to the soil provided an increase of the K levels in the root, whose values were
311 6.4, 8.6 and 9.6 g kg-1 DB, for the doses corresponding to 100, 200 and 400 K mg kg-1 soil, [F16] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
312 respectively (Figure 12 B). There was also an inter-genotypic difference (p<0.05) for the Ca TRECHO EM DESTAQUE: Esses
313 content in root DB (Figure 13). PA 13 genotype presented higher Ca content (2.8 g kg-1 DB) valores são médios para as duas
cultivares? E quanto a biomassa
314 at the root in relation to CC 40, which content was 2 g Ca kg-1 DB. On the other hand, PA 13 vegetal, eficiência de uso de K,
315 genotype had the highest Mn and Zn contents in root tissues, when compared to CC 40 geno- recuperação de K? Mais dados são
necessários nesta parte
316 type (Figure 14 A and B).
317
318 DISCUSSION
319
320 The PA 13 genotype, unlike of CC 40, accumulated more K in the root. This probably
321 contributed to the increase in water absorption efficiency of the root system of this genotype.
322 Differences in K requirement in plant tissues are generally attributable to differences in K+ ion
323 requirements for vacuoles and the apoplast, and the ability to maintain certain K content in
324 cytosol (Fricke et al., 1994). Diffusion rates are influenced by the ability of the plant to as-
325 similate K on the root surface and create a diffusion gradient from the soil toward the root.
326 According to some authors, there are steps that are considered critical in the selection of gen-
327 otypes efficient in use of K, such as: (i) identify the differences and efficiency of the use of K
328 in germoplasm of agricultural species (Trehan et al., 2005; Damon and Rengel, 2007;), (ii)
329 clarifying the main driving mechanisms that drive the observed differences in the K use effi-
330 ciency (Damon and Rengel, 2007) and (iii) identify suitable molecular markers or tread pro-
331 cedures by which the desirable characteristics can be efficiently recognized. For the nutrients
332 provided by diffusion, as in the case of K, it is important to have a large surface area of con-
333 tact between the roots and soil. Then, genotypes efficient in the absorption of K may have a
334 higher proportion of fine roots around the root system, compared to inefficient genotypes
335 (Barber, 1995; Fageria, 2013). In this case, the root volume can be very important for geno-
336 types exposed to low K availability. Furthermore, quick turnover or root renewal may be im-
337 portant in K absorption efficiency in exposed species to low K availability in soil (Barber,
338 1995; Steingrobe, 2005). The formation of fine roots is different between genotypes of the
339 same species (Jungk, 2001; Fageria, 2013). Plant types with the fine roots tend have higher
340 absorption factor for the K (Hogh-Jensen and Pedersen, 2003). Drought tolerant T. cacao [F17] Comentário: LEVANTA O
341 genotypes have higher proportion of fine roots when grown under water deficit conditions SEGUINTE QUESTIONAMENTO NO
TRECHO EM DESTAQUE: Discussão
342 (Santos et al., 2014). deve ser melhorada, a maior parte da
343 The dispersion of ѰWsoil classes for the two T. cacao genotypes evaluated showed a vis- literatura não é relevante para discutir
os resultados.
344 ible variation of A as a function of gs. The moisture ѰWsoil classes affected both T. cacao dif-
[F18] Comentário: LEVANTA O
345 ferently in expression of gs. However, the PA 13 genotype, unlike the CC 40 genotype pre- SEGUINTE QUESTIONAMENTO NO
346 sented a clear distinction between ѰWsoil classes with the variation of A as function of gs (Fig- TRECHO EM DESTAQUE: Como os
autores podem explicar o que significa
347 ure 2). This demonstrates the ability of PA 13 to keep A and gs values more constant, irre- a dispersão da umidade do solo?
348 spective of ѰWsoil classes. In general, plants under water deficit have decreased A and gs (Ni
[F19] Comentário: LEVANTA O
349 and Pallardy, 1992), which was not observed for PA 13 genotype. Rapid stomatal closure can SEGUINTE QUESTIONAMENTO NO
350 be associated with rapid acclimation of plant to water deficit (Mencuccini et al., 2000). On the TRECHO EM DESTAQUE : Os autores
podem explicar o que significa variação
351 other hand, the distribution of gs values, presented in Figure 3A for the CC 40 genotype fol- visível?
 8

352 lows woody plants behavior that commonly have gs values in the same range when the stoma-
353 ta are open (Nobel, 1991). However, the PA 13 genotype (Figure 3 B) showed distribution of
354 gs values far below than found for the CC 40 genotype, but these values are similar in magni-
355 tude reported for Acacia arabica (Lassouane et al., 2013)
356 The significant reduction of the gs values from dose K control applied to the soil (200
357 mg dm-3) and maintenance of its values at a dose of 400 mg dm-3 (Figure 4 A) are consistent
358 with studies developed by Gattward et al. (2012) that used two doses of K in the soil (97.5 mg
359 dm-3 and 156 mg dm-3) where K was applied in single dose, and gradually replaced by Na.
360 The doses applied by Gattward et al. (2012) are close to the first and second doses used in
361 this experiment. In addition, the cultivation times in both experiments were similar. The de-
362 crease in gs values can restrict CO2 fixation rate, with the consequent decrease in its concen-
363 tration in the substomatic cavities and intercellular spaces (Daley et al., 1989). The accumula-
364 tion and release of K by stomatal guard cells promote changes in their turgor pressure, result-
365 ing in the stomatal opening and closure. In stressed plants, the increase in abscisic acid (ABA)
366 content stimulates the release of K from the guard cells, promoting stomatal closure (Assmann
367 and Shimazaki, 1999).
368 For the CC 40 genotype, there were reductions of gs between ѰWsolo classes I (- 0.01 to -
369 0.03 MPa) and II (- 0.04 to - 0.07 MPa), followed by maintenance of their values to class IV
370 (- 0.25 to - 0.51 MPa). On the other hand, the gs values of PA 13 genotype remained constant
371 in the class I to IV (Figure 4 B). These data demonstrate that the PA 13 genotype has an effec-
372 tive strategy response to water deficit in the soil, since gs remained constant in different clas-
373 ses of ѰWsoil, an intrinsic feature of this genotype adjustment to water stress. According Bal-
374 asimha et al. (2013), some T. cacao genotypes can maintain high gs values even under
375 drought conditions in soil, showing that such genotypes are relatively less affected by water
376 stress factor. Stomata closure is the first response of the plant to water deficit and at the same
377 time it is dominant limitation to photosynthesis in mild to moderate stresses (Flexa and
378 Medrano, 2002). According to Lawlor and Tezara (2009), a water deficit slowly imposed on
379 plants can cause little change in gs, while a water deficit quickly imposed can cause large re-
380 ductions in gs. Typically, gs decrease rapidly with a drastic drop in soil moisture, because the
381 stomatal opening responds more quickly to changes in the water content in the soil (Sassaki
382 and Machado, 1999). The same fact was observed with the decrease of E as function of ΨWsoil,
383 due to the increased stomatal closure promoted by reducing of ΨWsoil (Figure 5). Rawat et al.
384 (1985) studying the variation of E in Eucalyptus species kept constantly at different moisture
385 levels in the soil, it was found that the plants were kept at field capacity showed high levels of
386 E.
387 The higher doses of K applied to the soil provided greater water use efficiency for CC [F20] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
388 40 genotype. However, with the increasing doses of soil K to 400 mg dm-3 soil intergenotypic TRECHO EM DESTAQUE: Eu não
389 difference were not observed. This shows the PA 13 genotype mained its water use efficiency consegui encontrar este resultado na
seção correspondente
390 by holding constants its intrinsic water use efficiency (A/gs) under low and high dose of K in
391 the soil (Figure 6 A). The same happened to the difference between the doses of K to the
392 same genotype, where the PA 13 showed superior results at the highest dose of K applied.
393 This difference between the genotypes and the doses of K suggests that increased K content in
394 plant tissues, associated with genotypic differences may contribute to a better reason A/gs. [F21] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
395 Moreover, higher instantaneous water use efficiency (A/E) (Figure 6 B) observed in CC 40 TRECHO EM DESTAQUE: Esta frase
396 genotype is due to the fact that this genotype also had a reduction in gs values between ΨWsoil deve ser reescrita.
397 classes (Figure 4 B), as a consequence there were an increase of A/E. However, for the PA 13
398 genotype, where gs remained relatively constant even in low soil moisture (ΨWsoil), the values
399 of A/E did not reflect their performance. According to Kozlowski and Pallardy (1997), sto-
400 matal closure causes relatively greater decreases in E than in A, in the face of additional re-
401 sistances associated with the diffusion of CO2 relative to water on the leaf. The decrease of gs
 9

402 not only suppresses the loss of water through transpiration, but also increases the efficiency of
403 water use by plant.
404 The higher leaf content of K found in the CC 40 genotype demonstrates the necessity
405 for this genotype of higher soil application of K to maintain higher levels of K in the cells of
406 leaf tissue in order to maintain constants values of A, gs and E, and, consequently, increase
407 CO2 assimilation and transport of other essential nutrients (Figure 9 B). According to Baligar
408 et al. (2001) the efficient use of nutrients is controlled by genetic and physiological factors
409 and modified by the interaction of the plant with environmental variables. The solute accumu-
410 lation in the early years of plant growth seems to be a tolerance mechanism that reduces mor-
411 tality rates during the establishment phase under drought condition (Araque et al., 2012). The
412 accumulation of K and other solutes keep turgidity of cells of the tissue, a condition that char-
413 acterizes the osmotic adjustment. Upadhyaya et al. (2012) observed that the accumulation of
414 K, Ca and Mn in Camellia sinenseis plants showed positive responses in growth and antioxi-
415 dative metabolism during the dry recovery process.
416 The higher value of Fv was observed in PA 13 genotype as compared to CC 40 (Figure
417 7 A) indicating an increased energy transfer capability from electrons for the formation of
418 reducing power (NADPH) and ATP production; and hence higher CO2 assimilation capacity
419 in biochemical phase of photosynthesis (Baker, 2008). The decrease in Fv may indicate the
420 existence of the damage on PS2 reaction centers, associated with the reduction of electron
421 transport for PS2 (Krause and Behrend, 1986). Moreover, the increase in the values of Fm,
422 observed for the CC 40 genotype, against the PA 13 (Figure 7 B), indicate that this genotype
423 releases or loses potential energy of electrons that would have achieved the quinone A (Q A)
424 stable primary receiving of PS2 electrons; or by some electronic flow blocker or a lack of
425 demand and the production of NADPH and, or ATP which return to their original molecules
426 (Baker, 2008). In contrast, PA 13 genotype has lower power dissipation, which reduces the
427 value of Fm. In this case, the excitation energy may exceed the dissipation capacity and
428 should not be a damage problem in the center of PS2 reaction (Martínez-Carrasco et al.,
429 2002). Reduction of Fm has been attributed to non-photochemical dissipation, principally
430 through the xanthophyll cycle, at which time the violaxanthin is converted into zeaxanthin
431 (Demmig-Adams and Adams, 1992). Thus, excess energy absorbed by the leaf of PA 13 geno-
432 type is drained to the carotenoid xanthophyll cycle, which dissipates as heat, thereby protect-
433 ing the PS2 against possible oxidative damage caused by full sunlight. [F22] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
434 By relating the observed values of the ratio Fv/Fm both genotypes T. cacao and K doses TRECHO EM DESTAQUE: Este pará-
435 applied to soil, it was observed that the PA 13 showed higher PS2 quantum yield at the high- grafo necessita de revisão. Considere
reescrevê-lo
436 est dose K (400 mg dm-3 soil) when compared to the CC 40 genotype (Figure 14). The reduc-
437 tion in the amounts of Fv/Fm would be an indicative of environmental stresses effects on pho-
438 tosynthesis (Baker, 2008). The absence of changes in the PS2 photochemical efficiency sug-
439 gests that the drought did not cause damage to PS2, with its effects observed indirectly. Simi-
440 lar effects were observed in Coffea sp, whose photochemical reactions were affected minimal-
441 ly or not affected at all by drought (Damatta et al., 1997; Praxedes et al., 2006). In contrast,
442 Silva et al. (2010) found a reduction in activity of PS2 in Jathropha curcas plants subjected to
443 the water deficit, and its relationship with oxidative stress. In the same way, many other au-
444 thors relate the decrease in PS2 photochemical capacity, and consequently, photosynthetic
445 activity, with the advance of drought (Oukarroum et al., 2007; Gonçalves et al., 2009). Ac-
446 cording to Li et al. (2013), the level of K in the soil have a significant effect on the concentra-
447 tion of Chl b and ratio Chl a/b. According to these authors, the increase in K dose in the soil
448 promotes the reduction ratio Chl a/b and increases the concentration of Chl b in leaves of T.
449 cacao.
450 The PA 13 genotype accumulated higher contents of macronutrients N, Ca and Mg in
451 the leaf level (Figure 9 A, C and D) than the CC 40 genotype. Nguyen-Queyrens and Bouchet-
 10

452 Lannat (2003) observed similar trend in Pinus pinaste, whose solutes accumulation was ap-
453 proximately 2.3 times higher in strains resulting from dry environments, when compared to
454 the humid environments. According to Santos et al (2014), the contents of N, P, K, Ca and
455 Mg in various genotypes of T. cacao, especially the CC 40, had significant reductions when
456 subjected to drought in the soil. A similar fact was observed in drought conditions in soil for
457 the tree species Fagus sylvatica, whose reductions in macro and micronutrients minerals con-
458 tents were highly significant (Peuke and Rennenberg, 2011). Serraj and Sinclair (2002) stated
459 that the osmolytes accumulation can be a crucial mechanism to increase the yield of crops
460 subject to drought. According Tischner (2000), nitrite in high contents is deposited on cell
461 vacuole and contributes significantly to the generation of turgor.
462 The leaf contents Ca and Mg (Figure 10 A and B) were higher in the most negative ΨW-
463 soil classes, due to greater accumulation of these nutrients in the older biomass, as in drought [F23] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO NO
464 conditions the plants are not producing new tissue and, hence the content of these minerals TRECHO EM DESTAQUE: Confusão
465 increase. In the case of Ca, considered as low mobile element in the plant, and its transloca- entre conteúdo e concentração.
466 tion from older tissues to the young, is minimal especially in drought condition (Figure 10 A).
467 This metallic element, absorbed by the roots of the plants in the form of cation (Ca 2+), is re-
468 sponsible for the stability and permeability of cell membranes (Marschner, 1986). In addition,
469 Ca2+ inhibits the entry of K+ into the plasma membrane of guard cells, acts in osmotic regula-
470 tion, active anion channels and is involved in the control of plant responses to adverse condi-
471 tions such as drought (Tuteja and Sopory, 2008; Geiger et al., 2009). It is assumed also that
472 Ca, in addition to structural support may act coordinating water molecules, providing rapid
473 access to the substrate to be transferred to the Mn sites and, finally, to promote water oxida-
474 tion in the photosystem 2 of photosynthesis (Pace et al., 2012). Furthermore, Ca is involved
475 in the signaling of oxidative stress enzymes participates in the perception of reactive oxygen
476 species, and induction of antioxidant genes in plants (Rentel et al., 2004) and is associated
477 with most cell signaling processes (Sanders et al., 2002). Moreover, although Mg is very mo- [F24] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO OU
478 bile in the plant, the dry soil also affects its translocation from older tissues to younger be- COMENTÁRIO NO TRECHO EM
479 cause the water potential within the plant is reduced. Furthermore, Mg is an essential element DESTAQUE: Esta parte precisa ser
revisada, provavelmente reescrita
480 for the activation of several enzymes, including ATPases, protein kinases and phosphatases
481 (Maathuis, 2009). According Shaul (2002), the stability of the ribosomes and photosynthesis
482 may be affected by Mg deficiency since this element is the central atom of the chlorophyll
483 molecule and takes part in the aggregation of ribosomes, besides acting on the stability of cell
484 membranes. According to this author, the most important enzymatic reactions, where Mg is
485 essential, are associated with the energy transfer and phosphorylation and dephosphorylation.
486 PA 13, a drought tolerant genotype, showed a very high Mn content as compared to the
487 CC 40 genotype, proving that drought tolerant genotypes have high nutritional demand for
488 Mn (Figure 11). This metallic element plays a key role in the photosynthetic apparatus, that
489 is, operates in ATP synthesis in the RuBP-carboxylase reactions, in the biosynthesis of fatty
490 acids, lipids and proteins, oxidation of the oxygen evolving complex of the water molecule
491 associated to the PS2, and contributes to transport electron of the water to the chlorophyll
492 molecule (Lidon et al., 2004). Mn also participates in protein synthesis such as ribosome
493 structural component and activator of RNA-polymerase, and cell elongation, besides influenc-
494 ing the content of 3-indole acetic acid (IAA) (Marschner, 1986). Moreover, Mn, in conjunc-
495 tion with Zn and Cu, presents a role as enzymatic cofactor in the activity of superoxide dis-
496 mutase, in protection of the plants against reactive oxygen species, and other enzymes
497 (Hänsch and Mendel, 2009), contributing to drought tolerance in plants.
498 The higher Ca content found in PA 13 genotype roots (Figure 13) may be an indication
499 that this genotype has a higher signaling efficiency in responses to abiotic stresses. Studies
500 have shown that high concentrations of ABA during periods of drought, cold, and salinity
501 results in an increase of Ca levels in the vegetative cells tissues (Mahajan and Tuteja, 2005;
 11

502 Tuteja, 2007). Takano et al. (1997) found changes in Ca content in the cytoplasm, followed
503 by perception of stress. Water stress and thermal induced changes in the levels of Ca (Takano
504 et al., 1997; Gong et al., 1998) and genetic expression (Kinight et al., 1997).Variations in re-
505 sponses to Ca between different species occur because of genetic factors related to plant effi-
506 ciency in the use of this nutrient (Clark, 1983). As dicotyledonous species, T. cacao has roots
507 with high cation exchange capacity, which provides higher capacity to absorb divalent cations
508 such as Ca.
509 PA 13 genotype accumulated more Mn and Zn in the root system than genotype CC 40
510 (Figure 14). Mn plays a catalytic role in the dismutase activity of superoxide, for protection of
511 plants against reactive oxygen species (ROS), and other enzymes (Hänsch and Mendel, 2009),
512 contributing to drought tolerance in plants. Zn is the amino acid tryptophan precursor, associ-
513 ated with auxin biosynthesis, hormone involved in the formation of lateral and adventitious
514 roots (Taiz and Zeiger, 2009) and may thus contribute to drought tolerance in plants (Bagci et
515 al., 2007). Moreover, its absorption by the roots can be difficult when the plants are subjected
516 to drought, which can promote deficiency of this element in the plant, exacerbating their re-
517 sponse to drought (Waraich et al., 2011). Diffusion is major mechanism of moving Zn to root
518 surface, and in low soil moisture conditions diffusion is greatly reduced there by affecting its
519 rate of movement to root rhizosphere and its absorption (Barber, 1995). Furthermore, Zn de-
520 ficiency reduces the osmotic adjustment capability under low water availability in the soil and
521 consequently the plants use less water inefficiently and are less able to respond to water defi-
522 cit in soil, different from plants that have an adequate supply of Zn (Khan et al., 2004).
523
524 Conclusions
525
526 The application of K to the soil caused both genotypes of T. cacao greater acclimation
527 to drought, especially for the drought tolerant genotype (PA 13). Overall there was a positive
528 interaction between increased K levels in soil and tolerance to drought. [F25] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO OU
529 The higher drought tolerance of PA 13 genotype compared to CC 40 genotype and fur- COMENTÁRIO NO TRECHO EM
530 ther potentiated by higher levels of soil K, was related, in part, to its higher absorption and DESTAQUE Ambas as cultivares eram
tolerantes? Em que nível K e
531 accumulation capacity of K, Ca, Mn and Zn in the roots and N, Ca, Mg and Mn in leaves; parâmetros fisiológicos de umidade
532 associated with higher capacity to keep the stomata partially opened in conditions of low wa- foram ótimos?
533 ter potential in the soil, thereby allowing it to maintain adequate leaf gas exchange and a posi-
534 tive carbon balance under drought conditions.
535
536
537
538
539 Acknowledgments
540
541 The second author gratefully acknowledges Brazil’s National Council for Scientific
542 and Technological Development (CNPq), Brazil, for the concession of a fellowship of scien-
543 tific productivity.
544
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875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
 19

897 List of Tables

898
899 Table 1 – Chemical analysis of soil. pH in water, KCl and CaCl - Ratio 1: 2.5; P - Na - K - [F26] Comentário: OBS: Já corrigido.
Indagação feita: Only pH in water is
900 Fe - Zn -Mn - Cu - Puller Mehlich 1, Ca - Mg-Al - Puller: KCI - 1 mol L-1, H + Al - Puller presented in the table, please add or
901 calcium acetate 0.5 mol L-1- pH 7.0; B - Puller hot water; S - Puller - monocalcium phosphate correct
902 acetic acid; Organic matter (OM) = Organic carbon x 1.724 -Walkley-Black; SB = sum of
903 exchangeable bases; CTC (t) - effective cation exchange capacity; CTC (t) - cation exchange
904 capacity at pH 7.0;V = base saturation level; m = aluminum saturation index; (AG) = coarse
905 sand.; (AF) = fine sand; Silt and Clay = density method.
906
pH (H2O) 5.32
-3
Ca (cmolc dm ) 0.31
-3
Mg (cmolc dm ) 2.27
P (mg dm-3) 0.30
-3
K (mg dm ) 11.0
S (mg dm-3) 14.6
-3
B (mg dm ) 0.17
-3
Cu (mg dm ) 1.21
Fe (mg dm-3) 254.80
Mn (mg dm-3) 9.00
-3
Zn (mg dm ) 3.53
Al+3 (cmolc dm-3) 4.20
-3
H + Al (cmolc dm ) 7.40
SB (cmolc dm-3) 2.92
-3
(t) (cmolc dm ) 7.12
-3
(T) (cmolc dm ) 10.32
V (%) 28.30
ISNa (%) 4.39
-1
MO (dag kg ) 0.52
P-rem (mg L-1) 10.40
AG (g kg-1) 190
AF (g kg-1) 130
-1
Silte (g kg ) 370
-1
Argila (g kg ) 310
907
908
909
910
911
912
913
914
 20

915 Table 2 – Rate of fertilization of macro and micro nutrients added at planting and as top
916 dress.
917
Fertilization at planting
Dose Amount
Nutrient -3
Source
(mg dm ) (mg pot-1)*
P 400 Ca(H2PO2)2H2 (Commercial) 26.285.0
**
B 0.4 H3BO3 (PA) 27.5

Cu 1.0 CuSO4.5H2O (PA) 47.2

Zn 3.0 ZnSO4.7H2O (PA) 158.4

Mo 0.15 (NH4)6Mo7O24.4H2O (PA) 3.3

Fertilization at top dressing

N 50 NH4NO3 (Commercial) 1818.2

B 0.6 H3BO3 (PA) 6.9

Cu 0.8 CuSO4.5H2O (PA) 9.4

Zn 2.2 ZnSO4.7H2O (PA) 26.4

Mo 0.1 (NH4)6Mo7O24.4H2O (PA) 0.7

918 *Pot containing 12 dm-3 soil. **(PA) – Pure for analysis.

919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
 21

943 Table 3 – Rate of soil potassium (K) applied and source at 45 days after planting the seeds
944 until the 150 days of growth to give total K in soil for each treatment 100 mg, 200 mg, and
945 400 mg K dm-3.
946
Dose Amount
Nutrient -3
Source
(mg dm ) (mg pot-1)*
K 100 KCl (PA)** 228.8

K 200 KCl (PA) 457.7

K 400 KCl (PA) 915.3

947 *Pot containing 12 dm-3 soil. **(PA) – Pure for analysis.

948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
 22

990 Table 4 - Soil water potential (ѰWsoil) classes associated with the percentage of water in the
991 soil and field capacity (FC).
992
FC
Class Ѱwsolo (-MPa) Water content in soil (%)
(%)

I 0.01 – 0.03 70 - 59 21.76 - 18.34

II 0.04 – 0.07 56 - 50 17.41 - 15.54
III 0.11 – 0.19 46 - 41 14.30 - 12.74
IV 0.25 – 0.51 39 - 34 12.12 - 10.57
993 *Dry soil weight = 16 kg each pot.
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
 23

1022
1023 List of Figures
1024

1025
1026 Figure 1 – Relationship between soil water potential and classes of soil water imposed.
 24

8
7
6
5
4
3 y = 0.1818 + 119.25x - 542.25x 2
A (µmol CO2 m-2 s -1)

R² = 0.89
2
Class I Class II
1 Class III Class IV
0

8
7
6
5
4
3
2 y = 0.523 + 82.889x
R² = 0.82
1
0
0 0.02 0.04 0.06 0.08 0.1 0.12
gs (mol H2O m-2 s-1)
1027
1028 Figure 2 – Net photosynthetic rate (A) as function of the stomatal conductance (gs) in two
1029 Theobroma cacao genotypes (CC 40 and PA 13) subjected to four water potential classes in [F27] Comentário: O revisor não fez
comentário, porém destacou este
1030 the soil (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and IV trecho. Comentar posteriormente.
1031 = - 0.25 to - 0.51 MPa), irrespective of doses of K, by 180 days of growth. The symbols
1032 representing the soil moisture classes. Mean values from 10 to 14 repetitions.
 25

0.120
A CC 40 B PA 13
0.100
Class I Class II
gs (mol H2O m-2 s-1)

Class I Class II
0.080 Class III Class IV Class III Class IV

0.060 y = -0.016ln(x) + 0.0008 y = 0.0426e-1.224x

R² = 0.5969 R² = 0.2115
0.040

0.020

0.000
C D
8
y = -1.361ln(x) + 0.6953 y = 4.3101e-1.784x
A (µmol CO2 m-2 s -1)

7 R² = 0.7381 R² = 0.4716
6
5
4
3
2
1
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6

Ѱwsoil (-MPa)
1033
1034 Figure 3 – Stomatal conductance (gs) (A and B) and net photosynthetic rate (A) (C and D) of
1035 two Theobroma cacao genotypes (CC 40 and PA 13) subjected to four classes of soil water
1036 potential (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and
1037 IV = - 0.25 to - 0.51 MPa) and three doses of K in the soil [100 mg dm-3, 200 mg dm-3 (con-
1038 trol) and 400 mg dm-3 by 180 days of cultivation. The symbols representing the soil water
1039 potential classes. Mean values from 10 to 14 repetitions. [F28] Comentário: LEVANTA O
SEGUINTE QUESTIONAMENTO OU
COMENTÁRIO NO TRECHO EM
DESTAQUE: Eu ainda não entendo
porque os níveis de K são usados
como variável independente para a
maioria dos números? De acordo com
o título é "Influência do potássio ..."
 26

0,054 [F29] Comentário: No primeiro gráfi-

A co ele pergunta sobre qual cor repre-
senta qual cultivar. No segundo ele faz
B A
gs ( mol H2O m-2 s -1)

0,045 B
uma marcação porém não faz nenhum
comentário

0,036

0,027

0,018

0,009

0
100 200 400
K (mg dm -3)
0,09
0,08 Aa B
gs (mol H2O m-2 s -1)

0,07
0,06 Ba
0,05 Aa
Ab Aa
0,04 Aa
Ab Ab
0,03
0,02
0,01
0
I II III IV
1040 Class of Ѱwsoil (-MPa)
1041 Figure 4 – Stomatal conductance (gs) as function of K doses in soil (A). Stomatal conduct-
1042 ance (gs) in two Theobroma cacao (genotypes (CC 40 and PA 13) as function of water po-
1043 tential class in the soil (ѰWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11
1044 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa) (B), by 180 days of growth. Capital letters
1045 compare the average K rates by Scott-Knott test (p<0.05). Uppercase compare the means of
1046 genotypes within of each class by Scott-Knott test (p<0.05). Lowercase letters compare the
1047 means of the same genotype between classes by Scott-Knott test (p<0.05). Each bar repre-
1048 sents the mean standard error (± SE). Mean values of five repetitions.
 27

1049
1050 Figure 5 - Leaf transpiration (E) in Theobroma cacao genotypes subjected to four classes of
1051 soil water potential (ѰWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to
1052 - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa), by 180 days of growth. Uppercase compare the
1053 means between classes by Skott-Knott test (p<0.05). Each bar represents the mean standard
1054 error (± SE). Mean values of five repetitions.
 28

140
CC 40 PA 13 A
mol H2O-1)

120 Aa Aa Aa
Aa
100 Bb Ab

80
2
A/gs ( µmol CO

60

40

20

0
100 200 400
K (mg dm-3)
A/E (µmol CO2 mmol H2O-1)

12
B
10 A
B
8

0
CC 40
CC-40 PA 13
PA-13

1055 Genotype
1056 Figure 6 – Intrinsic water use efficiency (A/gs) (A) and instantaneous water use efficiency
1057 (A/E) (B) in two Theobroma cacao genotypes subjected to four water potential classes (ѰW- [F30] Comentário: No segundo
gráfico (B) ele levanta a seguinte dúvi-
1058 soil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and IV = - da sobre a barra do genótipo PA 13:
1059 0.25 to - 0.51 MPa) and three doses of K in the soil, by 180 days of growth. Upper case let- Essas barras de erro parecem seme-
lhantes, talvez seja um erro?
1060 ters compare the means of the genotypes within each dose (A) by the Skott-Knott test (p
1061 <0.05). Lowercase letters compare the means of the same genotype between the different dos-
1062 es (A) by the Scott-Knott test (p <0.05). Upper case letters compare the means between the
1063 genotypes (B) by the Skott-Knott test (p <0.05). Each bar represents the mean standard error
1064 (± SE). Mean values of five repetitions.
 29

1065
1066 Figure 7 – Variable fluorescence (FV) (A) and maximal fluorescence (Fm) (B) of leaf of two
1067 Theobroma cacao genotypes subjected to four water potential classes (ѰWsolo) (I = - 0.01 to -
1068 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa)
1069 and three doses of K in the soil [100 mg dm-3, 200 mg dm-3(control) and 400 mg dm-3], by180
1070 days of growth. Uppercase compare the means of genotypes by Scott-Knott test (p<0.05).
1071 Each bar represents the mean standard error (± SE). Mean values of five repetitions.
 30

0.8
Aa CC 40 PA 13
Aa
Aa
0.78 Aa

Aa
0.76
Fv/Fm

0.74 Bb

0.72

0.7

0.68

0.66
100 200 400
K (mg dm-3)
1072
1073 Figure 8 – Quantum yield potential maximum of photosystem 2 (PS2) (Fv/Fm) in leaf of two
1074 Theobroma cacao genotypes (CC 40 and PA 13) subjected to four water potential classes
1075 (ѰWsolo) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and IV
1076 = - 0.25 to - 0.51 MPa) and three doses of K in the soil, by 180 days of growth. Uppercase
1077 compare the means of genotypes in each K dose. Lowercase letters compare the means of the
1078 same genotype in different doses by Scott-Knott test (p<0.05). Each bar represents the mean
1079 standard error (± SE). Mean values of five repetitions.
 31

27 A 13
A C
26 A
12

Ca (g kg -1 DB)
N (g kg-1 DB)

25
B
11 B
24

10
23

22 9
CC-40 PA-13 CC-40 PA-13

12 12
B A D
10 10 A
B
Mg (g kg -1 DB)

B
K (g kg-1 DB)

8 8

6 6

4 4

2 2

0 0
CC-40
CC 40 PA-13
PA 13 CC-40
CC 40 PA-13
PA 13
1080 Genotype Genotype

1081 Figure 9 – Nitrogen (N) (A), potassium (K) (B), calcium (Ca) (C) and magnesium (Mg) (D)
1082 content in leaves of two Theobroma cacao genotypes subjected to four classes of water po-
1083 tential (ѰWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa;
1084 and IV = - 0.25 to - 0.51 MPa) and three levels of K in the soil [100 mg dm-3, 200 mg dm-3
1085 (control) and 400 mg dm-3, by 180 days of growth . Uppercase compare the means of the gen-
1086 otypes by Scott-Knott test (p<0.05). Each bar represents the mean standard error (± SE).
1087 Mean values of five repetitions.
 32

13
12.5
A A
A
12
Ca (g kg-1 DB)

11.5
B B
11
10.5
10
9.5
9
I II III IV
10 A
B
9 B
B B
8
Mg (g kg-1 DB)

7
6
5
4
3
2
1
0
I II III IV

1088 Class of Ψwsolo (-MPa)

1089 Figure 10 – Overall leaf calcium (Ca) (A) and magnesium (Mg) (B) content as function of
1090 water potential classes in soil (ѰWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III
1091 = - 0.11 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa), by 180 days of growth. Uppercase
1092 compare the means of Ca and Mg between classes by Scott-Knott test (p<0.05). Each bar rep-
1093 resents the mean standard error (± SE). Mean values of five repetitions.
 33

1100

1000
A
900

800
Mn (mg kg -1 DB)

700 B

600

500

400

300

200

100

0
CC-40
CC 40 PA-13
PA 13
Genotype
1094
1095 Figure 11 – Leaf manganese (Mn) content of two Theobroma cacao genotypes (CC 40, PA
1096 13) subjected to four classes of water potential (ѰWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04
1097 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa) and three doses of K
1098 in the soil [100 mg dm-3, 200 mg dm-3 (control) and 400 mg dm-3], by 180 days of growth.
1099 Uppercase compare the means of the genotypes by Scott-Knott test (p<0.05). Each bar repre-
1100 sents the mean standard error (± SE). Mean values of five repetitions.
 34

12
A A
10
B
K (g kg-1 DB)

0
CC 40 PA 13
Genotype
12
A B
10 B
K (g kg-1 DB)

8 C

0
100 200 400
K (mg dm-3)
1101
1102 Figure 12 –.Content of K in the root in function of K doses applied in soil [100 mg dm-3, 200
1103 mg dm-3 (control) 400 mg dm-3] in Theobroma cacao genotypes subjected to four water po-
1104 tential classes in the soil (ѰWsoil) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III = -
1105 0.11 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa), by 180 days of cultivation. Uppercase
1106 compare the means of the K content in root between doses of K applied in the soil by Scott-
1107 Knott test (p<0.05). Each bar represents the mean standard error (± SE). Mean values of five
1108 repetitions.
 35

3.5
A

2.5
B
Ca (g kg-1 DB)

1.5

0.5

0
CC-40
CC 40 PA-13
PA 13

1109 Genotype
1110 Figure 13 – Calcium (Ca) content in the roots of two Theobroma cacao genotypes subjected
1111 to four water potential classes (ѰWsolo) (I = - 0.01 to - 0.03 MPa; II = - 0.04 to - 0.07 MPa; III
1112 = - 0.11 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa) and three doses of K in the soil [10 mg
1113 dm-3, 20 mg dm-3 (control) and 40 mg dm-3], by 180 days of growth. Uppercase compare the
1114 means of genotypes by Scott-Knott test (p<0.05). Each bar represents the mean standard error
1115 (± SE). Mean values of five repetitions.
 36

100
A A
90
Mn (mg kg-1 DB)

80
B
70
60
50
40
30
20
10
0

80 B
A
70
Zn (mg kg-1 DB)

B
60
50
40
30
20
10
0
CC 40
CC-40 PA
PA 13
-13
1116 Genotype
1117 Figure 14 – Manganese (Mn) (A) and zinc (Zn) (B) content in roots of two Theobroma cacao
1118 genotypes subjected to four water potential classes (ѰWsolo) (I = - 0.01 to - 0.03 MPa; II = -
1119 0.04 to - 0.07 MPa; III = - 0.11 to - 0.19 MPa; and IV = - 0.25 to - 0.51 MPa) and three doses
1120 of K in the soil [100 mg dm-3, 200 mg dm-3 (control) and 400 mg dm-3], by 180 days of
1121 growth. Uppercase compare the means of genotypes by Scott-Knott test (p<0.05). Each bar
1122 represents the mean standard error (± SE). Mean values of five repetitions. [F31] Comentário: Ao final ele faz o
seguinte comentário: Há um abuso de
1123 gráficos de barras, a maioria dos gráfi-
1124 cos não tem a lenda de reconhecer
qual cor representa qual cultivar. Em
um número muito limitado de gráficos /
análise, os níveis de K são usados. Os
autores devem se concentrar nessa
variável independente para traçar seus
parâmetros. Se a interação for detecta-
da, um gráfico tri-dimensional poderá
ser desenhado. Olhando para os gráfi-
cos, diferenças de cultivares relaciona-
das ao estresse hídrico são dificilmente
observadas. Os autores poderiam usar
outro tipo de gráfico para comprovar o
contrário?