The amino acid distribution in rachis xylem sap and phloem exudate of Vitis vinifera ‘Cabernet Sauvignon’ bunches

Highlights

• The vascular sap of the grape bunch rachis differs in composition from leaf petiole sap.

• Glutamine/glutamate were the predominant amino acids in the xylem sap.

• Arginine and glycine were the principal amino acids of the phloem sap.

• The amino acid composition of vascular sap alters during the day and over the season.

Abstract

Amino acids are essential to grape berry and seed development and they are transferred to the reproductive structures through the phloem and xylem from various locations within the plant. The diurnal and seasonal dynamics of xylem and phloem amino acid composition in the leaf petiole and bunch rachis of field-grown Cabernet Sauvignon are described to better understand the critical periods for amino acid import into the berry. Xylem sap was extracted by the centrifugation of excised leaf petioles and rachises, while phloem exudate was collected by immersing these structures in an ethylenediaminetetraacetic acid (EDTA) buffer. Glutamine and glutamic acid were the predominant amino acids in the xylem sap of both grapevine rachises and petioles, while arginine and glycine were the principal amino acids of the phloem exudate. The amino acid concentrations within the xylem sap and phloem exudate derived from these structures were greatest during anthesis and fruit set, and a second peak occurred within the rachis phloem at the onset of ripening. The concentrations of the amino acids within the phloem and xylem sap of the rachis were highest just prior to or after midnight while the flow of sugar through the rachis phloem was greatest during the early afternoon. Sugar exudation rates from the rachis was greater than that of the petiole phloem between anthesis and berry maturity. In summary, amino acid and sugar delivery through the vasculature to grape berries fluctuates over the course of the day as well as through the season and is not necessarily related to levels near the source.

Introduction

The reproductive structures of plants are particularly important targets of carbon (C) and nitrogen (N) partitioning. In the grape berry mesocarp, the N is found predominantly as free amino acids, but proteins, ammonium and nitrate are also present (Roubelakis-Angelakis and Kliewer, 1992). Among their diverse functions, the amino acids serve as precursors to anthocyanins and aroma compounds (Rapp and Versini, 1991) and their concentrations, along with sugars, are important for attracting birds to aid in seed dispersal. From a winemaking perspective, free amino acids in the juice of grape berries are required for successful alcohol and/or malolactic fermentation (Spayd and Andersen-Bagge, 1996). It is, therefore, important to understand how the concentration and composition of amino acids within the berry is affected by developmental stage, soil type, rootstock vigour, weather conditions (Huang and Ough, 1989) and vineyard cultural practises, especially N fertilisation (Holzapfel and Treeby, 2007) and irrigation (Matthews and Anderson, 1988). Amino acids are present in all parts of the berry with the highest concentration located in the pulp, followed by the skin and finally the seeds (Stines et al., 2000). More than 20 amino acids have been detected in grape juice, including alanine, arginine, aspartic acid, glutamic acid, proline, serine and threonine (Kliewer, 1968, Stines et al., 2000). Cultivar differences are apparent, however, with arginine being the most predominant amino acid in the juice of Semillon and Pinot Noir and proline in Cabernet Sauvignon and Chardonnay (Spayd and Andersen-Bagge, 1996).

Whilst a portion of the amino acids within the grape berry is derived from in situ synthesis, most of the amino acids are imported through the vascular system (Wermelinger, 1991). The amino acids in the phloem are mainly derived from synthesis in the leaves while those in the xylem sap are primarily produced in the roots. Inorganic N is taken up from the soil as nitrate or ammonium, and may then be reduced to amino acids in the roots before transport to other plant components. Root derived N is transported to the leaves, and then later remobilised to the other sink organs through the phloem (Rentsch et al., 2007). The phloem is thought to be the major contributor to the accumulation of amino acids in the ripening berry (Gholami, 1996). This is because, even though the amino acid distribution of grapevine phloem and xylem saps are similar, generally higher concentrations are found in the phloem at approximately 100 mmol L⁻¹ (Glad et al., 1992b) as compared to the xylem at 3–20 mmol L⁻¹ (Andersen and Brodbeck, 1989, Glad et al., 1992a, Andersen et al., 1995, Peuke, 2000). In other crops, seasonal changes in phloem composition were observed in response to growth stage (Weibull, 1987, Karley et al., 2002) and time of day (Sharkey and Pate, 1976, Smith and Milburn, 1980, Winter et al., 1992).

Historically, phloem sap was collected either through natural exudation properties of a limited number plant species (Pate et al., 1974) or stylectomy (Kennedy and Mittler, 1953, Weatherley et al., 1959). However, natural exudation is very brief in Vitis vinifera and there are no known aphids feeding on phloem in grapevines. Another method using radioactive labelled substances has been applied to the grapevine. It can be used to trace the transport of photoassimilate, however it does not allow the measurements of phloem components (Candolfi-Vasconcelos et al., 1994). The EDTA-facilitated exudation technique was used successfully in Vitis vinifera to assess phloem sap components (King and Zeevaart, 1974) and consists of dipping the excised organ in a buffered solution comprising of HEPES and EDTA. The latter is required to chelate calcium, and thus avoid callose formation in the sieve plates, a phenomenon that occurs naturally in the plant to prevent the loss of phloem sap after damage. By using phloem collection techniques such as these, it has been established that the composition of the vascular sap of the leaf is often quite different from that of the sink (Van Bel, 1990).

With the onset of ripening, grape bunches become a strong sink for sugars and amino acids. The composition of phloem sap in the bunch rachis could be expected to adjust according to changes in partitioning throughout the season and also alterations in the mechanism of phloem unloading from symplastic to apoplastic within the fruit around veraison (Zhang et al., 2006). Similarly, the xylem contribution to berry growth declines after veraison (Rogiers et al., 2000, Bondada et al., 2005) and this may be accompanied by alterations in xylem sap composition. The objective of this work was to assess seasonal and diurnal trends in rachis xylem and phloem amino acid composition of grapevine bunches in order to gain a better understanding of underlying factors driving berry composition. An assessment of the xylem and phloem saps of the leaf petiole was also undertaken to compare the composition at one potential source with that of the sink.