2019 Volume 88 Issue 2 Pages 263-269
Internally brown (IB) tomato fruit is a physiological disorder in which the inside of the fruit turns brown or black. The mechanisms underlying the development of IB are not well understood. In this study, we examined the incidence of IB using hydroponics, and investigated the anatomical features and the ratio of dimeric rhamnogalacturonan II-borate (dRG-II-B) to total rhamnogalacturonan II (RG-II) (boron cross-linking ratio) in cell walls that decreased with boron deficiency. IB fruit developed when the growth of the stem and leaves were normal and the micro-element concentrations were low. The IB region was observed to be brown inside the fruit at an early stage, and this changed to black in the mature fruit. It was detected around the pericarp, locular gel, placenta and columella in the tomato fruit. An abnormality was observed around the vascular bundle in IB fruit. The interior of the cells near the vascular bundle was changed to a substance with a high electron density, and a disintegrated image was observed. Few Ca precipitates were observed on plasma membranes or cell walls in the collapsed cells by an antimonite precipitation method. It is suggested that Ca deficiency appears to be related to the cell collapse in IB. There was no significant difference in the boron cross-linking ratio between the IB and normal fruit. This suggested that IB was not related to B deficiency directly. We concluded that IB was caused by necrosis of cells around the inner vascular bundle due to Ca deficiency.
Internally brown (IB) tomato fruit is a physiological disorder in which the inside of the fruit turns brown or black (Fig. 1). At production sites in Japan, other names for IB in tomatoes are heart rot (Iwamoto et al., 1993), heart-rot fruit (Ishizuka et al., 2000), browning of the tomato locules (Terabayashi et al., 2008), internally brown tomato (Norimitsu et al., 2017), and blackheart. IB (which cannot be identified as a symptom from the outside of the fruit) can cause a great deal of reputational and economic damage to farms and farm operators because it often leads to complaints from consumers (Fig. 1). It is therefore important to decrease the incidence of IB in tomato production.
An internally brown (IB) tomato fruit. A: outside. B–D: inside of mature fruit. E–H: inside of immature fruit. Arrow: IB region.
In Shizuoka prefecture, the incidence of IB tomato fruit grown hydroponically was observed in the autumn season. In hydroponic tomatoes, a high concentration of ammonium nitrogen and a low concentration of calcium (Ca) induced IB (Norimitsu et al., 2017). Defoliation treatment of tomatoes reduces the incidence of IB (Miyama and Terabayashi, 2013) and that of blossom-end rot (BER) (Sato et al., 2004). Ishizuka et al. (2000) suggested that IB was related to the concentration of Ca and boron (B) in the tomatoes. However, the mechanisms underlying the development of IB are not well understood. B deficiency and Ca deficiency are thought to be involved in the development of IB, but the details are unknown, and the causative conditions are not well understood either. Therefore, in this study, we investigated the conditions in which IB is likely to occur, and analyzed the characteristics of the IB fruit by an anatomical method and determination of B deficiency using the ratio of dimeric rhamnogalacturonan II-borate (dRG-II-B) to total rhamnogalacturonan II (RG-II) (boron cross-linking ratio) in the cell walls.
When the symptoms of IB become serious, blackening necrosis appears on the fruit surface. If necrosis occurs at the end of the fruit, it is difficult to distinguish IB from BER. There have been many studies of BER (de Freitas et al., 2012; Ho and White, 2005; Saure, 2014) and it was reported that a local deficiency in Ca induces cell necrosis. In recent studies about BER, it was suggested that Ca2+ transport was an essential factor in the lower incidence of BER using a tomato introgression line (Ikeda et al., 2017); the incidence of BER under salinity was reduced by organo-mineral fertilizer (Kataoka et al., 2017); the cultivar difference in the susceptibility to BER was likely explained by the difference in the growth rate of young fruit affecting water-soluble Ca in the distal part of tomato fruit (Vinh et al., 2018); the involvement of reactive oxygen species as a major protagonist in BER appearance; and BER-resistant cultivars showed a larger increase in their ROS scavenging capacity (Rached et al., 2018). We investigated the ultrastructural features of BER, and observed that there were no visible Ca precipitates on the traces of the plasma membrane near the cell wall of the collapsed cells in BER fruit (Suzuki et al., 2003). We examined the anatomical changes that may be associated with the development of IB and compared the changes with those observed in the development of BER in the present study.
Matsunaga and Ishii (2006) explored the boron cross-linking ratio in the cell walls of pumpkins grown hydroponically under various low-B conditions. Their results indicated that the degree of B shortage in plant tissues can be predicted based on the boron cross-linking ratio in the cell walls. In the present study, we determined the boron cross-linking ratio in the cell walls of IB fruit to clarify whether or not IB is related to B deficiency.
Tomato (Solanum lycopersicum, ‘CF Momotaro York’; Takii & Co., Ltd., Kyoto, Japan) seeds were sown in seed trays on March 9, 2015, and grown in a nursery chamber (Nae-Terrace; Mitsubishi Chemical Agri Dream Co., Ltd., Tokyo, Japan). The seedlings were transplanted into an extremely low-volume pot system (Zhang et al., 2015) filled with 250 mL of granular rockwool on April 2 in an experimental plastic greenhouse (7.2 × 15 m) at Shizuoka University in Japan (34°57'55.46'' N, 138°25'54.12'' E). The irrigation frequency was controlled using an environmental controller (DM-one; Double M Ltd., Shizuoka, Japan) based on the accumulated solar radiation. As a result, on completely sunny days approx. 50–70 mL of irrigation was supplied approx. 20–25 times·day−1.
Ten plants were kept in each container and irrigated with 35 L of recirculated nutrient solution. The solution in each container was refilled with original solution to compensate for crop water consumption every day and renewed weekly. Plants were trained vertically with a single stem, pinched at the first upper leaf above the fifth truss, and elongation was stopped. Cultivation was ended on June 25 when harvesting of the second truss fruit was finished. The average air temperature was 21.2°C during greenhouse cultivation.
The basal composition of the Enshi formula nutrient solution was (NO3-N: 8 me·L−1, NH4-N: 0.7 me·L−1, PO4-P: 2 me·L−1, K: 4 me·L−1, Mg: 2 me·L−1, Ca: 4 me·L−1, Fe: 3 ppm, Mn: 0.5 ppm, Zn: 0.05 ppm, Cu: 0.02 ppm, B: 0.5 ppm, and Mo: 0.05 ppm) as a control solution (CS) and a 1/2 micro element solution was (NO3-N: 8 me·L−1, NH4-N: 0.7 me·L−1, PO4-P: 2 me·L−1, K: 4 me·L−1, Mg: 2 me·L−1, Ca: 4 me·L−1, Fe: 1.5 ppm, Mn: 0.25 ppm, Zn: 0.025 ppm, Cu: 0.01 ppm, B: 0.25 ppm, and Mo: 0.025 ppm) (1/2 ME). Micro-elements were also present in the well water, in ppm: 0.12 Fe, 0.1 Mn, 0.06 B, 0.02 Cu. The solution was changed from April 2 and continued until June 25. Each solution with three replicates was adopted, and each replicate contained 10 plants. Thus, 60 plants were used in this study.
We assessed the total fruit yield, fruit number, and fruit weight over the course of the ripening stage for the first and second trusses. To detect IB fruit, all of the tomato fruit were cut and the inside was observed.
Fruit fragments including the brown region of an IB fruit and the same region of a normal fruit in 1/2 ME plants for transmission electron microscopy (TEM) observation on May 26, and for Ca precipitates on June 10, were sampled. Three fruit were used for each. They were fixed in a mixture of 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 6 h at room temperature. After washing with rinsing buffer (0.2 M phosphate buffer, pH 7.2), the samples were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 2 h at 4°C. They were then dehydrated in a graded alcohol series and embedded in epoxy resin.
For light microscopy evaluation, we prepared 2 μm thick sections using glass knives on an ultra-microtome (EM US7; Leica Microsystems Ltd., Wetzlar, Germany) and stained them with toluidine blue O. Three samples from normal fruit and three samples from IB fruit were also observed after TEM observation by light microscopy.
For the TEM evaluation, ultrathin sections were prepared with diamond knives on the microtome. The sections on grids were stained with TI Blue (Nisshin EM Co. Ltd., Tokyo, Japan) for 20 min followed by a lead electron staining solution (Katayama Chemical Industries Co., Ltd., Osaka, Japan) for 5 min. The sections were observed using a JEM-1400Plus electron microscope (JEOL Ltd., Tokyo, Japan).
Ca precipitates were observed as electron-dense deposits by an antimonite precipitation method that we used in a previous study (Suzuki et al., 2003).
Exp. 2. Investigation of the maturation period and determination of the boron cross-linking ratio in the cell walls of IB fruitThe tomato seeds were sown on July 26, 2016 and transplanted on September 2. The seedling, culture methods and conditions were the same as those used in Exp 1. The Enshi formula nutrient solution was the same as the control solution in Exp 1, and a double concentration of the control solution was used. Cultivation was ended on December 20. The average air temperature was 21.2°C during greenhouse cultivation. We measured the fruit number and fruit weight from the first to third trusses. The flowering day and harvesting date were examined. The harvested fruit were cut and examined, and their status as normal or IB, as well as their °Brix were determined. Normal young fruit and IB fruit with diameters of about 7 cm were sampled to determine the boron cross-linking ratio on October 20 and November 25 in 2016. Fresh 1 g tissue pieces were cut from the normal region and brown region of IB fruit and from the normal region of normal fruit, and freeze-dried for analysis. Three fruits were used for each experiment.
We determined the boron cross-linking ratio in the cell walls as described (Matsunaga and Chishaki, 2014; Matsunaga and Ishii, 2006). Freeze-dried material (20 mg of fruit) was placed in a 1.5 mL tube. The tube was suspended in 800 μL of aqueous 80% (v/v) ethanol, mixed in a mixer (MicroMixer E-36; TAITEC CORPORATION, Koshigaya, Japan), shaken for 20 min and then centrifuged (14000 rpm for 10 min at room temperature; KUBOTA1910; KUBOTA CORPORATION, Tokyo, Japan). The residue was washed with 80% (v/v) ethanol, 99.5% (v/v) ethanol, acetone and deionized water and used as a cell wall sample. The cell wall samples (5–8 mg) in 1.5 mL tubes were suspended with 1000 μL of 0.1 M sodium hydroxide at 4°C for 4 h. After treatment, 70 μl of 10% (v/v) acetic acid solution was added, it was centrifuged and the supernatant discarded. Next, 800 μL of acetate buffer which was 100:7 mixture of 0.1 M sodium hydroxide and 10% acetic acid solution was added, suspended, mixed with 4.5 μl of endo-polygalacturonase (EPG), and reacted in a refrigerator overnight. After the treatment, the supernatant obtained by centrifugation was taken into a syringe (Terumo Syringe 1 ml; Terumo Corporation, Tokyo, Japan), filtered (Minisart Syringe Filter 0.2 μm; Sartorius AG, Göttingen, Germany), and transferred to a new 1.5 mL tube.
The supernatants were subjected to HPLC equipped with a refractive index detector (RID-6A; Shimadzu Corporation, Kyoto, Japan). Size-exclusion chromatography was conducted using a Guardpack Diol-120 guard column of 8 × 30 mm (YMC Co. Ltd., Kyoto, Japan) and a Diol-120 column of 8 × 300 mm (YMC Co. Ltd., Kyoto, Japan). The HPLC conditions were as follows: eluent, 0.2 M NaCl; flow rate, 1.0 mL·min−1; and injection volume, 25 μL. The boron cross-linking ratio was calculated from the peak area of dRG-II-B and monomeric RG-II (mRG-II).
The growth and analysis data were analyzed by t-test and ANOVA using JMP 11.0 software (SAS Institute Inc., Cary, NC, USA).
There was no significant difference in terms of growth, leaf color SPAD value or solution consumption between the control and 1/2 ME plants (data not shown). We investigated the fruit of the first and second trusses. The number of fruit in each treatment was lower in the second truss than in the first truss. There was no significant difference in fruit number, fresh weight or fruit yield between the control and 1/2 ME (Table 1). About 13% of the fruit in the 1/2 ME developed IB. The control treatment also showed IB. The incidence of IB in the 1/2 ME was higher than the control (Table 1).
Fruit number, weight, °Brix and incidence of IB in the control and 1/2 ME in Experiment 1.
IB could not be distinguished from outside (Fig. 1A), so it was necessary to cut the fruit, check inside and determine IB. The IB region changed to black in the mature fruit (Fig. 1B), and IB was observed as brown flesh at the early stage inside fruit (Fig. 1E–H). It was detected around the pericarp, locular gel, placenta (Fig. 1E, G, H) and columella (Fig. 1F) in the tomato fruit. There was a case in which the locular gel did not develop (Fig. 1C, G), and IB fruit was detected as a hollow fruit from the outside (Fig. 1D). The epidermis was changed in some IB fruit (Fig. 1D, H). In this case, the IB region spread from inside to outside. Inside of about half the fruit was black in fruit with severe symptoms (Fig. 1D).
We conducted a histological examination of the peripheral region of the slightly blackened pericarp of IB fruit. An abnormality was observed around the vascular bundle. The xylems that had a secondary cell wall in the vascular bundle of IB fruit were not clearly observed compared to the vascular bundles of normal fruit, and other cells and cells surrounding the vascular bundle in IB fruit were different compared to those in the normal fruit (Fig. 2A, B). The cells surrounding the vascular bundle in IB fruit were destroyed (Fig. 2C).
Light micrographs of the pericarp vascular bundle in normal fruit (A), and IB tomato fruit (B, C). Bar: 100 μm.
TEM examination revealed that the interiors of the cells near the vascular bundle were changed to a substance with a high electron density, and a disintegrated image was observed. The inside of the collapsed cells was stained homogeneously with electron-dense materials (Fig. 3A, D) compared to the normal fruit (Fig. 3B). Ca precipitates were observed as electron-dense deposits by an antimonite precipitation method (Fig. 3B–D). Some Ca precipitates were localized in the cytosol, nucleus, plastids, and vacuoles, and were mainly localized in the plasma membrane and cell wall in the vascular bundle in the normal fruit (Fig. 3B) and the normal regions of the IB fruit (Fig. 3C). Marked amounts of Ca precipitates were observed on the secondary cell walls of the xylems (Fig. 3B, C). High amounts of Ca precipitates were observed on the plasma membranes and cell walls near collapsed cells (Fig. 3D). In contrast, few Ca precipitates were observed on plasma membranes and cell walls in the collapsed cells (Fig. 3D).
TEM micrographs of the vascular bundle in the pericarp from a normal fruit (B) and IB fruit (A, C, D). An antimonate precipitation method was used in panels B–D. Asterisks: Collapsed cells in an IB area. Arrows: calcium precipitates. X: xylem. A and C, Bar: 5 μm. B and D, Bar: 2 μm.
There was no significant difference in tomato plant height, leaf weight, stem weight, or total fresh weight of fruit in the control and double concentration solution (data not shown). There was no significant difference in fruit number (Table 2). The fruit weight was not significantly different in the first truss, that of the double concentration solution was heavier than the control in the second truss, but that of the control was heavier than the double concentration solution in the third truss. The °Brix of tomato fruit with the double concentration solution were higher than the control in all trusses. There was no significant difference in the incidence of IB between them (Table 2).
Fruit number, weight, °Brix and incidence of IB in the control and double concentration solution in Experiment 2.
Among the fruit that grew in the greenhouse, the number of days from flowering until harvesting in the normal fruit was greater than that in the IB fruit (Table 3). There was no difference in fresh fruit weight between the normal and IB fruit. The °Brix was significantly higher in the IB fruit compared to the normal fruit (Table 3).
Days from flowering until harvesting, fruit fresh weight, °Brix, and boron cross-linking ratio of normal and IB fruits in Experiment 2.
We investigated the boron cross-linking ratio in the normal region and brown region of IB fruit and in the normal region of normal fruit. We detected only the peak of dRG-II-B, but not the peak of mRG-II in the normal region and brown region of IB fruit and in the normal region of normal fruit. There was no significant difference in the boron cross-linking ratio between the IB and normal fruit and the ratio for both was 100% (Table 3).
In our cultivation study, incidence of IB in the 1/2 ME occurred more frequently than in the control. The development of the shoots was almost the same between the 1/2 ME and control plants. It therefore seems that the incidence of IB increases when shoots grow normally and the levels of micro-elements in the nutrient solution are low.
Our morphological examination revealed that the cells near the vascular bundle were locally disintegrated. The inside of the collapsed cells was similar to the images we obtained of BER epidermal cells (Suzuki et al., 2000). This cell collapse was not due to features of apoptosis, such as swelling of mitochondria or necrosis of the nucleus, but rather was due to necrosis of the whole cell due to changes in the high electron density inside the cell. We therefore speculate that the cells around the vascular bundle developed necrosis locally, without being supplied with nutrients around them, and the inside of the cells blackened.
There were low levels of Ca precipitates on the plasma membranes and cell walls in collapsed cells as shown by an antimonite precipitation method. Similarly, there were no visible Ca precipitates on the traces of plasma membranes near the cell walls of the collapsed cells in BER fruit. The amount of Ca precipitates on plasma membranes near collapsed cells was smaller than that in the cells of the normal fruit and that of normal regions of BER fruit, but this phenomenon was not observed in IB fruit. Ca deficiency appears to be related to the cell collapse in IB, but this is somewhat different from BER. It was reported that IB occurred due to a Ca deficiency (Norimitsu et al., 2017). Our anatomical research also showed that IB was due to Ca deficiency, the same as BER. BER is caused by the effects of abiotic stress, resulting in an increase in reactive oxygen species, high oxidative stress and finally cell death (Saure, 2014). Further study is necessary to clarify what causes cell death around the vascular bundle in IB fruit.
The boron cross-linking ratio has been proposed as a method to determine whether B is deficient. In this study we investigated the boron cross-linking rate in IB fruit, and detected only the peak of dRG-II-B, not the peak of mRG-II in normal and IB fruit. This indicates that the cell walls of IB fruit were cross-linked with B. Both the IB fruit and the control fruit had a 100% boron cross-linking ratio, so it seems that B deficiency does not occur in IB fruit. This result indicates that IB is not directly caused by B deficiency.
Krug et al. (2009) suggested that the symptoms caused by B deficiency and Ca deficiency occurred in the same young organ, in which the initial symptoms were similar, but thereafter the symptoms clearly differed. B deficiency slightly increased the total Ca uptake by the plant and inhibited the Ca translocation to the upper leaves in the tomato (Yamauchi et al., 1986). We found that B deficiency was not a direct cause of IB in this study. When we cultivated tomatoes under B-deficient conditions, we could observe other physiological fruit disorders (data not shown). The difference in physiological disorders caused by B deficiency requires further study in tomatoes in the future.
Our comparison of the maturation timing between IB fruit and normal fruit revealed that the IB fruit matured approx. 10% faster and their °Brix tended to be higher. We suspect that this was due to interior necrosis, and that ethylene was generated by the disorder, leading to maturation being accelerated. When tomato fruit undergo stress, the °Brix may rise, and it seems that the °Brix rises even in IB fruit. It is not possible to confirm the incidence of IB as these fruit apparently matured faster than the surrounding fruit.
In light of the above results, we speculate that IB develops when the growth of the stem and leaves is normal and the amount of micro-elements is low. IB seems to be caused by necrosis of cells around the inner vascular bundle. In order to reduce the incidence of IB, it appears important to balance the development of the shoots and fruit, as in the suppression of the BER occurrence.
We thank Dr. Matsunaga and Dr. Ohwaki in NARO for advice on the analysis technique.
