Journal of Applied Botany and Food Quality 94, 75 - 81 (2021), DOI:10.5073/JABFQ.2021.094.009 1Facultad de Ciencias, Departamento de Biología, Laboratorio de Fisiología y Bioquímica vegetal. Universidad Nacional de Colombia, Bogotá, Colombia 2Facultad de Ciencias Agrarias, Departamento de Agronomía. Universidad Nacional de Colombia, Bogotá, Colombia Photosynthesis, biochemical activity, and leaf anatomy of tree tomato (Solanum betaceum Cav.) plants under potassium deficiency Claudia Helena Ramírez-Soler1,2,*, Stanislav Magnitskiy2, Sandra Esperanza Melo Martínez2, Fagua Álvarez-Flórez1, Luz Marina Melgarejo1,* (Submitted: February 16, 2021; Accepted: March 28, 2021) * Corresponding author Summary The effects of potassium (K) deficiency on the physiological, bio- chemical, and anatomical parameters of leaves in the tree tomato plants (Solanum betaceum Cav.) were evaluated during vegetative growth. The experiment was carried out for 135 days after treatment applications under greenhouse conditions, employing the nutrient solutions with the following treatments: control plants (without K de- ficiency) and the plants with K deficiency. The light response curve, photosynthesis at light saturation (Amax), light compensation point (Ic), transpiration rate (E), stomatal resistance (SR), and pigment contents in leaves were evaluated. Additionally, the maximum pho- tochemical efficiency of PSII (Fv/Fm), contents of malondialdehyde (MDA), total soluble sugars, proline, and leaf anatomy parameters were assessed. In the K-deficient plants, the reduction in Amax (66%), Ic (63.7%), E (66%), Fv/Fm (17.3%), contents of total chlorophyll (77.4%) and chlorophyll a (52%), thickness of leaf blade L (28.5%), palisade parenchyma PP (6.5%), and spongy parenchyma SP (9.5%) were ob- served, compared to the control plants. In contrast, the variables that increased significantly were SR (65%), MDA (52%), Upper epidermis thickness (Ue) (27.1%), and Lower epidermis thickness (Le) (22.3%). The potassium deficiency caused alterations in the plant development due to the influence on physiological, biochemical, and anatomical parameters, which suggests the importance of mineral nutrition with K for this plant. Introduction The tree tomato (Solanum betaceum Cav.), also known as tamarillo, belongs to the Solanaceae family, originating from the Andean fo- rests of southern Bolivia and northern Argentina (ACOSTA-QUEZADA et al., 2015). It is a perennial plant that reaches around 2 to 3 m height in its natural habitat and has a semi-woody stem that ramifies and forms the crown. The tree tomato is one of the most important fruit crops in the Colombian Andean region, with a national production of 196,558 t (ASOHOFRUCOL, 2020), due to its potential for food pro- cessing, pigment, cosmetics, and pharmacy industries, fresh con- sumption, and antioxidant content (ACOSTA-QUEZADA et al., 2015; MOHD NOR et al., 2018). Chemical and biological factors can affect crop development, includ- ing nutrient management (GARZA-ALONSO et al., 2019). Mineral nu- trition plays a key role in the growth and development of plants and, consequently, in crop production. Potassium (K) is one of the crucial nutrient elements for meristem functioning, defense, signaling, and transport processes (ARMENGAUD et al., 2009; DEMIDCHIK, 2014). In addition, this element participates in stomatal opening, movement of solutes via phloem, cellulose synthesis, osmoregulation, induced re- sistance to pathogen attack (DEMIDCHIK, 2014), and water absorption (HAWKESFORD et al., 2012). It participates in about 60 enzymatic re- actions, involving photosynthesis, respiration, protein synthesis, and carbohydrate metabolism (DONG et al., 2010). It is also a key element for the establishment of transmembrane pH gradient required for ATP synthesis (HAWKESFORD et al., 2012; ZÖRB et al., 2014). Various experiments in hydroponics systems and in pots with a sub- strate lacking K show a negative effect of the K deficiency on the physiological and growth parameters of plants (WANG et al., 2015; SRINIVASARAO et al., 2016; DU et al., 2019). The absence of K altered the distribution of assimilates and this translated into the changes in metabolite contents in vegetative organs (HAWKESFORD et al., 2012). However, in the K-deficient leaves, a concentration of internal CO2 increased, indicating that the reduction in photosynthesis was more affected by the resistance of leaf mesophyll than by the stomatal resistance (RÖMHELD and KIRKBY, 2010; ZÖRB et al., 2014). In addi- tion, along with a decrease in the K+ concentration in leaves, not only the photosynthesis rate and RuBP carboxylase activity decreased but also the photorespiration rate (HAWKESFORD et al., 2012). In this way, the diagnostics of the nutrient status of K in plants is important for the optimal production and quality of the crops (MATTIELLO et al., 2015; LU et al., 2016; SANADI et al., 2018). Given its importance as an exotic fruit crop on the international markets (ACOSTA-QUEZADA et al., 2015), the tree tomato has positioned itself as one of the main fruit and vegetable crops produced in Colombia (ASOHOFRUCOL, 2020) due to its high potential for bioprospecting. To our knowledge, there were no published reports on the effects of K deficiencies in the tree tomato at the physiological, biochemical, and anatomical levels. Due to the above, it would be necessary to characterize the effects of K deficiency on photosynthesis, biochemical composition (malondi- aldehyde (MDA), proline, total sugars), and leaf anatomy in the tree tomato plants. Therefore, the objective of this study was to evalu- ate the effect of K deficiency on the physiological, biochemical, and anatomical parameters in the tree tomato plants during vegetative growth. Material and methods Plant material and growth conditions The experiment was carried out under the 7-gauge polyethylene plas- ticized greenhouse, with daily average air relative humidity of 70%, average temperature of 20.3 °C, and photosynthetically active radia- tion (PAR) of 200 μmol photons m2 s-1. Three-month-old Common Red ecotype tree tomato (Solanum beta- ceum Cav.) seedlings were used, which were subjected to root wash with distilled water to remove soil particles from the substrate. Sub- sequently, these were transplanted into the black plastic bags with 8 kg capacity (0.40 × 0.60 m) containing quartzite sand of two par- 76 C.H. Ramírez-Soler, S. Magnitskiy, S. Esperanza Melo Martínez, F. Álvarez-Flórez, L.M. Melgarejo ticle sizes (0.7 and 1.5 mm) in a 1:1 v/v ratio, electrical conductivity (EC) of 0.012 dS m-1 s-1, and pH of 6.7. Each bag was positioned 1 m apart to avoid the effects between the plants. Once transplanted, these were subjected to pretreatment (acclimatization) for one month, by supplying the complete modified nutrient solution of HOAGLAND et al. (1938), where the control treatment received the complete nutri- ent solution without mineral deficiencies (Tab. 1). Gas exchange and chlorophyll a fluorescence In two fully expanded leaves of the middle-third stratum of four plants per treatment, the maximum photochemical efficiency of PSII was measured (Fv/Fm) at 0, 15, 30, 45, 60, 75, 90, 105, 120 and 135 dat with a HandyPEA unmodulated Fluorometer (HansaTech instruments, Norfolk, UK) with a saturating PAR of 3000 μmol photons m2 s-1. In the same plants and leaves, the transpiration rate (E) and stomatal resistance (SR) were determined between 7:00 and 10:00 am, using a porometer Li-cor Li-1600 Steady (Lincoln, Nebraska, USA). Biochemical parameters These were analyzed at the end of the experiment (135 dat). Photosynthetic pigment contents In three individual plants per treatment, the leaves were collected from the middle-third stratum for extraction of the total chlorophyll content (total chl) according to LICHTENTHALER and WELLBURN (1983). The 0.05 g leaf tissue without ribs was used and the extrac- tion was carried out with 80% acetone previously kept at -4 °C. Absorbance at 470, 646 and 633 nm was measured using a BIO-RAD Smart SpecTM 3000 spectrophotometer (BIO-RAD, Philadelphia, USA) expressing the results obtained in fresh weight mg g-1 (fw), for the concentration of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Total Chl), and carotenoids. Malondialdehyde content The content of malondialdehyde (MDA) was determined with thio- barbituric acid based on that described by WANG et al. (2012). The 0.05 g of fresh leaf tissue was obtained from the middle-third stratum of three individual plants per treatment, then used for maceration and extraction, homogenized with 2 ml of 10% trichloroacetic acid (TCA) solution. To 1 mL of the supernatant, 4 mL of 0.5% TBA solution (prepared in 10% TCA) was added and stirred. Subsequent- ly, it was heated at 95 °C for 30 min and then cooled on ice. It was centrifuged at 5000 rpm for 15 minutes. Absorbance was read with a spectrophotometer (BIO-RAD Smart SpecTM 3000, Philadelphia, USA) at 450, 532 and 600 nm. The blank was 10% TCA. The MDA contents was expressed in μmol mL-1 fw. Total sugars The soluble sugars were extracted according to DUBOIS et al. (1956) modified by MELGAREJO et al. (2010). The 0.05 g of fresh plant material was weighted from leaves obtained from the middle-third stratum of three plants per treatment. Subsequently, it was macerated with liquid nitrogen, then 5 mL of distilled water were added, and it was stirred at room temperature for 60 min. It was centrifuged at 6,000 rpm for 30 min at 12 °C. The 30 μL of the supernatant were taken per sample, 180 μL of distilled water were added, homo- genized, and then 200 μL of 80% phenol were added. Subsequently, 1.0 mL of concentrated sulfuric acid was added, stirring in vortex for 1 min. Finally, absorbance was read at 490 nm with a spectro- photometer (BIO-RAD Smart SpecTM 3000, Philadelphia, USA). The content of total sugars was expressed in μg mg-1 fw. Proline content The proline content was determined according to BATES (1973), with adjustments (MELGAREJO et al., 2010). The 0.05 g of fresh plant ma- terial was obtained from the leaves taken from the middle-third stratum of three individual plants per treatment. Subsequently, 5.0 mL of 3% (w/v) sulfosalicylic acid extracting solution was intro- duced, stirred for 60 minutes at 10 °C, and centrifuged at 6,000 rpm for 30 minutes at 10 °C in darkness. Next, in dark glass tubes, 1.0 mL of the supernatant was placed and 1.0 mL of freshly prepared ninhy- Tab. 1: Concentration of mineral elements (ml of the stock solutions 1 M to be applied to 7.5 L H2O) modified based on the stock solutions (HOAGLAND et al., 1938) and the nutrient requirements reported for the species (FISCHER and MIRANDA, 2012). MACRONUTRIENTS (ml) Treatments Source Without K Control NH4NO3 69.72 55.78 KH2PO4 0.00 13.02 Ca(H2PO4)2.H2O 32.16 32.16 KCl 0.00 43.94 KNO3 0.00 37.86 CaCl2 2.70 2.70 Ca(NO3)2.4H2O 23.00 23.00 CaSO4.2H2O 20.96 12.58 Mg(NO3)2.6H2O 41.19 51.48 MgSO4 4.83 0.00 K2SO4 0.00 31.83 MICRONUTRIENTS H3BO3 0.28 0.28 MnCl2.4H2O 0.70 0.70 ZnS04 0.24 0.24 CuSO4.5H2O 0.19 0.19 FeSO4.7H2O 0.73 0.73 Treatments The Hoagland solution (HOAGLAND et al., 1938) was modified and adjusted based on the needs of the crop (FISCHER and MIRANDA, 2012) (Tab. 1); this was prepared in distilled water with an EC < 3 μS m-1 to generate the stock solutions; later it was diluted in 7.5 L of water to carry out the applications. The following two treatments were used: without K deficiency (control) and with K deficiency (without K), which were applied to the plants two times per week. A volume of 100 ml of the nutrient solution was supplied per plant (according to the substrate moisture retention curve) during vegeta- tive growth. Physiological parameters Photosynthesis At the end of the experiment (135 days after the treatment application (dat)), three plants were taken per treatment and light response curves (A/PFD) were obtained with an IRGA LCiPro + kit (BioScientific Ltd. Hoddesdon, UK). The measured radiation points were 1,200, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, and 0 μmol photons m2s-1 between 7:00 - 10:00 h (a range of time previ- ously determined, where a highest rate of photosynthesis was ob- served). The curves were made using the 4th leaf fully expanded of the middle-third stratum of the plants. The data obtained were adjusted using the Mitscherlich hyperbolic model (ALERIC and KIRKMAN, 2005; MELGAREJO et al., 2010; BARRERA et al., 2012). The parameters derived from the A/PFD curves were determined as Amax = photosynthesis at saturation by light or maximum photo- synthesis (μmol CO2 m-2 s-1), Ic = light compensation point (μmol photons m-2 s-1). Analysis of tree tomato plants under potassium deficiency 77 drin and 1.0 mL of warm glacial acetic acid were added, which were brought to the boil for 60 min. Then these were left at room tempera- ture and 3mL of toluene was added. Finally, the upper phase was col- lected and the absorbance was read at 520 nm in a spectrophotometer (BIO-RAD Smart SpecTM 3000, Philadelphia, USA), expressing the content of proline in μg g-1 fw. The concentration of proline was de- termined using the standard curve of L-proline from Sigma®. Leaf anatomy At the end of the experiment (135 dat), leaf sections (approximately 30 mm2) were collected from the middle region of the same leaf used for the gas exchange and Fv/Fm measurements. The sections were fixed in a mixture (formalin: 10, ethanol: 5, glacial acetic acid: 85), later they were subjected to different solutions of ethanol and HistoChoice® according to MEGÍAS et al. (2018). The paraffin blocks were cut with a model 820 Spencer rotary microtome (American Optical, Delhi, USA), making transverse cuts in the leaves and covering the midrib with a thickness of 13 μm. The cross sections were arranged and fixed on slides, later they were stained with the double stain of Astra-Blue, basic Fuchsin (KRAUS et al., 1998). Digital images with a resolution of 100X were obtained from the assemblies in Olympus® CX31 microscope (New York Microscope Company, NY), with camera adaptation. Using the Image-ProPlus® program (Media Cybernetics, Rockville, MD), the measurements were made of the leaf thickness (L), spongy parenchyma thickness (PS), pali- sade parenchyma thickness (PP), Upper epidermis (Ue), and Lower Epidermis thickness (Le). Experimental design and statistical analysis A completely randomized design and two repetitions of the same trial were used in time; in the present study, the data of the second repetition in time are presented. Each trial had four replicates and twenty individual plants per treatment. The data were statistically analyzed using ANOVA to identify the presence of significant differences between the treatment means due to their effects on the evaluated physiological, biochemical, and anatomical variables. The SAS version 9.2 package was used. Once the significant differences between treatment means were found, the Tukey ś multiple comparison test was used (p≤0.05). Results Physiological parameters Light response curve (A/PFD) The response curves to light A/PFD indicate the response that plants present under different light intensities (PÉREZ and MELGAREJO, 2014). The tree tomato plants during vegetative growth under po- tassium deficiency (without K) had a decrease in Amax under the different radiation points (PFD), presenting a final value of 2.3 μmol CO2 m-2 s-1 and differing from the control plants with 6.8 μmol CO2 m-2 s-1 at 135 dat (Fig. 1 and Tab. 2). The light compensation point (Ic) decreased in the plants deficient in K with a value of 12.17 μmol photons m-2 s-1 as compared with the control, which obtained a value of 35.1 μmol photon m-2 s-1 (Fig. 1, Tab. 2). Transpiration (E), stomatal resistance (SR) and fluorescence of chlorophyll a (Fv/Fm) The tree tomato plants without K registered a significant reduction (P≤0.05) in the transpiration rate (E) at 90, 120, and 135 dat by 45, 58 and 66%, respectively, compared to the control plants (Fig. 2a). The stomatal resistance (SR) presented an inverse behavior to the tran- spiration, that is, the lower was the transpiration, the higher was the stomatal resistance, as a physiological adaptation of plants to avoid water loss. The SR significantly increased in the plants without K at 120 and 135 dat by 55 and 65%, respectively, compared to the control plants (Fig. 2b). The plants without K registered a significant decrease in Fv/Fm at 105, 120 and 135 dat (0.74, 0.72 and 0.67, re- spectively) unlike the control plants, which remained with an average Fv/Fm of 0.81 (Fig. 2c). Photosynthetic pigment contents The tree tomato plants grown without K at 135 dat showed a sig- nificant reduction in the content of Chl a and total Chl (by 51.7 and 77.4%, respectively) as compared to the control (Tab. 3). However, the plants without K had the tendency to decrease the Chl b content by 33% and a slight increase in the carotenoid content by 14%, unlike the control plants (Tab. 3). The lipid peroxidation, measured as MDA contents, indicates a pos- sible damage at the cellular membrane level. The plants without K presented significant increases of 0.85 ± 0.07 μmol mL-1 MDA, com- pared to the control, which registered 0.52 ± 0.03 μmol mL-1 MDA (Tab. 3). The contents of total soluble sugars in leaves did not present sig- nificant differences between the treatments, however, the tendency to decrease was evidenced in the tree tomato plants cultivated with- out K (24.87 ± 0.85 μg mg-1 leaf fw), unlike of the control plants (29.35 ± 0.98 μg mg-1 leaf fw) (Tab. 3). The content of free proline did not present statistical differences be- tween the treatments; however, an increasing trend was registered for the treatment without K (0.20 ± 0.05 μg g-1), unlike the control (0.16 ± 0.01 μg g-1) (Tab. 3). Leaf anatomy The differences were observed in the plants with K deficiency, com- pared to the control at 135 dat. In contrast to the control, the leaves of the plants grown without K exhibited less thickness of leaf lamina (L), which was 28.5% thinner (Tab. 4). The leaves with K deficiency ZnS04 0.24 0.24 CuSO4.5H2O 0.19 0.19 FeSO4.7H2O 0.73 0.73 350 351 352 Fig. 1: Light saturation curves of photosynthesis in the tree tomato plants during vegetative 353 growth (A vs. PPFD). Control (without K deficiency) (R2 = 0.94); Plants grown without 354 potassium K (potassium deficiency) (R2 = 0.99). At the bottom of each figure the equation is 355 derived from the fit to a Mitscherlich model. n = 3 plants per treatment. 356 357 Tab. 2: Parameters obtained through the light response curve in the tree tomato plants during 358 vegetative growth at 135 dat. Control (without K deficiency) and plants grown without K 359 (potassium deficiency). These were calculated by fitting to a Mitscherlich model. A max = 360 photosynthesis at saturation by light or maximum photosynthesis, Ic = light compensation 361 point. n = 3 plants per treatment. 362 Treatment R2 Amax (μmol CO2 m-2s-1) Ic (μmol photons m-2s-1) Control 0.94 6.8±0.044 ±35.1 Without K 0.99 2.3±0.047 ±12.17 363 -8,0 -6,0 -4,0 -2,0 0,0 2,0 4,0 6,0 8,0 10,0 12,0 0 100 200 300 400 500 600 700 800 A (µ m ol C O 2/ m -2 s- 1 ) PFD (µmol m2 s of photons) Control Without K 7 6 5 4 2 1 0 -1 -2 -3 Control: P= 6.787*(1-Exp(-4.25E-03*(PFD-35.133))) Without K: P= 2.2286*(1-Exp(-0.0127*(PFD-12.170))) Fig. 1: Light saturation curves of photosynthesis in the tree tomato plants during vegetative growth (A vs. PPFD). Control (without K deficiency) (R2 = 0.94); Plants grown without potassium K (potassium deficiency) (R2 = 0.99). At the bottom of each figure the equation is derived from the fit to a Mitscherlich model. n = 3 plants per treatment. Tab. 2: Parameters obtained through the light response curve in the tree tomato plants during vegetative growth at 135 dat. Control (without K deficiency) and plants grown without K (potassium deficiency). These were calculated by fitting to a Mitscherlich model. A max = photosynthesis at saturation by light or maximum photosynthesis, Ic = light compensation point. n = 3 plants per treatment. Treatment R2 Amax (μmol CO2 m-2s-1) Ic (μmol photons m-2s-1) Control 0.94 6.8±0.044 ±35.1 Without K 0.99 2.3±0.047 ±12.17 78 C.H. Ramírez-Soler, S. Magnitskiy, S. Esperanza Melo Martínez, F. Álvarez-Flórez, L.M. Melgarejo showed a reduction by 6.5% in the thickness of the palisade paren- chyma (PP) (Tab. 4) and the cells were irregular (Fig. 4d), unlike in the control (Fig. 4b). The thickness of the spongy parenchyma (SP) was reduced by 9.5%, compared to the control (Tab. 4), and also ex- hibited an irregular shape of the cells (Fig. 4d). On the contrary, the K deficiency increased the thickness of the Upper epidermis (Ue) and Lower Epidermis (Le), surpassing the control plants by 27.1 and 22.3%, respectively (Tab. 4). The leaves of the K-deprived plants presented variations in the size of the middle vein (Midrib) (data not shown) and had irregular distribution of the vascular bundles (V) and irregular parenchyma cells (P), unlike the control leaves (Fig. 4c, a, respectively). Discussion Our results show that the tree tomato plants during vegetative growth under K deficiency severely reduced the photosynthetic rate (Fig. 1 and Tab. 2), presenting a behavior similar to that reported for Arabi- dopsis plants (ARMENGAUD et al., 2009), soybean (DONG et al., 2010) and corn (DU et al., 2019) under deficiency of this element. LU et al. (2016) pointed out that the K deficit affects the photosynthetic rate due to the lower activity of RuBisCO and the activation of the py- ruvate kinase, which catalyzes the phosphoenolpyruvate to produce ATP and pyruvate in glycolysis (WANG and WU, 2010). In the same way, the activity of RuBP carboxylase could be negatively affected due to the fact that K maintains a high pH in the stroma and its defi- ciency alters the photorespiration (HAWKESFORD et al., 2012). In this study, as the K deficiency progressed, the transpiration rate (E) decreased and stomatal resistance (SR) increased, which indi- cates that starting from 90 dat the plants experienced a stress, which was reflected with the reduction of Fv/Fm (Fig. 2) and was further translated into a reduction in photosynthesis (Tab. 2). Similar results were reported by TANG et al. (2015) in tea, WANG et al. (2015) in soy- bean, and DU et al. (2019) in corn, where K deficiency significantly reduced the variable E, unlike in the control plants. This response is due to the important role of K in maintaining turgor pressure, a fun- damental variable for stomatal opening (HAWKESFORD et al., 2012). It was, possibly, due to the fact that the reductions in E (Fig. 2a) and Amax (Tab. 2) are caused by the stomatal limitations (RÖMHELD and KIRKBY, 2010; JIN et al., 2011). ANDRÉS et al. (2014) pointed out that the K deficiencies affect the opening of stomata, decreasing the photosynthetic rate due to the limitation in the assimilation of CO2 from the atmosphere to the internal spaces of leaves, causing a nega- tive effect on the carboxylation sites within the chloroplasts (FLEXAS 364 365 366 0 2 4 6 8 10 12 0 15 30 45 60 75 90 105 120 135 E (m ol H 2O m -2 s- 1 ) dat (days after transplanting) Control Without K * a) * * 0 1 2 3 4 5 6 7 8 0 15 30 45 60 75 90 105 120 135 St om at al R es is ta nc e (S .c m ) dat (days after transplanting) Control Without K * b) 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0 15 30 45 60 75 90 105 120 135 Fv /F m dat (days after transplanting) Control Without K * * * * * c) Fig. 2: Physiological parameters in the tree tomato plants during vegetative growth. Treatments: Control (without deficiency) and plants grown without K (potassium deficiency). a) Transpiration rate (E), b) Sto- matal resistance (SR); and c) Maximum photochemical efficiency of photosystem II (Fv/Fm). Bars represent the standard error (n = 4 plants per treatment). Significant differences at the 0.05 level of significance are indicated by * (the Tukey’s test). Tab. 3: Contents of photosynthetic pigments (mg g-1 leaf fw), malondialdehyde (MDA) (μmol mL-1), total sugars (μg mg-1 leaf fw), and proline (μg g-1) in the leaves of tree tomato plants during vegetative growth. Means ± standard error (n = 3 per treatment) are presented. Control (without deficiencies) and plants grown without K (potassium deficiency). Different letters indicate significant statistical differences with a P value <0.05 (the Tukey’s test). Biochemical parameter Chl a Chl b Total Chl Carotenoids MDA Total sugars Proline Control 0.58 ±0.029 A 0.31 ±0.004 A 0.89 ±0.025 A 0.56±0.060 A 0.52±0.03 A 29.35±0.98 A 0.16±0.01 A Without K 0.30±0.064 B 0.24±0.042 A 0.54±0.100 B 0.64±0.195 A 0.85±0.07 B 24.87±0.85 A 0.20±0.05 A Tab. 4: Anatomical parameters registered in the cross sections of the tree tomato leaves during vegetative growth. Control (without deficiencies) and plants grown without K (potassium deficiency). Means ± standard error (n = 3 per treatment) are presented. Different letters indicate significant statistical differences with a P value <0.05 (the Tukey’s test). Treatment Leaf blade Palisade parenchyma Spongy parenchyma Upper epidermis Lower epidermis thickness (L) thickness (PP) (μm) thickness (SP) (μm) thickness (Ue) (μm) thickness (Le) (μm) Control 247.6±13.8 B 51.9±2.5 A 87.6±7.8 A 9.6±0.7 B 10.3±0.4 B Without K 177.1±10.7 A 48.5±2.83 A 79.3±6.1 A 12.2± 0.5 A 12.6±1.21 A Analysis of tree tomato plants under potassium deficiency 79 et al., 2008). Similarly, as K is important for the opening of stomata, the lack of this element would reduce its accumulation in the vacu- ole, affecting the stomatal opening (JORDAN-MEILLE and PELLERIN, 2008) and altering the stomatal and non-stomatal regulation of CO2 (TANG et al., 2015). The response of E was opposite to SR (Fig. 2a, b) as a strategy of the plants to avoid the loss of water inside the leaf towards the at- mosphere (QUEZADA et al., 2002). The K deficiency generated a reduction in Fv/Fm (Fig. 2c), suggesting a possible photoinhibition starting from day 105 (MAXWELL and JOHNSON, 2000). This photo- inhibition was, probably, related to the chlorophyll content (Tab. 3), which agrees with that reported by HU et al. (2016). KITAJIMA and BUTLER (1975) proposed that Fv/Fm alterations generate changes in the photochemical conversion of energy on the reaction centers of PSII and induce a possible photoinhibition. According to our results, it can be inferred that the reduction in the content of pigments, such as Chla, Chlb and total Chl (Tab. 3), due to the treatment without K were related to the low photosynthetic rate (Amax) (Tab. 2) and possible stomatal limitations. Results similar to those found in this study have been reported by JIN et al. (2011) and CAVALCANTE et al. (2015), indicating that the K deficiencies could be associated with the degradation of chlorophylls, biochemical altera- tion of chloroplasts and, therefore, less absorption of light. JIN et al. (2011) and WANG et al. (2012) suggested that the K deficiency gene- rates production of reactive oxygen species (ROS) due to the null or low consumption of ATP and NADPH in the Calvin cycle due to the absence of the electron acceptor NADP+, causing the degradation of the chloroplast pigments (CAVALCANTE et al., 2015). Additionally, the reduction in the chlorophyll contents was, possibly, due to the fact that the K deficiency is associated with the nitrogen deficiencies, which is key element in the synthesis of proteins destined for growth and de- velopment. Potassium is involved in the protein synthesis in roots as well as in the root uptake and assimilation of nitrogen (COSKUN et al., 2017). In particular, K participates in the regulation of NRT2 nitrate transporters in roots and the activity of nitrate reductase (COSKUN et al., 2017; HU et al., 2016; ARMENGAUD et al., 2009). Tab. 3 shows the increase in the MDA content in leaves with the K deficiency. These results agree with that reported by HU et al. (2016) in cotton and DU et al. (2019) in corn, which, under the K deficiency, presented the high MDA contents and were associated with high H2O2 production (a variable not measured in the present study). Similarly, HERNANDEZ et al. (2012) found significant differ- ences in tomato with low K deficiency, presenting high accumulation of ROS, simultaneously increasing the amount of MDA, which sug- gests that K deficiency caused the oxidative damage to lipids and, finally, caused a chlorosis in the plants. Our results show that the K deficiency decreased the leaf thickness including the thickness of the spongy and palisade parenchyma (Tab. 4) resulting in a decrease in Amax (Tab. 2), which shows a close relationship between the leaf anatomy and the photosynthetic pro- cess. The diffusion of CO2 during photosynthesis can be regulated by carbon allocations, which is determined by the leaf thickness and the size of the mesophyll cells (HU et al., 2020). This is in accordance with data reported in corn by DU et al. (2019) who observed that the K deficiency negatively affected the anatomical structure of leaves, significantly reducing the size of the cells. In the same way, LIN and YEH (2008) indicated that the thickness and size of leaf cells are reduced in plants because the water storage tissues in cells decreases along with the increment of K deficiency. The damage to the leaf anatomy, apparently, influences the photosynthetic process, the trans- port of nutrients and water (MATTIELLO et al., 2015). Faced with the stress due to the K deficiency, the leaves tend to be smaller, and the growth and cell expansion decrease (ARMENGAUD et al., 2009; ELISE et al., 2020). It has been reported that the mesophyll cells reduce the rate of cell division due to the K deficiency (TOSHIO et al., 2009; HU et al., 2020) and K regulates cell expansion and proliferation of the mesophyll, which affects the leaf area (BATTIE-LACLAU et al., 2014; LU et al., 2020). For this reason, the less thickness was observed in the spongy and palisade parenchyma of the tomato tree plants grown without K (Tab. 4). In general, it was observed that the K deficiency negatively affected the physiology, biochemical components, and anatomical parameters in the tree tomato plants. Fig. 4: Cross-sections of leaves in the tree tomato plants at 135 dat. Control plants (without deficiencies) (a, b) and plants grown without K (K deficiency) (c, d). Midrib (a, c), same magnification, Bar = 100 μm. Leaf blade (b and d), same magnification, Bar = 50 μm. P: parenchyma; V: vascular bundles; Co: Colenquima; Midrib: rib; PP: palisade parenchyma; SP: spongy parenchyma, Ue: Upper epidermis, Le: Lower Epidermis, T: trichomes, and Cu: cuticle. 381 Fig. 4: Cross-sections of leaves in the tree tomato plants at 135 dat. Control plants (without 382 deficiencies) (a, b) and plants grown without K (K deficiency) (c, d). Midrib (a, c), same 383 magnification, Bar = 100 𝜇𝜇m. Leaf blade (b and d), same magnification, Bar = 50 𝜇𝜇m. P: 384 parenchyma; V: vascular bundles; Co: Colenquima; Midrib: rib; PP: palisade parenchyma; 385 SP: spongy parenchyma, Ue: Upper epidermis, Le: Lower Epidermis, T: trichomes, and Cu: 386 cuticle. 387 388 389 390 391 392 393 80 C.H. Ramírez-Soler, S. Magnitskiy, S. Esperanza Melo Martínez, F. Álvarez-Flórez, L.M. Melgarejo Conclusions The tree tomato plants grown with the K deficiency reduced the physiological parameters Amax, Ic, E, Fv/Fm, content of chlorophylls a, b and total. On the other hand, these plants increased the SR, and the MDA contents in leaves. Compared with the control plants, the deficiency of K altered the leaf anatomy, reducing the thickness of the leaf along with the thickness of spongy and palisade parenchyma. In addition, an increase in the thickness of the adaxial and abaxial epidermis was evidenced. This research presents the physiological, biochemical and anatomical indicators of the tree tomato plants sub- jected to potassium deficiency during vegetative growth, which can be used for monitoring and adjusting fertilization programs of this species and, thus, be useful for the tree tomato growers. Acknowledgments The authors thank Colciencias (Minciencias) and the Universidad Nacional de Colombia for funding the project “Ecophysiology, mi- neral nutrition and integrated management of pests and diseases in avocado, curuba, gulupa and tree tomato oriented towards their agro- nomic management, as raw material for the development of products of commercial interest” led by Dr. L.M. Melgarejo, of the National Network for the bioprospecting of tropical fruits (contract 459/2013) and for financing C. Ramírez-Soler MSc thesis. Conflicts of interest No potential conflict of interest was reported by the authors. References AcostA-QuezAdA, P.G., RAiGón, M.d., RiofRío-cuencA, t., GARcíA- MARtínez, M.d., PlAzAs, M., BuRneo, J.i., fiGueRoA, J.G., VilAnoVA, s., PRohens, J., 2015: Diversity for chemical composition in a collection of different varietal types of tree tomato (Solanum betaceum Cav.), an Andean exotic fruit. Food Chem. 169, 327-335. 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Ciudad universitaria, Carrera 30 #45-03, Bogotá, Colombia E-mail: lmmelgarejom@unal.edu.co © The Author(s) 2021. 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