Developmental Changes in Xylem Functionality in Kiwifruit Fruit: Implications for Fruit Calcium Accumulation B. Dichio Dipartimento di Produzione Vegetale, Università della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy D. Remorini Dipartimento di Coltivazione e Difesa delle Specie Legnose, Università di Pisa Via del Borghetto, 80-56124 Pisa, Italy S. Lang HortResearch, Private bag 11 030, Palmerston North, New Zealand Keywords: xylem conductivity, dye infiltration, fruit growth, and microscopy imaging analysis Abstract In kiwifruit, as in most fruits, storage quality is related to calcium concentration and many disorders are associated with low fruit calcium status. Previous studies show that after an early rise, fruit calcium concentration decreases because calcium influx ceases by the mid-growth stage whereas volume growth continues till harvest. Calcium transport to the fruit is exclusively via the xylem - calcium is not phloem mobile. We postulate that declining fruit xylem functionality is responsible for this pattern of calcium accumulation. Xylem functionality was measured from fruit setting to harvest in Zespri Gold kiwifruit. The applied measurement technique consists in transpiring an apoplast-mobile dye into detached fruit through the stalk and recording the percentage of dyed (functional) bundles in sections cut along the length of the fruit. For the middle part of the fruit analysed, the dye method revealed high bundle functionality till about d10 (after bloom). A drastic reduction in the number of functional bundles occurred around d20 and, again, around d55 and d90. Some recovery was observed between these time steps. An almost complete permanent dysfunction was evident nearly at d120 up to harvest time. Fluctuations in bundle functionality are interpreted as being due to vessel breakage (resulting from stretching caused by fruit expansion) and temporary functional recovery resulting from early season differentiation of new vessels. The length growth rate is very high in the first 8 weeks from full bloom with a maximum value of 1.3 mm d -1 at the 4 th week. In this period, the fruit achieves 70% of the total length at harvest time. We conclude that patterns of xylem dysfunction in Zespri Gold provide a satisfactory explanation of the well-known patterns of fruit calcium accumulation. INTRODUCTION In kiwifruit, as in most fruits, storage quality is related to calcium concentration and many disorders are associated with low fruit calcium status. While much attention has been paid to study mineral nutrient absorption and partitioning in different kiwifruit plant organs during the season, (Clark and Smith, 1988; Clark and Smith,1991; Fergurson, 1980) the transport of water in the fruit has not been investigated. Many studies have been carried out on the accumulation of mineral nutrients, calcium in particular, in apple fruit and in grape which are respectively badly affected by physiological disorders like bitter-pit and bunch stem necrosis which are correlated to calcium deficiency (Saure, 1996; Lang et al., 1994). Low calcium concentration in kiwifruit has been found to be involved in premature fruit softening (Prasad and Spiers, 1992). In kiwifruit, previous studies show that after an early rise, fruit calcium concentration decreases because calcium influx ceases by the mid-growth stage whereas volume growth continues till harvest (Xiloyannis et al., 2001), while potassium as well as Proc. IS on Kiwifruit Ed. H. Huang Acta Hort. 610, ISHS 2003 191
phosphorus continued to move into the fruit over the whole growing season thus making their total amount in the fruit increase. It is well known that calcium transport to the fruit is exclusively via the xylem - calcium is not phloem mobile (Marschner, 1983, White, 2001). Therefore, the amount of calcium into the fruit is related to the fruit transpiration rate, and the vascular xylem efficiency. It has been suggested that the low Ca and K/Ca imbalance observed in bitter-pit affected apples may be related to the imbalance between xylem and phloem supplies (Fergurson and Watkins, 1989). For better understanding this phenomenon, the contribution of phloem and xylem stream to fruit growth was studied (Lang, 1990). In kiwifruit fruit, the same pathway has been found using a mass flow-vascular transport model based on carbon and water influxes to predict the relative contribution of phloem and xylem to the total mineral nutrient supply to the fruit (Clark and Smith, 1988). The question is why the xylem contribution to growth is very small or negligible in late fruit growth stage. Many studies have highlighted that calcium accumulation is related to fruit transpiration rate. In fact, the reduction of the transpiration rate in kiwifruit is coincident with the calcium accumulation stoppage (Xiloyannis et al., 2001). The transpiration rate of fruits is certainly dependent on climate, weather, orchard management etc., and their seasonal decrease has been correlated with the loss of surface conductance (Smith et al. 1995) probably due to the development of a suberized periderm (Schmid, 1978). No studies on fruit xylem development and efficiency of kiwifruit are available. We postulate that declining fruit xylem functionality during the season is responsible for the fruit transpiration pattern, the changes in phloem and xylem balance, and implications for calcium accumulation. The purpose of this study is to verify the hypothesis that fruit growth stretches the vascular bundles in the flesh that causes the disruption of xylem vessels. This mechanism is involved in the reduced calcium import via xylem to the fruit. In this paper, the preliminary results on xylem functionality on Zespri Gold TM are presented. MATERIAL AND METHODS Plant Material The trials were conducted in a 10 years old mature kiwifruit orchard of Massey University in Palmerston North, New Zealand. We used a commercial variety Zespri Gold TM (Actinidia chinensis) that is a new variety that has been widely spread in many countries, in particular in New Zealand, in the last 5 years. Orchards were trained at T-bar and a spacing of 5x6, with N-S row orientation, organically managed with no-tillage and supplemental pollination. At the start of flowering, on November 4 th for Zespri Gold TM, about 700 flowers on determinate fruiting shoots located in the horizontal part of the canopy were selected. Once flowers were wide open, they were labelled indicating the exact day of full bloom. This was a very accurate measurement to be sure about the age of each flower and therefore reduce variability during sampling. Dye Infiltration During the experiment and throughout the growing season, the fruits were collected and analysed by a dye-infiltration technique. The total measurements were 26 from full bloom to harvest time with different sampling frequencies throughout the growing season. For the first 65 days FFB 5 fruits every 4-5 days were collected, from 65 to 90 days 5 fruits every 7 days and from 90 to 170 days (harvest time) 10 fruits every 14 days. For each measurement, the fruits were collected from the orchard before dawn, when the xylem water potential was close to zero. To avoid air embolism, each fruit was cut at the base of the peduncle in water, immediately placed in a polyethylene bag that 192
was promptly sealed to avoid fruit transpiration and then transferred to the lab. In the laboratory, the fruits were prepared to start the dye-infiltration technique; the proximal end of the peduncle was cut again in water with a sharp blade to expose a fresh, uncrushed surface. After that, the distal end of the peduncle was put a ring of melted paraffin which, once solidified proved to be a good insulation material to prevent surface capillary movement of the dye solution throughout the fruit. The fruit was transferred to a small vial containing the dye solution (Fig. 1) and placed for 75 minutes in wind tunnel equipment which provides standard and high transpiration conditions. The apoplastic dye (toluidine blue, 0.5% aqueous) moved upward into the fruit through the stalk carried by the fruit transpiration flow. Determination of Xylem Functionality After being treated with dye infiltration, all the fruits sampled were cut into 10 slices starting from the stylar end (the 10 th ) till the basal end (the 1 st ), the 5 th slice being in the middle. For the purpose of this preliminary work, the analysis was performed on the fifth slice of each fruit using an optical microscopy and image-analysis tools. The median dorsal carpellary bundles were scored and the stained ones counted. The percentage of functionality was calculated as 100x (no. of stained bundles/ total number of functional bundles). RESULTS Fruit Growth The Zespri Gold TM is an early flowering variety, under the same climatic conditions its flowering time was about 30 days earlier than Hayward and its full duration was almost 1 week. As for the fruit growth, fig. 2 illustrates quite well the double sigmoid curve characteristics of other kiwifruit species. The length growth rate is very high in the first 8 weeks from full bloom with a maximum value of 1.3 mm d -1 at the 4 th week. In this period the fruits achieve 70% of the total length at harvest time. From the 8 th week on, the fruits exhibit a more gradual length growth. Xylem Functionality The xylem functionality of fruits changed throughout the season (Fig. 3). In the first 10 days FFB (from full bloom) the percentage of functional bundles was high (between 95-100%) while a total dysfunction occurred at d20 and, again, around d55, d90 and d120. Between these time steps some recovery on bundles functionality was observed (value from 50% up to 90%) while a permanent and total dysfunction occurred after at d120 up to harvest time. DISCUSSION It is well know that in developing fruit there is a shift in the phloem:xylem supply balance. In particular, starting from mid-season up to the end of the growing season, the contribution to growth is almost phloem dominant (Lang, 1990). Certainly, the xylem supply depends on vessels functionality and so its conductivity efficiency in the sap transport. The results of the dye experiment confirm the hypothesis that xylem functionality decreases during the growth season. Figure 3 shows a sharp reduction in the percentage of functional bundles after d90 FFB till complete total dysfunction around d120 and towards the end of harvest time. The xylem dysfunction is caused by vessel breakage upon stretching resulting from fruit elongation (data not shown). Functional recovery of the xylem occurs at the early fruit growth stage, supposedly due to metaxylem activity and thus to the formation of new xylem vessels. After the last dysfunction peak observed at d90 and d120 FFB, metaxylem is no longer active and dysfunction becomes permanent. We may conclude that xylem vessel dysfunction during the season, together with other factors, may cause a sharp reduction in the fruit transpiration rate 8 9 weeks 193
after flowering and consequently a decline in calcium accumulation in kiwifruit, as already reported by Xiloyannis et al (2001) and Ferguson (1980). Our results indicate that to increase calcium concentration in fruits, calcium has to be supplied in the early weeks that correspond to the period of maximum functionality of the xylem transport system of the fruit. However, for a better knowledge of xylem functionality, it is suggested to further investigate the conductivity of the peduncle fruit pathway and study the effects of the radiative flux available in the canopy on xylem functionality and on calcium accumulation in the fruit. ACKNOWLEDGEMENTS The authors wish to acknowledge Dr. L. Drazeta for helpful discussions. This study was supported by COFIN-2001 National Project. Literature Cited Prasad, M. and Spiers, T.M. 1992. The effect of nutrition on the storage quality of kiwifruit (a review). Acta Hort. 297: 579-585. Xiloyannis, C., Celano, G., Montanaro, G., Dichio, B., Sebastiani, L., Minnocci A. 2001. Water relations, calcium and potassium concentration in fruits and leaves during annual growth in mature kiwifruit plants. Acta Hort., 564:129-134 Smith, G.S. Klages, K.U., Green, T.G.A, Walton, E.F. 1995. Changes in abscisic acid concentration, surface conductance, and water content of developing kiwifruit. Scientia Horticulturae 61, 13-27 Clark, C.J. and Smith, G.S. 1988. Seasonal accumulation of mineral nutrients by kiwifruit. 2.Fruit. New Phytol. 108, 399-40 Clark, C.J. and Smith, G.S. 1991. Seasonal changes in the form and distribution of calcium in the fruit of kiwifruit vines. Journal of Horticultural Science. 66 (6) : 747-753. Saure, M.C. 1996. Reassessment of role of calcium in development of bitter pit in apple. Aust. J. Plant Physiol. 23, 237-43 White, P.J. The pathways of calcium movement to the xylem. 2001. J. of Exp. Bot. Vol. 52, n0. 358, pp. 891-899. Düring, H., Lang, A. and Oggionni, F. 1987. Patterns of water flow in Riesling berries in relation to developmental changes in their xylem morphology. Vitis, 26: 123-131. Ferguson, I.B. and Watkins, C.B. 1989. Bitter pit in apple fruit. Horticultural Reviews, 11: 289-355. Ferguson, I.B. 1980. Movement of mineral nutrient into developing fruit of the kiwifruit (Actinidia Chinensis Planch). N.Z. Journal of Agricultural Research. 23: 349-353. Lang, A. 1990. Xylem, phloem and transpiration flows in developing apple fruits. Journal of Experimental Botany, 41: 645-651. Lang, A. and Düring, H. 1991. Partitioning control by water potential gradient: Evidence for compartmentation breakdown in grape berries. Journal of Experimental Botany, 42: 1117-1122. Lang, A. and Ryan, K.G. 1994. Vascular development and sap flow in apple pedicels. Annals of Botany, 74: 381-388. Marschner, H. 1983. General introduction to the mineral nutrition of plants. In: Encyclopedia of plant physiology, New Series, Volume 15A. Eds A. Läuchli and R.L. Bielski. Springer-Verlag, Berlin, Heidelberg, New York. Pp 5-60. 194
Figures Fig. 1. Diagram to show the arrangement used for dye infiltration experiment. Fruit lenght (mm) 80 70 60 50 40 30 20 10 Lenght Regression Growth rate 0 0 20 40 60 80 100 120 140 160 Time from full bloom (d) Fig. 2. Fruit length (solid line) and length growth rate (open circle) during the growing season. All points represent the average of values recorded on 10 fruits. 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Lenght growth rate (mm d -1 ) Fraction of boundles with dye (%) 100 80 60 40 20 0 0 20 40 60 80 100 120 140 160 Time from full bloom (d) Fig. 3. Fluctuation of percentage of bundle stained during the growing season in the middle part of the fruit. Each point represents the mean value of 5-10 fruits. 195