Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Chelator used in pectin extraction triggers ethylene production by tomato fruit

In our search for an endogenous ethylene trigger from tomato ( Lycopersicon esculentum Mill. ev. Rutgers) fruit cell wall alkaline soluble pectin (ASP), we purified an active component using DEAE-Sepharose chromatography followed by elution on Bio-Gel P-100 or Superose 12. The purified active fraction produced a single band on silver-stained SDS-PAGE of approximately M_r 20000. Using two-dimensional proton-proton and proton-carbon correlation spectroscopy, we identified the repeating sub-unit as trans-1,2-diamino-cyclohexane- N,N,N',N'-tetraacetic acid (CDTA), a chelator used to extract ASP. Although the ASP undergoes extensive dialysis during its extraction which should remove CDTA, the CDTA apparently forms a large molecular weight polymer which does not diffuse out of the dialysis tubing. Infiltration of commercially prepared CDTA into mature green tomato fruit stimulated ethylene production. The ethylene stimulatory effect of CDTA was not affected by the presence of equimolar amounts of CaCl₂, or nmol g^-1 amounts of the calcium channel blockers, nifedipine or verapamil. EDTA, EGTA, and diethylenetriaminepentaacetic acid, other divalent cation chelators, also stimulated ethylene production when they were infiltrated into tomato fruit. Neither the purified material nor commercial CDTA stimulated ethylene production when they were infiltrated into leaf tissue.     

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato β-galactosidase.     

Changes in free polyamine levels induced by salt stress in leaves of cultivated and wild tomato species

The effects of N_aCl on endogenous free levels of the poluamines putrescine, spermi dine and spermine, and the relationships between polyamines, K⁺ levels and Na⁺ accumulation were determined in leaves of the cultivated tomato ( Lycopersicon esculentum Mill.) and its wild, salt-tolerant relative L. pennellii (Correll) D' Arcy at different exposure times during a 32-day period. Both stress treatments (100 and 200 m M NaCl) decreased the levels of putrescine and spermidine, although to a different degree for the cultivated and wild tomato species. The spermine levels did not decrease with salinity in L. pennellii over the salinization period, whereas they decreased in L. esculentum , except at the first application of the 100m M NaCl treatment. In both species, the changes induced by salinity in total polyamines and K⁺ were very similar, with the accumulation of Na⁺ in the leaf being concomitant with a decrease in both total polyamines and K⁺. This suggests that the main role of the polyamines in the leaf tissues. In this sense, a direct relationship between total polyamines and K⁺, and inverse relationship between polyamines and Na⁺ and between K⁺ and Na⁺ were found for both species. In the short term (up to 4 days) a peculiar physiological behavior was found in L. pennellii , as the total polyamine and K⁺ levels decreased at 100 m M but not at 200 m M NaCl, while after this time the latter plants had values lower than those of the 100 m M NaCl-treated plants at day 11.     

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall-bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 m M NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five m M CaCl₂ did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca²⁺ levels and ionic strength of the apoplast.     

Biochemical changes associated with the ripening of hot pepper fruit

Hot pepper ( Capsicum annuum L. cv. Chooraehong) fruit underwent a respiratory climacteric during ripening. However, the rate of ethylene production was low, reaching a maximum of approximately 0.7 μl kg⁻¹ h⁻¹ at the climacteric peak when the surface color was 30 to 40% red. Ripening was accompanied by a loss of galactose and arabinose residues from the cell wall. The content of uronic acid and cellulose in the wall changed only slightly during ripening. The average molecular weight of a cell wall hemicellulosic fraction shifted progressively toward a lower molecular weight during ripening. Total β-galactosidase (EC 3.2.1.23) activity increased 50-fold from the immature green to the red ripe stage. No polygalacturonase (EC 3.2.1.15) activity was detected at any stage of ripeness. Thus, the loss of galactose and arabinose residues from the cell wall, as well as the observed modification of hemicelluloses during ripening, seem to be unrelated to active polygalacturonase. Soluble polyuronide content remained relatively constant at approximately 60 μg (g fresh weight)⁻¹ as fruit ripended.     

The HAL1 function on Na+ homeostasis is maintained over time in salt-treated transgenic tomato plants, but the high reduction of Na+ in leaf is not associated with salt tolerance

To achieve a deeper knowledge on the function of HAL1 gene in tomato ( Solanum lycopersicum ) plants submitted to salt stress, in this study, we studied the growth and physiological responses to high salt stress of T3 transgenic plants (an azygous line without transgene and both homozygous and hemizygous lines for HAL1 ) proceeding from a primary transformant with a very high expression level of HAL1 gene. The homozygous plants for HAL1 gene did not increase their salt tolerance in spite of an earlier and higher reduction of the Na⁺ accumulation in leaves, being moreover the Na⁺ homeostasis maintained throughout the growth cycle. The greater ability of the homozygous line to regulate the Na⁺ transport to the shoot to long term was even shown in low accumulation of Na⁺ in fruits. By comparing the homozygous and hemizygous lines, a higher salt tolerance in the hemizygous line, with respect to the homozygous line, was observed on the basis of fruit yield. The Na⁺ homeostasis and osmotic homeostasis were also different in homozygous and hemizygous lines. Indeed, the Na⁺ accumulation rate in leaves was greater in hemizygous than in homozygous line after 35 days of 100 m M NaCl treatment and only at the end of growth cycle did the hemizygous line show leaf Na⁺ levels similar to those found in the homozygous line. With respect to the osmotic homeostasis, the main difference between lines was the different contribution of inorganic and organic solutes to the leaf osmotic balance. Taken together, these results suggest that the greater Na⁺ exclusion ability of the homozygous line overexpressing HAL1 induces a greater use of organic solutes for osmotic balance, which seems to have an energy cost and hence a growth penalty that reverts negatively on fruit yield.