Partitioning of ¹³C-photosynthate from Spur Leaves during Fruit Growth of Three Japanese Pear (Pyrus pyrifolia) Cultivars Differing in Maturation Date

Abstract

• Background and Aims In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date.

• Methods Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to ¹³CO₂ and measurement of the change in ¹³C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit.

• Key Results Compared with the earlier-maturing cultivars ‘Shinsui’ and ‘Kousui’, the larger-fruited, later-maturing cultivar ‘Shinsetsu’ had a greater total leaf area per spur, greater source strength (source weight × source specific activity), with more ¹³C assimilated per spur and allocated to fruit, smaller loss of ¹³C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp.

• Conclusions Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.

INTRODUCTION

Regulation of fruit size is of major importance in plant development (Gillaspy et al., 1993), with large effects on fruit quality; size at harvest is important commercially in determining orchard profitability. Knowledge of fruit growth patterns of cultivars and the factors that affect them can be particularly helpful in horticulture, for example in selecting superior genotypes, establishing breeding programs, and in orchard management (Westwood and Blaney, 1963). As a consequence, there is extensive research aimed at understanding mechanisms of fruit growth and the genetic and cultural influences (Westwood et al., 1967; Hayashi and Tanabe, 1991; Scorza et al., 1991; Gillaspy et al., 1993; Grossman and DeJong, 1995; Famiani et al., 2000; Richings et al., 2000; Jackson, 2003; Zhang et al., 2005). Although the potential size of fruit is genetically determined, actual size depends on the interaction between genetics and environment (Grossman and DeJong, 1995). However, it is still unclear how plants regulate their fruit size.

There is wide variation in fruit size in many species of fruit-bearing plants, both wild and cultivated, the latter crops including Japanese pear (Pyrus pyrifolia) (Laney and Quamme, 1975; Hayashi and Tanabe, 1991; Grandillo et al., 1999). Fruit growth of Japanese pear is characterized by an initial period of rapid cell division, followed by a long period of cell expansion, primarily by vacuolation (Hayashi and Tanabe, 1991; Jackson, 2003). Thus, cell number and cell size are very important factors determining final fruit size and any environmental factors that affect them will affect final fruit size. Consequently, it is very important to address each phase in order to clarify the mechanisms that control final size.

There have been numerous studies of the effects of genetic and environmental factors on fruit development, such as growth characteristics (Hayashi, 1960; Jackson, 2003), hormonal regulation (Hayashi and Tanabe, 1991), sink–source interactions (Zhang et al., 2005) and carbohydrate partitioning (Hayashi, 1960; Teng et al., 1998a, b, 1999, 2001, 2002; Yamamoto, 2001). Partitioning, that is the amount and proportion of photosynthates distributed to fruit at different stages in development, is critical for final fruit size and has attracted more and more attention in many fruit crops (Darnell and Birkhold, 1996; Escobar-Gutierrez and Gaudillere, 1997; Amano et al., 1998; Flore and Layne, 1999).

In fruit trees, the early stage of fruit development is particularly important, since strong competition exists between the vegetative and reproductive organs both before and after anthesis (Hayashi and Tanabe, 1991; Jackson, 2003). Cell division of the mesocarp is closely correlated with final fruit size in peach (Prunus persica; Scorza et al., 1991) and apple (Malus domestica; Westwood et al., 1967). It has been suggested that carbon deficits limit cell division and thus affect final fruit size. In general, the carbohydrate supply from reserves has been found to be closely related to early fruit development in many fruit crops (Hayashi and Tanabe, 1991; Jackson, 2003), so that defoliation in the previous autumn decreases assimilate reserves and thereby early growth in the spring (Hayashi, 1960). In other words, the availability of carbohydrates early in the season is crucial for larger fruit production. The fate of photosynthates from spur leaves during the period of rapid fruit growth was partially investigated in ‘Nijisseiki’ pear: most ¹³C assimilated in the spur at 87 DAA was either found in the fruit (approx. 48 %) or lost to respiration and export (41 %) by the time of fruit harvest (Teng et al., 2001). However, the time-course of photosynthate partitioning from spur leaves during fruit development has not been well documented. Moreover, there is little information about genotypic differences in photosynthate partitioning from spur leaves in any fruit, including Japanese pear.

Later-maturing cultivars usually have larger fruits than earlier-maturing cultivars in many crops, including Japanese pear (Hayashi and Tanabe, 1991), but no general explanation or model for the genotypic differences in fruit size has been advanced. Therefore in order to understand fruit development better, and to promote the production of larger fruit, this study focuses on the fruiting spur unit without bourse shoots in Japanese pear. To establish the relative importance of the supply of photosynthates and the demand from the fruit, in relation to the developmental stages and maturation period of the fruit, the time-course of partitioning of photosynthates from spur leaves during fruit development was investigated together with the structure of the mesocarp in Japanese pear cultivars differing in maturation date.

MATERIALS AND METHODS

Plant material

Three Japanese pear Pyrus pyrifolia Nakai cultivars, ‘Shinsui’, ‘Kousui’ and ‘Shinsetsu’ propagated on Pyrus betulaefolia Bunge rootstocks, were used for experiment. ‘Shinsui’ is an early-maturing cultivar with small-sized fruit (about 200 g). ‘Kousui’ has medium-sized fruit (about 350 g), which ripens around 1 week later than ‘Shinsui’. ‘Shinsetsu’ is a late-maturing cultivar with large fruit (more than 1000 g). All cultivars were 20 years old and cultured with a flat-canopied pergolar system (Teng et al., 1998a) and hand-pollinated with pollen of ‘Chojuro’ at anthesis.

Fresh weight of fruit, cell number and cell length of the mesocarp

Fifteen fruit of each cultivar were sampled after anthesis and weighed, then ten fruits were immediately preserved in formalin–acetic–alcohol (80 % ethanol: acetic acid: formalin = 90 : 5 : 5) for histological analysis. The fruit was cut along the equatorial region. Then the mesocarp width was calculated from the difference between the longest width of a transverse section of fruit and core. Subsequently, a transverse slice of mesocarp was taken along the equatorial region and stained by rubbing softly with a cloth soaked in blue ink. The stained surface was observed under a digital HF microscope system (VH-8000, Keyence, Tokyo, Japan) and an image from a CCD camera displayed on a monitor. Cell length, as an indicator of cell size, was measured from the length of seven contiguous cells from the core to the fruit surface: from these the average cell length was calculated. Ten observation zones per section were measured. Cell number of the mesocarp along the equatorial region was then calculated by dividing the mesocarp width by average cell length, and this was taken as an indicator of total cell number per fruit.

Total leaf area and leaf number per spur

After anthesis, seven fruiting spurs on each cultivar were harvested randomly on each sampling date, and leaf number and total leaf area per spur were measured with a LI-3000A portable leaf area meter (LI-COR, Lincoln, NE, USA).

Net photosynthesis of spur leaves

At 7, 14, 28, 63, 104 and 184 d after anthesis (DAA), net photosynthesis (P_n) of healthy spur leaves was measured between 1100 h and 1200 h by a Shimadzu portable photosynthesis system (Analytical Development Co. Ltd. Hoddesdon, Hertfordshire, UK). The third or fourth leaf from the base of spur was used for P_n measurement. Each leaf was a replicate, with four replicates for each cultivar.

¹³C labelling and sampling

The earlier maturing cultivars ‘Shinsui’ and ‘Kousui’ were labelled at 7, 28, 63 and 104 DAA, and ‘Shinsetsu’ was also labelled 184 DAA because of its late maturation. Healthy, uniform 2-year-old fruiting spurs without bourse shoots on lateral branch were selected for ¹³C labelling. Fruit were hand-thinned to one per spur, according to commercial practice, at 30 DAA on earlier maturing cultivars, and 40 DAA for the later one (‘Shinsetsu’). The number of fruit per spur was adjusted to three and one just before ¹³C labelling at 7 and 28 DAA, respectively. Statistically, one tree was a replicate, with four replicate trees for each cultivar. Three fruiting spurs per tree were selected. One of them was girdled by removing a 2-mm-wide section of bark immediately before ¹³C labelling; this prevented export of photoassimilate from the spur to enable determination of the total amount of ¹³C assimilated by the spur. The fruit were covered with aluminium foil to prevent them from fixing carbon dioxide.

Individual spurs were exposed to ¹³CO₂ enclosed in a polyethylene bag that contained a 25 mL glass vial fixed on the frame of the bag. The ¹³CO₂ was generated by injecting 3 mL of 70 % lactic acid on to 0·8 g Ba¹³CO₃ with an abundance of 99 % ¹³C (Cambridge Isoptope Laboratories, Andover, Massachusetts, USA). To ensure uniform labelling among the spurs, 1·5 h after the start of ¹³C labelling, unlabelled CO₂ was produced by injecting lactic acid into another vial containing 1 g of BaCO₃ in the polyethylene bag. Labelling was under ambient field conditions with clear skies and lasted for 2 h between 0800 h and 1000 h for the 7 and 28 DAA labelling periods during the period of initial fruit growth. For the labelling periods 63, 104 and 184 DAA, the labelling was carried out between 0730 h and 0930 h because of high temperatures during the period of rapid fruit growth and before harvest. The four girdled spurs in each sample per cultivar were harvested immediately after labelling. Four spurs on each cultivar were harvested 7 d after ¹³C labelling (DAL) and the remaining four were collected at the final (commercial) harvest. Harvested spurs were immediately separated into leaves, current shoot, and old wood and fruit, then stored on ice and brought to the laboratory. Fruit was further divided into pedicel, flesh (exocarp + mesocarp) and core (pith of receptacle + pericarp + seeds). The parts were freeze-dried and then weighed. Current shoot, old wood and leaves were oven-dried at 65 °C for 10 d to determine dry weight. The dried material was finely ground in a coffee mill and stored in glass vials for ¹³C analyses.

Measurement of ¹³C

¹³C abundance and carbon contents were determined using an infrared ¹³CO₂ analyser (Model EX-130S, Japan Spectroscopic Co. Ltd., Tokyo, Japan) after combustion of a sample at 900 °C in an O₂ stream according to Okano et al. (1983) and Kouchi and Yoneyama (1984). The absolute amounts (mg) of labelled ¹³C recovered in each organ were calculated as total carbon in each organ × ¹³C atom %. The total amount of ¹³C recovered from the girdled spurs harvested immediately after ¹³C labelling was the basis for calculating the amount and proportion of ¹³C lost in respiration and exported from the spur. The losses due to respiration and export from the spurs were estimated from the difference between total ¹³C recovered from girdled spurs harvested immediately after ¹³C labelling and the amount of ¹³C in spurs harvested on each sampling date.

Calculation of source strength and relative sink strength

In this study, the ¹³C abundance of each organ was expressed as atom %. Due to variations in the amount of ¹³C recovered between treatments and the different weight of plant organs, comparison of photosynthate partitioning was made on sink strength and relative sink strength. Sink strength is considered as the product of sink size and activity and expressed as: sink strength=sink size × sink specific activity (Ho, 1988). The ¹³C abundance in each organ was regarded as sink specific activity, and dry weight as sink size. Based on the sink strength at 7 DAL, the relative sink strength (RSS) was calculated by dividing the sink strength of an individual organ by the sum of sink strengths of the whole spur, and the result expressed as a percentage of the total (Zhang et al., 2005). The leaf is considered the only source in the spur, so source strength refers to the rate at which photosynthate is produced. Sources and sinks are closely in balance in terms of carbon flux, therefore source strength affects the partitioning of photosynthate to sink organs (Farrar, 1996). The source strength in all cultivars was calculated as source weight × source specific activity 2 h after ¹³C labelling (HAL).

Statistical analysis

Data were analysed by Duncan's multiple range test (P < 0·01, 0·05). Since the earlier-maturing cultivars were harvested before 184 DAA, ¹³C labelling 184 DAA was done only in the late-maturing cultivar. The data from ‘Shinsetsu’ at 184 DAA and from ‘Shinsui’ and ‘Kousui’ at 104 DAA were used for the statistical analysis at maturity. Sigmoid curves were fitted to the data of fresh fruit weight by Sigmaplot software (Jandel Scientific, San Rafael, California, USA). To estimate the length of the period of cell division, the increasing patterns of cell number of the mesocarp in all cultivars were fitted by logarithmic curves. The critical point was calculated according to Higashi et al. (1999) and the period from pollination to the critical point was regarded as the period of cell division for each cultivar.

RESULTS

Leaf growth and fruit development

The three cultivars had similar patterns of leaf and fruit growth, but clearly varied significantly in important aspects with the late-maturing ‘Shinsetsu’ having more leaves and greater total leaf area per spur than earlier-maturing cultivars (Figs 1–3). ‘Shinsetsu’ required about 3 weeks to approach a relative stable total leaf area per spur, compared with ‘Shinsui’ and ‘Kousui’ with about 1 and 2 weeks, respectively. Number of leaves per spur was not significantly different between ‘Shinsui’ and ‘Kousui’, but ‘Kousui’ had a greater total leaf area per spur than ‘Shinsui’. As spur leaves grew and matured, P_n of spur leaves increased and remained relatively large subsequently (see Fig. 5). At 63 DAA, ‘Shinsui’ and ‘Kousui’, with rapid fruit growth (Fig. 2), had a higher P_n than ‘Shinsetsu’. However, ‘Shinsetsu’ had a higher P_n at 184 DAA than the other cultivars in which fruit had been harvested.

Fig. 1.

Changes in total leaf area (A) and leaf number (B) per spur after anthesis in three Japanese pear cultivars. Vertical bars represent s.e. ^**P < 0·01; n = 7.

Fig. 2.

Patterns of fruit fresh weight during fruit development in three Japanese pear cultivars, (A) 1–10 weeks after anthesis, (B) 1–26 weeks after anthesis. The S-shaped curves of fruit fresh weight are those of the fitted sigmoid equations: y = a/{1 + exp[−(x − x₀)/b]} where a is the maximal value of y and x₀ is the abscissa at inflexion point. For ‘Shinsui’, a = 322, b = 2·1842, x₀ = 14·3. For ‘Kousui’, a = 355, b = 1·8317, x₀ = 11·8. For ‘Shinsetsu’, a = 1246, b = 3·2882, x₀ = 20·2. ^*P < 0·05, ^**P < 0·01; n = 7.

Fig. 3.

Changes of cell number (A) and cell length (B) of the mesocarp along the equatorial region during fruit development in three Japanese pear cultivars. The patterns of increasing cell number (A) were fitted by logarithmic curves: ‘Shinsui’, y = 19·1ln(x) + 40·5; ‘Kousui’, y = 18·5ln(x) + 67·3; ‘Shinsetsu’, y = 24·6ln(x) + 60·9. The duration of cell division is indicated on the graph. Vertical bars represent s.e. ^**P < 0·01; n = 10.

Fig. 5.

Seasonal changes in net photosynthetic rate (P_n) of spur leaves in three Japanese pear cultivars. The data were obtained between 1100 h and 1200 h. Vertical bars represent s.e. Values followed by different letters are significantly different at P < 0·05; n = 4.

Fruit development was described by an approximately sigmoid growth pattern based on fresh weight, irrespective of cultivar (Fig. 2). From 0 to 17 weeks after anthesis, the growth rate of ‘Kousui’ was higher than that of ‘Shinsui’ and ‘Shinsetsu’. ‘Shinsetsu’ had a much longer linear stage than the other cultivars. The critical point when the slope of the fitted curve was bellow 0·5 cells d⁻¹ was calculated and the period from pollination to the critical point was regarded as the period of cell division for each cultivar. The duration of cell division was cultivar-dependent and the critical points in ‘Shinsui’, ‘Kousui’ and ‘Shinsetsu’ were 34, 36 and 49 d after pollination, respectively (Fig. 3). Histological studies of fruit showed that ‘Shinsetsu’ had significantly more cells than other cultivars (Fig. 3A). However, the final cell length was not significantly different between ‘Shinsetsu’ and ‘Kousui’, but was smaller in ‘Shinsui’ (Fig. 3B).

Amount of ¹³C in spur leaves

The total amount of ¹³C recovered in spurs at 2 HAL (hours after labelling) indicated the amount of ¹³C assimilated by spur leaves; it depended on the stage of fruit development and cultivar (Fig. 4A). At 7 DAA, more ¹³C was recovered in spurs of ‘Shinsetsu’ than ‘Kousui’ and ‘Shinsui’ (2- and 3-fold, respectively), but there was no significant difference between ‘Shinsui’ and ‘Kousui’. At 28 DAA, the amount of ¹³C in spur leaves of all cultivars was greater than at 7 DAA, since leaf growth was completed (Fig. 1). Moreover, there were significant differences among cultivars at this stage. However, there were no significant differences 63 DAA. At 104 DAA, as well as at 184 DAA, ‘Shinsetsu’ assimilated more ¹³C than the other cultivars, which did not differ between each other.

Fig. 4.

Amount of ¹³C recovered in spurs 2 h after ¹³C labelling (A) and 7 d after ¹³C labelling (B), and losses via respiration and export 7 d after ¹³C labelling (C) in three Japanese pear cultivars. The amount of ¹³C recovered in the spur includes leaves, current shoot, old wood and fruit. The losses of ¹³C via respiration and export from the spur 7 d after ¹³C labelling was calculated as a percentage of the total ¹³C recovered from girdled spurs harvested 2 h after ¹³C labelling. Vertical bars represent s.e. Values followed by different letters are significantly different at P < 0·05; n = 4.

¹³C recovered in spurs at 7 d after labelling

There were variations in the amounts of ¹³C recovered in spurs 7 DAL in all cultivars during fruit development (Fig. 4B). At 14 DAA (7 DAA plus 7 DAL), most ¹³C from young spur leaves was recovered in spurs and only a small portion of ¹³C was used for respiration and export. The proportion of losses in ‘Shinsetsu’ and ‘Shinsui’ was smaller than in ‘Kousui’ (Fig. 4C). At 35 DAA (28 DAA plus 7 DAL), almost half of the ¹³C was lost in respiration and export in ‘Shinsui’ and ‘Shinsetsu’ but only about 30 % in ‘Kousui’ (Fig. 4C). Compared with results at 35 DAA, more ¹³C was recovered at 70 DAA (63 DAA plus 7 DAL) in all cultivars. A very high proportion (approx. 93 %) of ¹³C was recovered in ‘Shinsetsu’ and only approx. 7 % was lost in respiration and export (Fig. 4B, C). Although there were no differences in proportion of losses via respiration and export among cultivars at 111 DAA (104 DAA plus 7 DAL) (Fig. 4C), ‘Shinsetsu’ retained more ¹³C in spurs than ‘Shinsui’ and ‘Kousui’ (Fig. 4B), because more ¹³C was assimilated by its spur leaves (Fig. 4A). Similarly, in comparison with ‘Shinsui’ and ‘Kousui’ at maturity, more ¹³C was recovered in ‘Shinsetsu’ at 191 DAA (184 DAA plus 7 DAL).

Amount of ¹³C allocated to individual organs

Distribution of ¹³C in individual organs is shown in Table 1. At 14 DAA, most of the ¹³C was retained in spur leaves, with ‘Shinsetsu’ retaining more than the other cultivars. Compared with ‘Kousui’ and ‘Shinsetsu’, very little ¹³C was recovered in fruit of ‘Shinsui’. At 35 DAA, the amount of ¹³C recovered in leaves was similar to that in fruit of ‘Shinsetsu’ and ‘Shinsui’, but more ¹³C was in leaves than fruit of ‘Kousui’. Subsequently, at 70 and 111 DAA, the amount of ¹³C retained in leaves was less than that incorporated into fruit in all cultivars. More ¹³C was allocated to fruit of ‘Shinsetsu’ than of the other cultivars at 35, 70, and 111 DAA, but at maturity there were no significant differences in amount of ¹³C allocated to fruit of different cultivars.

Table 1.

Amount of ¹³C allocated to individual organs of the spur 7 d after ¹³C labelling in three Japanese pear cultivars

					Fruit (mg)
DAA*	Cultivar	Leaf (mg)	Current shoot (mg)	Old wood (mg)	Flesh	Core	Pedicel	Total
14†	Shinsui	4·8b	0·2b	0·0b	–	–	–	0·2c
	Kousui	4·2b	0·5a	0·0b	–	–	–	2·7a
	Shinsetsu	15·5a	0·6a	0·1a	–	–	–	1·8b
35	Shinsui	3·9c	0·4b	0·1b	2·8b	0·8a	0·2b	3·8b
	Kousui	10·7a	1·6a	0·5a	3·0b	1·2a	0·4a	4·8b
	Shinsetsu	7·0b	0·4b	0·4a	6·0a	1·4a	0·7a	8·1a
70	Shinsui	7·7a	0·7a	0·6a	8·8b	4·3a	0·1a	13·2b
	Kousui	4·4b	0·5a	0·4a	8·8b	3·3a	0·1a	15·2b
	Shinsetsu	8·6a	0·6a	0·7a	15·4a	3·5a	0·1a	19·0a
111	Shinsui	1·9b	0·3a	0·1b	11·5b	1·0b	0·0a	12·5b
	Kousui	1·4b	0·4a	0·3a	11·6b	2·3a	0·1a	14·0b
	Shinsetsu	3·3a	0·5a	0·5a	15·3a	2·9a	0·0a	18·2a
194	Shinsui	nd‡	nd	nd	nd	nd	nd	nd
	Kousui	nd	nd	nd	nd	nd	nd	nd
	Shinsetsu	3·6	0·3	0·2	13·3	1·6	0·0	14·9

Data are the mean of four replicates.

^*DAA = days after anthesis.

^†The fruit at 7 DAA was not divided into flesh, core and pedicel for measurement because of its small size.

^‡nd = not detected.

^{a b c}Values followed by different letters are significantly different at P < 0·05 according to Duncan's multiple range test.

Further analysis of ¹³C distribution in fruit showed that most of the ¹³C was recovered in the flesh on each measuring date except at 14 DAA (because of the practical difficulty in division of small fruit). However, relatively more ¹³C was in the core at 70 DAA. Generally, ‘Shinsetsu’ had more ¹³C in flesh and fruit than the other cultivars at 35, 70, and 111 DAA. The proportion of the total ¹³C partitioned to current shoots and old wood was small in all cultivars during fruit growth. More ¹³C was allocated to current shoots in ‘Kousui’ than in the other cultivars at 14 and 35 DAA.

Effects of labelling date on ¹³C recovery at harvest

At harvest, the spurs labelled at different stages of fruit growth were sampled and separated to determine ¹³C distribution in individual organs (Table 2). The losses due to respiration and export at the time of harvest were higher than those at 7 DAL in all cultivars (Fig. 4C, Table 2). The proportion of ¹³C assimilated in spurs and recovered in leaves 7 DAA was 42–55 % in all cultivars: a considerable proportion of assimilated ¹³C was lost in respiration and export particularly in ‘Shinsui’ (47 %) compared to approx. 27 % in the other cultivars. However, only a very small proportion (approx. 3–5 %) of ¹³C incorported was recovered in leaves on other labelling dates. The proportion of ¹³C allocated to fruit at harvest varied with the labelling date, but increased as fruit grew. ‘Shinsui’ had the smallest proportion of ¹³C recovered in fruit of all three cultivars. A much larger proportion of ¹³C was incorporated into fruit in ‘Shinsetsu’ at 7 DAA than ‘Shinsui’. As mentioned earlier, P_n of mature spur leaves increased and remained relatively high during most of the period of fruit growth (Fig. 5). ‘Shinsui’ and ‘Kousui’, with rapid fruit growth (Fig. 2), had a higher P_n than ‘Shinsetsu’ during early growth; as the fruit matured the rate of P_n decreased in the three cultivars, with ‘Shinsetsu’ maintaining P_n much longer than the others.

Table 2.

Effects of labelling date on percentage of ¹³C distribution in individual organs of the spur at harvest in three Japanese pear cultivars

					Fruit
DAA*	Cultivar	Leaf	Current shoot	Old wood	Flesh	Core	Pedicel	Total	Loss
7†	Shinsui	42·5b	0·0c	0·9a	0·0b	9·2a	0·6b	9·8b	46·9a
	Kousui	55·5a	8·7a	1·1a	0·0b	6·1b	0·9b	7·0b	27·8b
	Shinsetsu	47·7b	0·4b	0·0b	20·4a	3·6b	1·1a	25·1a	26·7b
28	Shinsui	3·0a	1·4b	0·1b	19·6b	4·9a	0·5a	25·0b	70·3a
	Kousui	3·1a	10·7a	0·5a	29·4a	4·3a	0·6a	34·3a	51·4c
	Shinsetsu	3·1a	2·0b	0·4a	25·4a	4·3a	0·5a	30·2a	64·4b
63	Shinsui	3·8a	1·3a	1·6a	23·9b	7·0a	0·3a	31·2b	62·1a
	Kousui	3·5a	0·8b	0·3b	43·6a	3·7a	0·6a	47·9a	47·6b
	Shinsetsu	3·3a	2·0a	0·6b	25·9b	7·6a	0·4a	33·9b	60·4a
104	Shinsui	5·0a	1·1a	2·1a	45·3a	0·6c	0·1b	46·0b	45·8a
	Kousui	5·0a	1·1a	2·0a	43·5a	11·7a	0·2a	55·4a	36·6b
	Shinsetsu	3·0a	1·6a	1·6b	41·3a	2·5b	0·3a	44·1b	49·8a
184	Shinsui	nd‡	nd	nd	nd	nd	nd	nd	nd
	Kousui	nd	nd	nd	nd	nd	nd	nd	nd
	Shinsetsu	3·9	1·8	1·4	52·3	5·0	0·3	57·6	35·2

Data are the mean of four replicates.

^*DAA = days after anthesis.

^†13C labelling date.

^‡nd = not detected.

^{a b c}Values followed by different letters are significantly different at P < 0·05 according to Duncan's multiple range test.

Source strength and relative sink strength

Changes of source strength in Japanese pear cultivars during fruit growth are shown in Fig. 6. The late-maturing ‘Shinsetsu’ exhibited greater source strength than the earlier-maturing ‘Shinsui’ and ‘Kousui’ at each labelling date. Although no significant differences were observed at 7 and 63 DAA between ‘Shinsui’ and ‘Kousui’, the latter had greater source strength than the former at 28 and 104 DAA.

Fig. 6.

Source strength (source weight × source specific activity) 2 h after ¹³CO₂ exposure to the spur during fruit development in three Japanese pear cultivars. Vertical bars represent s.e. Values followed by different letters are significantly different at P < 0·01; n = 4.

During the initial period of fruit growth, spur leaves had greater RSS than other organs (Fig. 7), although a smaller RSS of spur leaves was observed in ‘Kousui’. The RSS of current shoots decreased with time after anthesis. Old wood also had small RSS; this increased from 14 to 35 and 70 DAA but then decreased again. The RSS of fruit was small at 14 DAA but increased progressively to over 80 % of the ¹³C assimilated.

Fig. 7.

Relative sink strength (RSS) of individual organs in the spur (without bourse shoots) during fruit development in three Japanese pear cultivars. Vertical bars represent s.e. ^*, RSS significantly different from that in ‘Shinsui’ at P < 0·05, as shown by one-way anova followed by Duncan's multiple test; n = 4.

DISCUSSION

Allocation of ¹³C assimilate during fruit growth

In Japanese pear, fruit growth was shown by Hayashi (1960) to depend on reserve carbohydrates before the spur leaves could provide photosynthate for early fruitlet growth within 3–4 weeks after anthesis. However, Teng et al., (1999) reported that unfolded leaves could export labelled photosynthate to fruitlets 7 DAA in young ‘Nijisseiki’ pear. This is confirmed by our study. The amount and proportion of ¹³C allocated to fruit were cultivar-dependent (Table 1) and can be mainly ascribed to differences in leaf growth (Fig. 1), photosynthetic ability (Fig. 5) and source strength (Fig. 6) among cultivars. The relatively poor allocation of ¹³C to fruit of ‘Shinsui’ may be explained by a comparatively higher RSS of the current shoot (Fig. 7, Table 1) which is related to active vegetative growth (unpublished results).

At 28 DAA, the fruit of ‘Shinsui’ and ‘Kousui’ are in a transitional phase when cell division ceases and cell enlargement starts, whereas ‘Shinsetsu’ fruits are still in the active cell division phase and have not reached the linear growth phase (Figs 2, 3). Although there was no difference in the RSS of fruit between ‘Shinsui’ and ‘Shinsetsu’ (Fig. 7), the differences in the total leaf area per spur (Fig. 1) and the total amount of ¹³C assimilated by spur leaves (Fig. 4A) resulted in differences in the amount of ¹³C allocated to fruits. Apparently more photosynthate is available and invested in the early fruit development of the later-maturing cultivar. In contrast, ‘Kousui’ fruit grew somewhat faster than ‘Shinsui’ (Fig. 2), but there was no difference between them in the amount of ¹³C allocated to fruit (Table 1). This could be partially interpreted as a consequence of the larger fruit in ‘Kousui’ at anthesis (data not shown).

At 63 DAA, there was no significant difference between the cultivars in total amount of ¹³C assimilated by spur leaves, but there was in total leaf area per spur. At this stage, ‘Shinsetsu’ fruit are just in the early phase of cell enlargement while the fruit of ‘Shinsui’ and ‘Kousui’ are in the phases of rapid growth. Enhanced leaf photosynthetic rate (Fig. 5) and RSS of fruit in ‘Shinsui’ and ‘Kousui’ supported the conclusion that, generally, photosynthesis and carbohydrate metabolism in source leaves respond to sink activity (Paul and Foyer, 2001). Regardless of fruit, RSS of ‘Shinsetsu’ was significantly lower than that of ‘Shinsui’ and ‘Kousui’, in which more ¹³C was translocated to fruit, and incorporated particularly into the flesh. This could be partially explained by a much smaller proportion of ¹³C loss in ‘Shinsetsu’.

At 104 DAA, fruit of ‘Shinsui’ and ‘Kousui’ were mature while those of ‘Shinsetsu’ were still in the linear growth phase, because of its longer duration of cell enlargement and therefore of fruit growth (Figs 2, 3). Although no significant differences occurred in the RSS of fruit between cultivars (Fig. 7), there was greater ¹³C allocation to fruit in ‘Shinsetsu’ than in the other cultivars (Table 1) because of its greater source strength (Fig. 6). ‘Shinsetsu’ still had greater source strength at 184 DAA (as well as at 104 DAA) because of the maintenance of active sinks and longer leaf area duration.

Losses in respiration and export

In addition to sink activity and sink size, carbon accumulation depends on sink characteristics such as photosynthesis and respiration of the fruit itself, as well as carbon supply via the peduncle attachment (Henton et al., 1999). As with apple (Bepete and Lakso, 1997), pear fruit has a high respiration rate early in development, when cell division is active, and this subsequently declines as fruit matures. By comparison with that 7 DAL at each developmental stage, the losses increased at final harvest and varied with labelling date and cultivar (Table 2). In ‘Shinsetsu’, a very low proportion of loss (approx. 7 %) occurred 7 DAL at 63 DAA, but it approached 60 % at final harvest (Fig. 4C, Table 2), attributable to its long period of fruit enlargement. As with avocado (Richings et al., 2000), different losses may partially contribute to the variation in fruit size between ‘Shinsui’ and ‘Kousui’, which have similar duration of fruit growth.

Effects of assimilate supply for fruit growth

Generally, it is accepted that if more photosynthate (carbohydrates) are available, they will be allocated to fruits and result in a large fruit size of later-maturing cultivars, with longer periods of fruit enlargement. However, the early stage of fruit growth and assimilate availability is also important in pear, as our data show. Flowers emerge earlier than spur leaves in Japanese pear (Jackson, 2003). Many studies have pointed out that the current photosynthate available during the initial period of fruit growth has an important role in cell division (Hayashi and Tanabe, 1991; Bepete and Lakso, 1998; Famiani et al., 2000; Jackson, 2003). Histology indicates that the cell number should be the primary determinant of final fruit size in Japanese pear, as has been shown in peach (Scorza et al., 1991) and apple (Westwood et al., 1967). Therefore, photosynthate availability is crucial for fruit growth especially during the period of cell division, and is closely correlated with final fruit size. The crop-load is very low in the flat-canopied pergolar system and no source limitation for fruit growth was observed after fruit thinning (Zhang et al., 2005). However, before fruit thinning there are more fruit per spur because of enhanced fruit set by hand-pollination compared with natural pollination. Furthermore, the earlier-maturing cultivars always have more flowers per cluster on spurs than the later-maturing cultivars (unpublished results). Thus, less photosynthate would be allocated to individual fruit in earlier-maturing cultivars than in the later-maturing ones, and early fruit growth of earlier-maturing cultivars (but not of later-maturing) was severely restrained by assimilate supply rather than by sink limitation. Grossman and DeJong (1995) have also pointed out that source limitation early in fruit development of peach results in permanent loss of growth potential. Therefore, early fruit thinning should be more important for large fruit production in the early-maturating cultivars.

As in apple, spur leaves of Japanese pear are a larger proportion of total leaf area early in the season than at full canopy (Forshey et al., 1987; Hayashi and Tanabe, 1991). For larger fruit production, therefore, earlier leaf emergence and leaf production are very important to ensure rapid fruit development in the early-maturing cultivars with shorter duration of fruit growth. Manipulation of assimilate supply may be achieved by pruning-out water shoots (one of the vegetative shoot types with excessive growth) in late October to promote leaf production and earlier leaf emergence, and sink capacity may be regulated by retaining as many flower buds as possible, by light spur pruning, and by altering fruit number and size by fruit thinning to optimize sink capacity in ‘Shinsui’ (Hayashi and Tanabe, 1991).

In conclusion, fruit growth of Japanese pear depends on the interaction of assimilate supply from source leaves (a function of leaf area and photosynthetic ability), and on the capacity (number, size, activity and duration of growth) of the sinks. We have shown that early assimilate supply as well as continued fruit growth and assimilate production are important for development of large fruit in Japanese pear.