Use of protein-based matrices as amino acids source in in-vitro grapevine

1 Use of protein-based matrices as amino acids source in in-vitro grapevine Mercedes Jiménez-Rosado1,*, Arleen Rodríguez Declet2, Paola Negri2, Antonio Guerrero1, Alberto Romero1, Adamo Domenico Rombolà2 1 Departamento de Ingeniería Química, Facultad de Química, Escuela Politécnica Superior. Universidad de Sevilla. 41012 Sevilla, Spain. [email protected], [email protected], [email protected] 2 Department of Agricultural and Food Sciences, Alma Mater Studiorum. University of Bologna. 40127 Bologna, Italy. [email protected], [email protected], [email protected] *MERCEDES JIMÉNEZ ROSADO Departamento de Ingeniería Química, Universidad de Sevilla (Spain) e-mail: [email protected] 2 Abstract 1 Proteins are sources of peptides and amino acids which are able to stimulate 2 plant growth through mechanisms such as enhancement of root growth and 3 increased availability of micronutrients. Protein-based matrices are a novel 4 source of these raw materials since they allow their availability in small, prolonged 5 doses, which could be of interest in in-vitro assays. Thus, this work aimed to 6 evaluate the use of soy protein-based matrices in in-vitro cultures of grape, cv. 7 Magliocco Canino. Their influence was assessed in different media conditions in 8 the presence or absence of zinc (an essential microelement for plant growth). 9 The shoots were evaluated after 35 days of growth based on their growth 10 parameters (weight increase, number of stems, number of leaves, stem and 11 internode length). A biochemical profile of the shoots cultivated in different media 12 was obtained by Fourier-Transform Infrared Spectroscopy (FTIR). The results 13 highlighted the benefits of using protein-based matrices in in-vitro culture as 14 shoots showed an increase in weight, number of leaves, and longer stems, also 15 in zinc-deficient media. In conclusion, this work emphasizes the potential of 16 protein-based matrices as stimulants for grapevine explants. This could have 17 important implications for the cultivation of these crops and could be the starting 18 point for further studies on the stimulating effect of different proteins on crops. 19 Keywords: Grapevine; In-vitro culture; Matrices; Proteins; Stimulants. 20 3 1. INTRODUCTION 21 Amino acids are organic molecules composed of amino and carboxylic groups 22 (Pink et al., 2018). The main route of amino acid supply to the plant is through the 23 roots, which take up both amino acids, inorganic and organic nitrogen from the 24 soil (Gioseffi et al., 2012; Jones et al., 2005). Plants transform inorganic nitrogen 25 (nitrate (NO3-) and ammonium (NH4+)) into amino acids for the consequent 26 synthesis of proteins (Mokhele et al., 2012). In this way, amino acids are present 27 in every metabolic reaction and plant structure (Bender, 2012). Highlighting the 28 potential use of amino acid-based products for crop production, which possess 29 the capacity to safeguard certain nutrients (Jones et al., 1994; Souri & Hatamian, 30 2019). In addition, they increase the mobility of fairly mobile nutrients (i.e., 31 calcium, zinc, iron, etc.), thereby reducing and preventing plant stress (Popko et 32 al., 2018; Teixeira et al., 2020). 33 The synthesis of amino acids from inorganic nitrogen requires a high amount of 34 energy (Gioseffi et al., 2012; Kang & Kang, 2021). This process is particularly crucial 35 during stressful situations (drought, disease, sudden temperature variations, etc.) 36 where free amino acids are required up to 100 times more than usual (Matysik et 37 al., 2002). This decreases the formation of proteins, therefore negatively impacting 38 plant growth (Ali et al., 2019; Chen et al., 2019). For this reason, the application of 39 peptides and free amino acids may favor all plant processes in which proteins are 40 involved. In this sense, many authors have used amino acids and peptides to 41 promote plant growth. For example, Noroozlo et al. (2019) evaluated the 42 stimulating effect of glycine and glutamine on lettuce growth (Noroozlo et al., 2019). 43 On the other hand, Ghasemi et al. (2012) provided a solution to iron deficiency in 44 tomato plants through the synthesis of iron-amino acid chelates (Ghasemi et al., 45 4 2012). It is worth mentioning that although the goal of these amino acids is the 46 synthesis of proteins, these proteins cannot be exogenously incorporated into the 47 plant due to their size and molecular weight (Olbrycht et al., 2020). 48 The use of protein as a source of amino acids has not been fully explored due to 49 its complex structure that requires protease digestion to obtain those amino acids 50 (Adamczyk et al., 2010). However, this digestion is ensured due to the enzymes 51 secreted by the microbes and the plants themselves (Godlewski & Adamczyk, 52 2007). Therefore, proteins could be used as amino acid source. In addition, it has 53 the advantage of slow digestion, so it can provide amino acids for a longer time, 54 being efficient throughout all the crop growth without needing more nutrients 55 (Schapira et al., 2019). 56 Agri-food wastes can be a great alternative to providing these proteins due to 57 their low price and high protein content (Chojnacka et al., 2022). Nevertheless, 58 these residues have great amounts of water, so it is necessary to stabilize them 59 in order to use them in plant production without detrimental effects (Rajkhowa et 60 al., 2019). In this sense, the formation of protein-based matrices, through the 61 application of temperature and pressure, can be an easy and economical solution 62 to obtain a product with a stable high protein concentration (Jiménez-Rosado et al., 63 2021a, 2023). 64 In-vitro culture aims to grow plants in an aseptic-controlled environment, reducing 65 the effect of factors that affect plant growth (Kozai & Smith, 1995). However, the 66 use of media presents challenges in replicating natural conditions in the lab and 67 may not fully supply plans with the nutrients they previously obtained from the 68 complete system (Greenway et al., 2012). The use of the protein-based matrices 69 5 asset in this work can represent a source of amino acids for in vitro cultivated 70 plantlets. 71 Zinc is a microelement, required for different plant enzymatic activity, metabolic 72 pathways, protein synthesis, and carbohydrate metabolism, and is also involved 73 in the synthesis of tryptophan, cell division, maintenance of membrane structure 74 and photosynthesis (Marschner, 1995, 2012). The most characteristic symptoms of 75 zinc deficiency in fruit trees, rosetting and little leaf, are the results of strong 76 inhibition of internode elongation and leaf expansion (Swietlik, 2002). In addition, 77 zinc can also be involved in plant-pathogen interactions. In this sense, zinc is 78 used by plants to combat pathogens by poisoning them and controlling their 79 proliferation (Escudero et al., 2022). 80 The main objective of this work was the evaluation of the use of soy protein-based 81 matrices in in-vitro culture. We aimed to verify if the application of protein-based 82 matrices could improve the performance of in-vitro cultivated plants. Magliocco 83 Canino 'Ninno' shoots were selected for this work. It is a biotype present in the 84 Calabria Region (Italy) displaying some peculiarities with respect to the standard 85 Magliocco Canino genotype, as it produces with high-frequency small berries 86 without seeds, which lower vine productivity (Rombolà, p.c.), therefore in-vitro 87 study can contribute to understanding the possible causes of its peculiarities. 88 Thus, these shoots were studied using different media with or without zinc 89 deficiency to evaluate the influence of zinc and protein in the shoot growth. To 90 this purpose, the parameters (weight increase, number of stems, number of 91 leaves, stem, and internode length) of the shoots were monitored throughout the 92 study. In addition, an estimation of the biochemical profile of these shoots was 93 performed by FTIR. 94 6 2. MATERIALS & METHODS 95 Materials 96 In this study micropropagated grape (cv. Magliocco Canino “Ninno”) shoots were 97 used, in order to assess different substrates in a completely randomized 98 experimental design. The culture employed as an explant source had been 99 previously stabilized on the media tested in this experiment, further referred to as 100 ‘Standard modQL’ (Badmus et al., 2020). The explant donor plants were more than 101 one hundred years old. Under laminar flow, with a scalpel apical shoot portions 102 (37.7 ± 13.6 mg) were aseptically detached, including the first four visible, sub- 103 apical internodes (2.8 ± 1.8 mm long). 104 A salt formulation previously tested on Magliocco Canino Ninno [28] was selected 105 for this study (Table 1): ‘mQL’, i.e. a modified Quoirin and Lepoivre (Leblay et al., 106 1991), with the ammonium nitrate (NH4NO3) concentration further increased to 107 7.5 mM (Negri, unpublished). It was tested either as its standard or Zinc-deficient 108 compositions. For all the tested media the same organic base was used at pH 109 5.7: sucrose (30 g L-1), myoinositol (100 mg L-1), thiamine (1 mg L-1), nicotinic (1 110 mg L-1), pyridoxine (1 mg L-1), glycine (2 mg L-1), 6-Benzylaminopurine (BA 1 mg 111 L-1), indole butyric acid (IBA, 0.05 mg L-1) and agar (7.5 g L-1). All the reagents 112 used in the media were analytical grade. 113 Soy protein-based matrices were used to supply proteins to the medium. They 114 were processed with a similar protocol to those described by Jiménez-Rosado et 115 al. (Jiménez-Rosado et al., 2021b). Briefly, soy and glycerol (ratio 1:1) were 116 homogenized in a rotating mixer (Polylab QC, ThermoHaake, Germany) at 50 117 rpm for 10 min, and they were subsequently injected in a MiniJet Piston Injection 118 7 Molding System II (ThermoHaake, Germany); parameters used: 40 °C and 90 ºC 119 in the cylinder and mold, 600 bar of injection pressure for 20 s, and 300 bar of 120 holding pressure for 300 s to obtain the bioplastic matrices. This system was 121 immersed in 300 mL of ethanol for 24 h to remove the glycerol. The protein-based 122 matrices were obtained after a freeze-drying process (LyoQuest, Telstar, Spain), 123 with a biodegradability of 40 days, providing nitrogen, which can also be taken up 124 by plants. The amino acid composition of the soy protein used is shown in Table 125 2. It should be noted that the matrices that are the object of study in this work do 126 not contain any other component apart from the protein. 127 In-vitro culture treatments 128 Four different treatments were imposed: (i) standard medium (control), (ii) zinc- 129 deficient medium (i.e., the same as the standard medium, but lacking zinc sulfate 130 heptahydrate (ZnSO4·7H2O)), (iii) standard medium with protein-based matrix 131 and (iv) zinc-deficient medium with the protein-based matrix. Once autoclaved 132 (20 min at 121 °C, 1 bar) into Pyrex bottles, the substrates were transferred to 133 the laminar flow and aliquoted (10 ml) into previously sterilized, 55 ml glass tubes 134 (30 mm diameter). After medium gelation a shoot was positioned in the center of 135 each tube. A matrix fragment (70 mg approximately) was introduced beside the 136 sample. Finally, each tube was non-hermetically sealed with an aluminum cap 137 and fastened with a single layer of polyethylene film. The process was carried out 138 under sterile conditions in a laminar flow hood. 139 Shoots were kept for 35 days in a growth chamber with 16 hours of light (intensity 140 of 35 µcd) at a temperature of 26 ± 2 ºC. Figure 1 shows the growth of the shoots 141 at different cultivation days. 142 8 Assessment of morphological and physiochemical plant traits 143 The different samples were hermetically preserved during their cultivation to 144 avoid contamination of the environment. After 35 days, the plants were first 145 dissected with a scalpel to facilitate their analysis. Later, their weight increase 146 (final fresh weight – initial fresh weight), the number of stems and leaves, as well 147 as the length of their stems and internodes were determined. In addition, the 148 morphology of the leaves was observed. 149 Tissue zinc concentration was also determined in the different samples. For this, 150 inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was 151 carried out. The plants were subjected to a previous digestion with 7:1 ratio of 152 HNO3:H2O2 to obtain an aqueous solution which contain the zinc present in the 153 samples. Then, the aqueous solutions were analyzed in an ICP SpectroBlue TI 154 (Spectro, Germany) to obtain the zinc concentration. 155 Finally, the leaves of the different treated shoots were also analyzed by Fourier 156 Transform Infrared spectroscopy (FITR) to evaluate the biochemical profile of 157 each shoot (Badmus et al., 2020). A MIR-ATR-XPM spectroscope (Bruker, USA) 158 was used to obtain the absorbance profile of each sample between 4000 and 400 159 cm-1, with a resolution of 4 cm-1. To obtain the profile, an average of 100 scans 160 was performed. 161 Statistical analysis 162 At least twelve replicates (twelve shoots with the same conditions in two different 163 experiment batches, twenty-four shoots in total) were performed for each analysis 164 and system. All the data were reported as average values. An analysis of 165 variance was performed. Tukey’s post hoc test with a confidence level of 95% (p 166 9 <0.05) was performed using the SPSS 18 statistical package (Excel, Microsoft, 167 USA) to evaluate the significant differences, which were reported with different 168 letters. 169 170 3. RESULTS & DISCUSSION 171 Table 3 shows the parameters obtained for the shoots submitted to the different 172 treatments i, ii, iii, and iv. The inclusion of soy protein-based matrices increased 173 weight and proliferation (higher number of stems and leaves). This behavior may 174 suggest that the matrices improve plant formation due to the stimulation 175 generated by the amino acids present in the matrix (Calvo et al., 2014; De Pascale 176 et al., 2018; Jiménez-Rosado et al., 2023). Soy protein presents high concentrations 177 of glutamic acid (21.7%), which promotes plant development (Iqbal et al., 2021); 178 alanine (10.4%), which increases metabolic activity (de Sousa & Sodek, 2003); 179 leucine (8.6%) and aspartic acid (8.2%), which are important in the growth, 180 development, and flowering of plants (Paleckiene et al., 2007). The proteins break 181 down into amino acids during biodegradation (Shlush & Davidovich-Pinhas, 2022). 182 These amino acids are an extra source of nitrogen and building blocks for 183 enzymes, allowing plants to have more energy to develop (Basha & El-Aila, 2015). 184 Zinc deficiency produces a slight decrease in weight (43% approximately) 185 obtained from the plants (Table 3, i (media without matrix) vs. ii (zinc deficiency 186 media without matrix)). In addition, zinc deficiency results into a lower tissue Zn 187 concentration and increases the percentage of deformed leaves. 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The letters denote the treatment in which the shoots were 452 grown: (i) standard medium (control), (ii) zinc-deficient medium, (iii) standard 453 medium with protein-based matrix and (iv) zinc-deficient medium with the protein- 454 based matrix. 455 18 Figure 2: Visual appearance of dissected Magliocco Canino plants cultivated 456 with different treatments for 35 days: (i) standard medium (control), (ii) zinc- 457 deficient medium, (iii) standard medium with protein-based matrix and (iv) zinc- 458 deficient medium with the protein-based matrix in modified Quoirin and Lepoivre 459 medium (mQL). 460 Figure 3: FTIR profile of the Magliocco Canino plants (leaves) cultivated with 461 different treatments: (i) standard medium (control), (ii) zinc-deficient medium, (iii) 462 standard medium with protein-based matrix and (iv) zinc-deficient medium with 463 the protein-based matrix. 464 19 TABLES 465 Table 1: Composition of macro and microelements in the standard media: Quoirin 466 and Lepoivre modified medium (mQL). 467 Salt Standard mQL (µM) CaCl2·2H2O - Ca(NO3)2·4H2O 4801.059 KH2PO4 2324.914 KNO3 17804.154 MgSO4·7H2O 1443.367 NH4NO3 7495.971 CoCl2·6H2O 0.105 CuSO4·5H2O 0.100 FeNaEDTA 465.559 H3BO3 100.275 KI 0.482 MnSO4·H2O 4.497 20 Na2MoO4·2H2O 1.033 ZnSO4·7H2O 29.907 468 Table 2: Amino acid composition of soy protein-based matrices. 469 Amino acids Concentration µmol L-1 % Amino acid in the protein Aspartic acid(Asp) 361.4 8.2 Threonine (Thr) 109.6 2.5 Serine (Ser) 46.2 1.0 Glutamic acid (Glu) 958.8 21.7 Glycine (Gly) 326.3 7.4 Alanine (Ala) 460.5 10.4 Cisteine (Cys) 73.7 1.7 Valine (Val) 261.3 5.9 Methionine (Met) 38.2 0.9 Isoleucine (Ile) 193.6 4.4 Leucine (Leu) 379.2 8.6 Tryptophan (Trp) 1.0 <0.1 Tirosine (Tyr) 131.3 3.0 Pheenilalamine (Phe) 186.5 4.2 Histidine (His) 59.8 1.4 Lysine (Lys) 248.1 5.6 Proline (Pro) 343.5 7.8 Arginine (Arg) 234.3 5.3 470 21 Table 3: Parameters (weight increase, number of stems, number of leaves, stem 471 and internode length and tissue zinc concentration) obtained for the Magliocco 472 Canino plants subjected to the different treatments ((i) standard medium (control), 473 (ii) zinc-deficient medium, (iii) standard medium with protein-based matrix and (iv) 474 zinc-deficient medium with the protein-based matrix). Different letters in the same 475 column mean significant differences (p <0.05). 476 Treatment Weight increase (mg) Number of stems Number of leaves Deformed leaves (%) Stem Length (mm) Internode Length (mm) Tissue Zn concentration (ppm) i 810.3 ± 83.4c 6 ± 1a 40 ± 7a 22 ± 1b 20.5 ± 1.5ab 2.9 ± 0.9a 25.2 ± 0.1b ii 350.5 ± 23.6a 6 ± 1a 39 ± 4a 92 ± 1c 19.0 ± 2.0a 2.1 ± 0.7a 8.1 ± 0.4a iii 1250.97 ± 139.0d 9 ± 2b 70 ± 16b 14 ± 1a 15.0 ± 8.9ab 3.4 ± 2.1a 29.3 ± 0.4c iv 498.8 ± 79.4b 7 ± 1ab 41 ± 10a 15 ± 1a 23.5 ± 1.2b 2.7 ± 1.1a 25.4 ± 0.2b 477