The objectives of this study were to evaluate the effect of Ca2+ binding to the cell wall 14 on fruit susceptibility to BER

FREITAS, S. T. de

HANDA, A. K.

MITCHAM, E. J.

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1 ROLE OF CELL WALL ON TOMATO FRUIT SUSCEPTIBILITY TO CALCIUM 1 DEFICIENCY DISORDER 2 Sergio Tonetto de Freitas1, Avtar K. Handa2, Elizabeth J. Mitcham 3 3 1 Embrapa Semiárido, Petrolina, PE, 56302-970, Brazil. E-mail: sergio.freitas@embrapa.br; 4 2 Purdue University, West Lafayette, IN 47907 USA. E-mail: ahanda@purdue.edu; 5  3 University of California, Davis, CA 95616 USA. Email: ejmitcham@ucdavis.edu 6 INTRODUCTION 7 Blossom-end rot (BER) is believed to be a Ca2+ deficiency disorder in tomato fruit (White & 8 Broadley, 2003). However, no threshold of fruit Ca2+ concentration has been used to accurately 9 predict BER (Saure, 2001). This suggests that BER may also be triggered by abnormal cellular Ca2+ 10 partitioning and distribution that leads to a cellularly localized Ca2+ deficiency. Since the cell wall 11 represents 60 to 75% of the total tissue Ca2+ content, it plays an important role in cellular Ca2+ 12 partitioning and distribution (De Freitas et al., 2010) and possibly in fruit susceptibility to BER 13 development. The objectives of this study were to evaluate the effect of Ca2+ binding to the cell wall 14 on fruit susceptibility to BER. 15 MATERIALS AND METHODS 16 Wild type and PME-silenced tomato plants (Solanum lycopersicum) cultivar Rutgers were grown in 17 9.5 L pots containing 0.3 kg of perlite as substrate in a greenhouse environment. The PME-silenced 18 plants (line 3781^) contain two copies of a PME type I antisense nucleotide sequence (GenBank: 19 U70676.1) under the control of the cauliflower mosaic virus 35S promoter. Both wild type and 20 PME-silenced plants were irrigated every day with a nutrient solution containing N (102 mg L-1), P 21 (26 mg L-1), K (124 mg L-1), Ca2+ (90 mg L-1), Mg2+ (24 mg L-1), S (16 mg L-1), Fe (1.6 mg L-1), 22 Mn (0.27 mg L-1), Cu (0.16 mg L-1), Zn (0.12 mg L-1), B (0.26 mg L-1), and Mo (0.016 mg L-1). 23 After tagging and manually pollinating the flowers at full bloom, the plants were irrigated everyday 24 with the same nutrient solution, but without Ca2+. There were four replications with four plants each 25 for wild type and PME-silenced plants. Fruit from the first and second clusters on each plant were 26 harvested and analyzed at 15, 30, and 45 DAP. All tissue analyses were accomplished in fruit 27 without visible BER symptoms using blossom end tissue. Fruit were analyzed for BER incidence, 28 electrolyte leakage of pericarp tissue, PME expression, Ca2+ concentrations in pericarp tissue and 29 soluble and insoluble pectins. Statistical differences between wild type and PME-silence tomato 30 samples were calculated with one-tailed unpaired Student’s t-test. P-values <0.05 were considered 31 significant. Data are presented as means ± standard error (SE). 32  33  34 2 RESULTS AND DISCUSSION 35 Throughout fruit growth and development, PME-36 silenced fruit had a lower BER incidence and 37 electrolyte leakage of pericarp tissue than wild type 38 fruit tissue (Figure 1A, 1B). While more than 80 % of 39 WT fruit exhibited BER, only about 30% of PME-40 silenced fruits were affected by this disorder by the 41 time fruit reached full size (45 DAP). Higher 42 electrolyte leakage is associated to higher tissue 43 susceptibility to BER (Saure, 2001). 44 The expression of the six PME genes PMEU1, 45 LOC544090, LOC544289, Les.9028, Les.10790, and 46 Les.10560 in the wild type pericarp tissue increased 62, 47 491, 220, 77, 40, and 57 fold, respectively, from 15 48 DAP to 45 DAP (Figure 2). PME-silenced fruit also 49 showed increased expression of all six PME genes 50 during growth and development. However, expression 51 of PMEU1, LOC544090, LOC544289, Les.9028, 52 Les.10790, and Les.10560 were 48, 474, 214, 63, 18, 53 and 42 fold lower in PME-silenced fruit, respectively, 54 than in wild type fruit at 45 DAP (Figure 2). 55  Figure 1. Blossom-end rot incidence (a) and electrolyte leakage of pericarp tissue (b) of wild type and PME-silenced tomato fruit cultivar Rutgers. Different letters on each day represent statistical difference between wild type and PME-silenced samples (P-value < 0.05). Data are means ± SE.  Figure 2. Changes in PME expression during wild type and PME-silenced fruit growth and development. Different letters on each day represent statistical difference between wild type and PME-silenced samples (P-value < 0.05). Data are means ± SE. 3 The total concentrations of Ca2+ in the 56 pericarp tissues of wild type and PME-57 silenced fruit decreased from 15 to 45 DAP, 58 but the values for each fruit type were similar 59 at each developmental time point (Figure 60 3A). The PME-silenced fruit pericarp showed 61 a slightly lower Ca2+ concentration in the 62 water soluble pectin fraction than wild type 63 fruit at 45 DAP (Figure 3B). The Ca2+ 64 concentrations in the water insoluble pectin 65 were similar in wild type and PME-silenced 66 fruit pericarp at 15 DAP and increased 67 steadily from 30 to 45 DAP in wild type fruit 68 while the Ca2+ concentration remained 69 unchanged in the PME-silenced fruit (Figure 70 3C). These results show that higher cell wall 71 Ca2+ binding capacity due to higher PME 72 expression increase fruit susceptibility to 73 BER. Accordingly, plants that have more 74 binding sites for Ca2+ in the cell wall are 75 known to require higher levels of Ca2+ for 76 normal growth and development. For 77 instance, dicotyledeonous plants require more 78 Ca2+ in their tissues than monocotyledonous 79 plants, a phenomenon attributed to the larger 80 cation exchange capacity of their cell walls 81 (Kirkby & Pilbeam, 1984). Therefore, 82 susceptibility of tomato genotypes to BER 83 development may be determined by the 84 capacity of their cell walls to bind Ca2+ 85 during rapid cell expansion and vacuolation 86 under conditions in which fruit Ca2+ uptake is 87 restricted.  88  89  Figure 3. Calcium concentration in pericarp tissue (a), as well as in water soluble pectin (b) and water insoluble pectin (c) fractions extracted from pericarp tissue of wild type and PME-silenced tomato fruit cultivar Rutgers. gdw = grams of dry weight. sol. = soluble, insol. = insoluble. Different letters on each day represent statistical difference between wild type and PME-silenced samples (P-value < 0.05). Data are means ± SE. 4 CONCLUSIONS 90 Suppressing expression of PMEs in tomato fruit reduces the amount of Ca2+ bound to the cell wall. 91 Decreasing Ca2+ binding to the cell wall decreases fruit tissue susceptibility to BER. 92 ACKNOWLEDGEMENTS 93 We acknowledge funding from CAPES Foundation, a federal agency under the Ministry of 94 Education of Brazil for supporting this study. 95  96  97  98  99  100 REFERENCES 101 DE FREITAS, S.T.; AMARANTE, C.V.T. DO; LABAVITCH, J.M.; MITCHAM, E.J. Cellular 102 approach to understand bitter pit development in apple fruit. Postharvest Biology and Technology, 103 v.57, p.6-13, 2010. 104 KIRBY, E.A.; PILBEAM, D.J. Calcium as a plant nutrient. Plant Cell and Environment, v.7, 105 p.397–405, 1984. 106 SAURE, M.C. Blossom-end rot of tomato (Lycopersicon esculentum Mill.) – a calcium or a stress-107 related disorder? Scientia Horticulturae, v.90, p.193-208, 2001. 108 WHITE, P.J.; BROADLEY, M.R. Calcium in plants. Annals of Botany, v.92, p.487-511, 2003. 109 

Role of cell wall on tomato fruit susceptibility to calcium deficiency disorder

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