Cell wall modifications in the pericarp of litchi (Litchi chinensis Sonn

Journal of Horticultural Science & Biotechnology (2006) 81 (2) 231–237

Cell wall modifications in the pericarp of litchi (Litchi chinensis Sonn.) cultivars that differ in their resistance to cracking By XU-MING HUANG1*, HUI-CONG WANG1, XUE-JUAN LU2, WEI-QUN YUAN3, JIE-MEI LU4, JIAN-GUO LI1 and HUI-BAI HUANG1 1 Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou 510642, China 2 Instrumental Analysis and Research Center, South China Agricultural University, Guangzhou 510642, China 3 Huizhou Agricultural Bureau, Huizhou, Guangdong, China 4 Guangzhou Agricultural Technology Extension Center, Guangzhou 510520, China (e-mail: [email protected]) (Accepted 22 October 2005) SUMMARY Changes in structural calcium and galacturonan concentrations, the degree of methylesterification of pectins, the activities of pectin methylesterase (PME) and soluble and wall-bound peroxidases (POD), and amino acid compositions of structural proteins in the fruit pericarp (skin) were studied in cracking-resistant ‘Huaizhi’ and cracking-susceptible ‘Nuomici’ litchi. Structural calcium concentrations were higher in ‘Huaizhi’ than in ‘Nuomici’ and decreased from 22 d to 52 d after anthesis (DAA), then increased. Galacturonan concentrations increased over time and were higher in ‘Huaizhi’. Methylesterification of pectins increased from 50% at 22 DAA, to 55% at 78 DAA in ‘Huaizhi’, but fluctuated around 48% in ‘Nuomici’. PME activity was significantly higher in ‘Nuomici’ than in ‘Huaizhi’ in the later stages of fruit development (> 50 DAA). The activities of soluble (SPOD) and ionically wall-bound POD (IWBPOD) increased with fruit development. Maximum SPOD activity was similar in both cultivars, whereas the peak activity of IWBPOD was higher in ‘Nuomici’ than in ‘Huaizhi’. Total concentrations of SDS-soluble amino acids were similar in the two cultivars. Total structural proteins (SDS-insoluble amino acids) decreased with fruit development. Among the SDS-insoluble amino acids, hydroxyproline was the only one that increased over time. The results suggest that higher levels of structural calcium and galacturonans may contribute to cracking resistance in ‘Huaizhi’, while a higher activity of IWBPOD, which catalyses the formation of phenolic cross-linkages and an irreversible increase in the rigidity of cell walls, may be associated with cracking susceptibility in ‘Nuomici’. Structural proteins may not be involved in resistance to cracking in litchi cultivars.

T

he strength and extensibility of the pericarp (fruit skin) play important roles in cracking in litchi (Litchi sinensis Sonn.). Both are higher in the cracking-resistant cultivar ‘Huaizhi’ than in the susceptible cultivar ‘Nuomici’ (Li and Huang, 1995; Huang et al., 1999; Huang, 2005). In previous studies, we compared the anatomy and the biochemistry of their cell walls, including calcium and polysaccharide concentrations in the two cultivars (Huang et al., 1999; 2001; 2004a; 2005a). The extensibility of litch pericarp during aril expansion was related to spongy tissue (Huang et al., 2004a) formed from localised cell death, possibly signalled by excessive accumulation of calcium in the early stage of fruit development (Huang et al., 2004b). However, the higher pericarp strength in ‘Huaizhi’ than in ‘Nuomici’ could not be explained anatomically (Huang et al., 2004a). An examination of pericarp cell walls, which are the basis of the tissue’s mechanical properties, showed that ‘Huaizhi’ had higher concentrations of structural calcium and polysaccharides compared with ‘Nuomici’ (Huang et al., 1999; 2001). However, more detailed studies on the cell walls are required to understand the physiology of cracking resistance. *Author for correspondence.

Cell walls are composed of a network of cellulose microfibrils connected to networks of hemicellulose, pectin and structural proteins (Brett and Waldron, 1990). The network of pectin together with calcium ions serves as a cohesive material to stabilise cell walls (Hudson and Buescher, 1986). Positively-charged calcium ions are bound to pectin through the negatively charged carboxyl groups in the galacturonic acid residues along the homogalacturonan, the most abundant pectic polymer (Brett and Waldron, 1990) to create structural cell wall calcium. Several factors determine the formation and the amount of structural calcium (Li, 1991). First, the number of galacturonic acid residues in the pectin that provide potential calcium-binding sites. Second, pectin methylesterification “shields” the negative charge and blocks the calcium-binding sites. Third, demethylesterification, mediated by pectin methylesterase (PME), provides a mechanism to release the calcium-binding site (Barnavon et al., 2001). However, once pectin is demethylesterified, the acidic pectin becomes more vulnerable to polygalacturonase, an enzyme that breaks pectic polymers and leads to tissue softening (Fischer and Bennett, 1991). If the calcium supply is not limited, the quantity of structural calcium is determined by the quantity of galacturonic

232

Cell wall components in litchi pericarp

acid residues, the degree of methylesterification of pectin and the activity of PME. Young, plastic cell walls have higher levels of methylesterified galacturonan and lower levels of acidic pectin compared with older cell walls, where a higher activity of PME was observed (Goldberg et al., 1986; Li et al., 1994; McCann and Roberts, 1996; Liberman et al., 1999). Liberman et al. (1999) also observed a gradient of structural calcium levels that increased from expanding to expanded cells. They suggested that higher structural calcium in old cell walls was attributable to the increased availability of calcium-binding sites resulting from demethyesterification as well as incorporation of de novo non-methylesterified galacturonan. A network of proteins also contributes to cell wall structure, especially hydroxyproline-rich proteins (the extensins; Cassab, 1998). Incorporation of large amounts of extensins is associated with cessation of growth (Ye and Varner, 1991). Extensins contribute significantly to the tensile strength of a tissue and play an important role in wound-healing and defence (Cassab, 1998). Cell walls are subjected to secondary modifications, which involve phenolic cross-linkages such as the diferulic bridge between polysaccharides, the isodityrosine bridge between structural proteins, as well as polymerisation of lignin precursors which are formed irreversibly by the action of peroxidase (POD; Li, 1991; Campa, 1991). Such secondary modification leads to a loss of cell wall extensibility. Because these modifications of cell walls affect the mechanical properties of tissues, we proposed that these modifications in the cell walls of litchi pericarp might be associated with cracking-resistance. In the present study, this hypothesis was examined by comparing cell wall modifications. These included the accumulation of structural calcium and galacturonans, methylesterification of pectins, the activities of PME as well as soluble and wall-bound POD, and changes in the amino acid compositions of structural proteins, in the pericarp of two cultivars differing in resistance to cracking. The aim of the work was to understand the biochemical basis for cracking resistance.

MATERIALS AND METHODS Fruit were harvested from 13–15 year-old litchi (L. chinensis Sonn.) trees of cvs. ‘Huaizhi’ and ‘Nuomici’ growing in an orchard in Dongguan, Guangdong, China. In 2002, samples were taken 28, 38, 45, 52, 59, 66, 73 and 80 d after anthesis (DAA) to analyse POD levels in the pericarp. In 2003, fruit were harvested 22, 36, 50, 64 and 78 DAA to analyse structural calcium, galacturonic acid, methylesterification of pectin and PME activities in the pericarp. In 2004, samples were harvested 38, 45, 52, 59, 66, 73 and 80 DAA for amino acid analysis. In all years, 20 litchi fruit were sampled from each of three-to-five trees of the same cultivar at each date (DAA). Each replicate for the following analyses consisted of three fruit sampled from the same tree. Analysis of structural calcium A sequential extraction procedure, based on Chen and Uemoto (1976), was adopted to extract the different forms of calcium. A 0.5 g sample (n = 4) of pericarp was

ground in distilled water and the homogenate was centrifuged at 5,000 g for 10 min at room temperatures (27°C). The sediment was resuspended in distilled water and centrifuged again at 5,000 g for 10 min. The waterinsoluble structural calcium in the sediment was released by 2% (v/v) glacial acetic acid. The calcium content in the solution was measured with an atomic absorption spectrometer (Hitachi Z-5000) using 1, 2, 5 and 10 mg l–1 CaCl2 in 2% (v/v) glacial acetic acid solution as standards. Extraction of pectin from litchi pericarp Extraction of pectin was based on the method of Bouranis and Niavis (1992). A sample (n = 5) of 0.5 g of pericarp was ground in water. The homogenate was centrifuged at 5,000 g for 10 min at room temperature. The supernatant was discarded and the sediment resuspended in distilled water and centrifuged at 5,000 g for 10 min, twice. The sediment was then re-suspended with pectin-extracting solution [0.24% (w/v) oxalic acid, 0.24% (w/v) ammonium oxalate] at 60°C. The mixture was centrifuged at 5,000 g for 10 min after 2 h in a water bath at 60°C. The sediment was re-suspended with hot pectin-extracting solution and centrifuged at 5,000 g for 10 min. The two supernatants were mixed and used in assays for galacturonic acid and methylesterification. Assays of galacturonic acid We used the method described by Scott (1979). A 50 µl portion of the above supernatant (n = 5) was pipetted into a test-tube, to which was added 50 µl 2 M NaOH and 50 µl 2% (w/v) NaCl. The mixture was left for 10 min at room temperature (27°C) to allow saponification of the pectin and to remove the methylester groups from the galacturonans, then 2 ml concentrated sulphuric acid was added. Ten min later, 0.1 ml 0.1% (w/v) 3,5-dimethylphenol dissolved in glacial acetic acid was added. The absorbance at 450 nm (OD450nm) and 420 nm (OD420nm) were measured within 15 min with a spectrophotometer (Model 752; Shanghai Precision and Scientific Instrument Co. Ltd., Shanghai, China). A standard curve of galacturonic acid concentration against the difference in absorbance at 450 nm and 420 nm (OD450nm – OD420nm) was plotted using galacturonic acid at 0, 25, 50, 100, 200 and 400 µg ml–1. The regressed standard curve was linear (y = 746.9 x; r2 = 0.9997). Assay of methylester groups in pectin Analysis was based on the procedure of Wood and Siddique (1971). A test-tube containing 0.5 ml of the above pectin extract (n = 5) had 0.25 ml 1.5 M NaOH added. Saponification occurred at room temperature for 30 min. The test tube was then placed in an ice bath and 0.25 ml 5.5 M sulphuric acid added, followed by 0.2 ml 2% (w/v) potassium permanganate to oxidise the methanol released in the previous step. Fifteen min later, 0.2 ml 0.5 M sodium arsenite solution and 0.6 ml distilled water was added and the mixture allowed to react for 1 h at room temperature before 2 ml 20 mM pentane-2,4-dione dissolved in 2 M ammonium acetate and 0.05 M acetic acid was added and the mixture placed in a water bath at 60°C for 15 min. After the mixture cooled down, the absorbance at 412 nm was measured using a Model 752 spectrophotometer. A standard curve was prepared with

X. M. HUANG, H. C. WANG, X. J. LU, W. Q. YUAN, J. M. LU, J. G. LI and H. B. HUANG

Extraction and enzyme assay of peroxidase (POD) A 0.5 g sample of pericarp (n = 4) was ground with 3 ml cold 20 mM phosphate buffer pH 6.8 in a mortar and centrifuged at 8,000 g for 10 min at 4°C. The precipitate was extracted with 3 ml phosphate buffer and centrifuged twice. The supernatant represented the soluble portion of the crude enzyme (soluble POD; SPOD). Ionically cell wall-bound POD (IWBPOD) was then extracted twice from the precipitate with 3 ml 20 mM phosphate buffer pH 6.8 containing 1 M NaCl. The reaction mixture included 0.05 ml enzyme extract and 2 ml 0.05% (w/v) guaiacol in the same buffer. The speed of guaiacol oxidation was measured colorimetrically to indicate peroxidase activity (units mg–1 protein). One unit of enzyme activity was taken as an increase in OD490nm of 0.01 in 1 min. Amino acid composition of cell wall structural proteins The analysis was based on the method of Huang et al. (1983). A 0.5 g sample of pericarp was ground in a mortar with 3 ml 1% (w/v) sodium dodecylsulphate (SDS). The extract was centrifuged at 5,000 g for 10 min. The precipitate was then re-suspended in 3 ml 1% (w/v) SDS, stirred for 5 min and centrifuged at 5,000 g for 10 min. This procedure was repeated, and the supernatants from the three centrifugations collected. This SDS-soluble fraction contained ionically cell wall-bound proteins as well as soluble amino acids and proteins. The precipitate, or SDS-insoluble fraction contained covalently wallbound structural proteins. Both fractions were hydrolysed with HCl at 110°C for 18 h. The hydrolysates were centrifuged at 4,000 g for 10 min and the supernatants analysed for of amino acid contents on an autoanalyser (Hitachi 834-50). The 18 amino acids measured included glycine (Gly), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), leucine (Leu), proline (Pro), serine (Ser), lysine (Lys), valine (Val), threonine (Thr), isoleucine (Ile), arginine (Arg), phenyalanine

Statistics Statistical analyses were conducted using SPSS 10.0. software. The significance of differences in structural calcium, PME, SPOD and IWBPOD activities, and amino acids in structural proteins between ‘Huaizhi’ and ‘Nuomici’ were analysed by one-way ANOVA.

RESULTS Concentrations of structural calcium decreased between 22 – 52 DAA, then increased during rapid aril growth. Structural calcium levels were significantly higher in ‘Huaizhi’ than in ‘Nuomici’ at 35 and 66 DAA, then at all other periods, but they were only slightly higher in ‘Huaizhi’ than in ‘Nuomici’ (Figure 1). Concentrations of galacturonic acid and methylester groups (methanol) increased with fruit development (Figure 2), indicating that partially methylesterified galacturonan was constantly being synthesised and incorporated into cell walls. The rates and average values were higher in ‘Huaizhi’ than in ‘Nuomici’, suggesting that the former cultivar had a higher rate of galacturonan synthesis and incorporation into cell walls. The degree of methylesterification of galacturonans increased from 22 DAA and peaked at 64 DAA in ‘Huaizhi’, but fluctuated at about 48% in ‘Nuomici’, with higher average values in the former cultivar (Figure 3A). These results suggest that the galacturonan incorporated into the cell walls of ‘Huaizhi’ was more highly methylesterified and, in ‘Nuomici’, the accumulated galacturonan was less methylesterified. The activity of PME increased in both cultivars and there was no significant difference between cultivars from 22 – 36 DAA (Figure 3B). PME activity increased following a period of relatively constant activity in ‘Nuomici’, and a drastic decline in ‘Huaizhi’ from 36 – 50 DAA. Consequently, PME activity in ‘Nuomici’ became significantly higher than in ‘Huaizhi’at > 50 DAA. 0.8 0.7 0.6

*

-1

Extraction and assay of pectin methyesterase (PME) PME was extracted according to Hagerman and Austin (1986). A sample of 0.5 g pericarp (n ≥ 3) was ground with 5 ml pre-cooled 0.1 M phosphate buffer solution pH 7.0 containing 8.8% (w/v) NaCl, and centrifuged at 20,000 g at 4°C for 10 min. The supernatant served as the crude enzyme and 0.l ml of this supernatant was pipetted into a test-tube containing 0.4 ml 0.5% polygalacturonan with 40% of galacturonic acid residues methylesterified (Sigma Chemical Co. Ltd., St Louis, MO, USA). The test-tube was placed in a water bath at 30°C for 2 h. Methanol released was measured based on Wood and Siddique (1971). The procedure was the same as the assay for methylester groups in pectin described above, but without the saponification and neutralisation steps.

(Phe), tyrosine (Tyr), histidine (His), hydroxyproline (Hpro), methionine (Met) and cysteine (Cys). Analyses were conducted on a single replicate (except at 66 DAA, during the critical period of fruit cracking, where n = 3).

Structural calcium (mg g FW)

0.5 ml methanol solutions at 0, 10, 20, 40, 60, 80 and 100 µg ml–1 treated as above. The standard curve of methanol concentration against OD412nm fitted a quadratic equation (y = –1460.2 x2 + 860.17 x + 0.1791; r2 = 0.9999).The degree of methylesterification of pectin was calculated as the percentage of the molar concentration of methylester groups against the molar concentration of galacturonic acid in the pectin extract.

233

*

0.5

'Huaizhi' 0.4 0.3

'Nuomici'

0.2

Rapid aril growth

0.1 0 0

10

20

30

40

50

60

70

80

90

DAA

FIG . 1 Changes in structural calcium concentrations in the pericarp of ‘Nuomici’ (open circles) and ‘Huaizhi’ (closed circles) litchi (n = 4) at different times (DAA). Asterisks indicate significant difference between cultivars at P = 0.05 based on one-way ANOVA for each date.


234

A

Galactouronic acid (mg g-1 FW)

16

Degree of methylesterification of pectin (%)

18

'Huaizhi' y = 0.2064x - 0.2692 2

r = 0.9814

14

'Nuomici'

12 10

y = 0.1284x + 2.4533 2

r = 0.9657

8 6 4

55

35

1600 30

40

50

60

70

80

0

10

B

90

1.6

20

30

40

50

60 'Nuomici' 70

*

*

* 80

90

-1

y = 0.0175x - 0.0776

PME activity ( mg g FW h )

1400

B

1.4

'Nuomici'

40

30 1800 20

'Huaizhi'

45

2

10

A

50

1.8 0

0

'Huaizhi'

2

r = 0.9906 1.2 1 0.8

'Nuomici' 0.6

'Huaizhi'

1000 800 600 400

y = 0.0083x + 0.2061

0.4

1200

-1

Methanol (mg g -1 FW)

60

2

r = 0.9715

200

0.2

0 0

0

0

10

20

30

40

50

60

70

80

10

20

30

90

40

50

60

70

80

90

DAA

DAA

FIG . 3 Changes in methyl-esterification of galacturonan (Panel A) and activity of PME (Panel B; n ≥ 3) in ‘Huaizhi’ (closed circles) and ‘Nuomici’ (open circles) litchi pericarp. Asterisks indicate significant differences between cultivars at P = 0.05 based on one-way ANOVA.

FIG . 2 Changes in the concentrations of galacturonic acid (Panel A) and methylester groups measured as released methanol (Panel B) in the cell wall pectin of litchi cvs. ‘Huaizhi’ (closed circles) and ‘Nuomici’ (open circles) litchi pericarp (n = 5). Linear regressions are shown (P < 0.005).

The concentration of structural proteins (total SDSinsoluble amino acids) fell over time. The decrease fitted a power curve (y = 12439x–0.9369; r2 = 0.93) and a linear curve (y = –3.1769 x + 449.98; r2 = 0.92) in ‘Nuomici’ and ‘Huaizhi’, respectively (Figure 5). The amount of structural protein was initially higher in ‘Nuomici’ than in ‘Huaizhi’, but there was almost no difference at ≥ 66 DAA.

In general, the activities of SPOD and IWBPOD increased with fruit development, but most rapidly from 38 – 60 DAA (Figure 4). SPOD activity was initially higher in ‘Nuomici’ than in ‘Huaizhi’ (at < 38 DAA), but was similar in the two cultivars at ≥ 45 DAA. IWBPOD activities were higher in ‘Nuomici’ than in ‘Huaizhi’ in five out of the seven periods measured, especially during rapid aril growth (≥ 60 DAA; Figure 4).

600

400

400 'Nuomici'

*

200

300 200

* 'Huaizhi'

100

100

I o n i c a l l y w all-bound POD (IWBPOD)

500 -1

500 POD activity (units g FW)

-1

POD activity (units g FW)

Soluble POD (SPOD) 500

300

600 1

600

400

* 300

*

*

'Nuomici'

*

200 100

'Huaizhi'

* 0 0

10

20

30

40

50

DAA

60

70

80

0 90

0

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10

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70

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DAA

FIG . 4 Changes in soluble POD and ionically wall-bound POD activities in ‘Huaizhi’ (closed circles) and ‘Nuomici’ (open circles) litchi pericarp (n = 4). Asterisks indicate significant differences between cultivars at P = 0.05 based on one-way ANOVA.

X. M. HUANG, H. C. WANG, X. J. LU, W. Q. YUAN, J. M. LU, J. G. LI and H. B. HUANG

235

TABLE I. Changes in the concentrations (nmol g–1FW) of amino acids in the SDS-insoluble fraction from the pericarp of litchi cvs. ‘Huaizhi’ (H) and ‘Nuomici’ (N) Amino acid

38 H

Met Tyr His Pro Hypro Phe Arg Ile Thr Ser Val Lys Ala Leu Gly Asp Glu Total

45 N

2.1 6.85 7.92 9.55 10.28 13.63 13.73 17.31 17.89 22.64 23.37 23.81 29.57 32.00 32.14 33.53 35.49 331.81

2.37 9.95 10.27 12.54 13.70 18.68 18.88 23.12 23.70 31.06 31.11 32.17 39.85 43.81 42.82 45.55 47.46 447.04

52

H

N

2.56 6.42 7.88 9.50 12.92 12.77 12.96 16.33 17.04 21.44 22.39 21.91 28.29 29.56 31.59 32.11 33.19 318.86

1.72 6.35 7.58 9.20 11.97 12.65 12.70 16.17 16.40 21.64 22.20 22.76 28.00 29.38 30.45 31.32 32.63 313.12

H 1.75 5.50 6.94 8.75 14.19 11.02 10.48 14.05 14.43 18.26 20.14 20.06 24.62 25.10 27.66 27.43 28.13 278.51

Days after anthesis (DAA) 59 N

H

1.72 6.47 8.09 9.33 14.47 12.15 12.07 16.06 16.01 21.86 22.01 22.63 27.39 28.34 30.22 30.56 31.98 311.36

1.63 5.00 6.33 7.88 14.72 9.55 8.99 12.30 12.67 16.45 18.22 18.17 21.68 21.53 25.45 23.76 24.48 248.81

N 1.79 5.41 7.27 8.61 15.64 10.67 10.30 13.92 14.02 19.41 19.72 20.45 23.96 24.37 27.06 26.94 28.07 277.61

H

66

73 N

1.64a 1.4 a 4.28a 4.45a 5.71a 5.57a 7.40a 7.48a 15.8a 15.2a 8.17a 8.23a 7.69a 8.15a 10.9a 11.3a 11.2a 11.4a 15.6a 16.7a 16.6a 17.3a 14.8a 14.8a 19.5a 20.7a 18.2a 19.1a 23.8a 23.0a 20.6a 21.3a 22.0a 24.1a 224.0a 230.2a

H 1.42 4.73 6.20 7.70 16.73 8.76 8.70 11.66 11.91 17.57 17.75 16.43 21.53 20.35 24.14 22.33 24.37 242.28

77 N

1.55 5.04 6.32 8.30 19.31 8.47 8.23 11.30 11.64 17.46 17.42 16.02 20.54 19.36 23.61 21.52 23.37 239.46

H 1.47 4.07 4.75 6.62 15.89 6.74 6.36 9.05 9.44 13.73 14.22 13.22 16.85 14.89 20.91 16.81 18.44 193.46

N 1.44 4.24 5.48 7.18 17.67 7.04 6.82 9.57 9.55 14.61 14.91 14.65 17.17 15.98 20.63 17.86 19.55 204.35

The analysis was conducted with a single replicate, except for 66 DAA where n = 3. Different lower-case letters adjacent to the 66 DAA values indicate significant differences between the two cultivars at P = 0.05 by one-way ANOVA.

Concentrations of SDS-soluble amino acids were about 10-times lower and relatively stable over the season in both cultivars (Figure 5). Concentrations of all SDS-insoluble amino acids decreased over time except for hydroxyproline, which increased. The SDS-insoluble fraction decreased with fruit development in both cultivars (Table I). Thus hydroxyproline-rich extensins were constantly being incorporated into the cell walls. As a result, the percentage of hydroxyproline increased with fruit development.

DISCUSSION Fruit cracking occurs when the stress exerted on the fruit skin (pericarp) from the expanding aril exceeds the strength of the skin (Huang et al., 2004a). The mechanical strength of the pericarp is largely due to its cell walls, which are composed of a network of cellulose microfibrils linked to networks of hemicellulose, pectin and structural proteins (Brett and Waldron, 1990). The physiological basis of cracking-resistance may be

500 450 y = 12439x

Total amino acids (nmol g -1 FW)

400

-0.9369

2

r = 0.93

SDS-insoluble

350 'Nuomici' 300 y = -3.1769x + 449.98 250

2

r = 0.92

'Huaizhi'

200 150 100 50

'Nuomici'

SDS-soluble

'Huaizhi'

0 0

10

20

30

40

50

60

70

80

90

DAA

FIG. 5 Changes in total amino acid concentrations in SDS-soluble and SDSinsoluble fractions in ‘Huaizhi’ (closed circles) and ‘Nuomici’ (open circles) litchi pericarp. Analyses were conducted with a single replicate, except for 66 DAA data where n = 3. Linear regressions for the SDSinsoluble fraction in ‘Huaizhi’ and ‘Nuomici’ are shown (P < 0.01)

revealed through the study of cell wall modifications in cultivars that differ in cracking-resistance. Modifications in the pectin-calcium network The network of pectin and calcium ions serves as a cohesive material to stabilise cell walls and add strength to tissues (Hudson and Buescher, 1986). Structural calcium levels decreased in litchi pericarp from 22 DAA to 52 DAA, corresponding to the period of spongy tissue formation and the release of structural calcium (Huang et al., 2004 b). Cell expansion in the pericarp during this period might also have caused a “dilution” of structural calcium (Huang et al., 2004a). Structural calcium increased during aril expansion, when pericarp extension was mainly by flattening of the protruberance of the skin segments instead of cell expansion (Huang et al., 2004a), suggesting that calcium was constantly incorporated into the cell walls. The increase in structural calcium could be due to an increased availability of calcium or to increased calcium-binding sites (free carboxyl groups) in the pectins. An increase in calcium-binding sites can be achieved by newly-synthesised galacturonans incorporating into the cell walls, or by demethylesterification of available galacturonan through the action of PME (Liberman et al., 1999). Both galacturonan (galacturonic acid) and its methylesters increased during fruit development (Figure 2) without a decrease in the degree of methylesterification of pectins (Figure 3), suggesting that de novo synthesised galacturonans incorporated into the cell walls play a more decisive role than demethylesterification in increasing calcium-binding sites. In contrast, in grape, galacturonic acid content increased while the degree of methylesterification decreased (Laurent et al., 2001). However, changes in structural calcium were not paralleled by those in galacturonans, especially in the early stages (≤ 50 DAA; Figures 1 and 2), indicating that calcium-binding sites were not the rate-limiting factor in structural calcium formation throughout fruit development. Alternatively, calcium availability became rate-limiting especially during the early stages (≤ 50 DAA).

236


Cracking-resistant ‘Huaizhi’ has a higher concentration of structural calcium than cracking-susceptible ‘Nuomici’ (Huang et al., 1999; 2005a; Figure 1). The former accumulated more galacturonans (Figure 2) as well as calcium (Huang et al., 2005a). In an unpublished study, ‘Huaizhi’ accumulated more calcium than its physiological needs, and the excess was stored as calcium oxalate, chiefly in the epidermis. In contrast, ‘Nuomici’ stored little calcium oxalate. Therefore, the higher levels of structural calcium in ‘Huaizhi’ are due to higher calcium availability and more calcium-binding sites (i.e., acidic galacturonans). With its higher concentrations of structural calcium and galacturonans, ‘Huaizhi’ has a stronger pectic network, which provides a biochemical basis for its cracking-resistance. POD-mediated modifications POD-mediated cell wall modifications include the irreversible formation of phenolic cross-linkages, such as the diferulic bridge between polysaccharides, the iso-dityrosine bridge between structural proteins, as well as polymerisation of lignin precursors (Li, 1991; Campa, 1991). These modifications make cell walls more hydrophobic, more rigid and less extensible (Li, 1991). The activities of SPOD and IWBPOD in the pericarp increased during litchi development (Figure 4). IWBPOD was more closely-related to cell wall modification than SPOD, and increased with fruit development. This provides for an increase in the formation of phenolic cross-linkages, as shown by the development of sclerenchyma with highly lignified cells in the pericarp (Huang et al., 2004a). IWBPOD activity was higher in ‘Nuomici’ than in ‘Huaizhi’ from 59 DAA, when fruits crack as the arils expand (Huang, 2005). With lower concentrations of structural polysaccharides and

calcium (Huang et al., 1999; Figures 1 and 2), the mechanical strength of ‘Nuomici’ pericarp is likely to be due more to irreversible phenolic cross-linkages catalysed by POD, especially IWBPOD, with a concomitant loss in extensibility. With a less extensible pericarp, ‘Nuomici’ is more susceptible to fruit cracking (Huang et al., 2004a). Modifications in structural proteins Structural proteins are among the most important components of cell walls. In litchi, the concentration of total structural proteins (SDS-insoluble amino acids) decreased over time (Figure 5), which was different from grape, where structural proteins accumulated during berry maturation (Huang et al., 2005b). However, the increase in hydroxyproline concentration over time in litchi (Table I) suggests that hydroxyproline-rich proteins (extensins) are actively synthesised and incorporated into the cell walls. In general, there was almost no difference in the concentrations of structural proteins (including extensins) between the two litchi cultivars (Figure 5; Table I). The result suggest that structural proteins may not be involved in crackingresistance in litchi.

CONCLUSION Higher levels of structural calcium and galacturonans may increase resistance to cracking, whereas a higher activity of IWBPOD may make fruit more susceptible to cracking. Structural proteins may not be involved in cracking-resistance in litchi cultivars. This study was supported by the National Natural Science Foundation of China (No. 30471199).

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