Plant Physiology Preview. Published on July 10, 2015, as DOI:10.1104/pp.15.00031
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Running Head: Water transport and xylem function of the pedicel
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Corresponding Authors:
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Thorsten Knipfer
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Department of Viticulture & Enology
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University of California
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1 Shields Ave.
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Davis, CA 95616
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Phone: (530)752-1762
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E-mail: [email protected]
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Mark A. Matthews
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Department of Viticulture & Enology
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University of California
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1 Shields Ave.
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Davis, CA 95616
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Phone: (530)752-2048
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E-mail: [email protected]
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Research Category: Ecophysiology and Sustainability
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Title: Water transport properties of the grape (V. vinifera L.) pedicel during fruit
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development: Insights into xylem anatomy and function using microtomography
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Authors: Thorsten Knipfer1*, Jiong Fei1, Gregory A. Gambetta1+, Andrew J. McElrone1,2,
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Kenneth A. Shackel3, Mark A. Matthews1*
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Institution Addresses:
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Department of Viticulture and Enology, University of California, Davis, CA 95616, USA
USDA-ARS, Crops Pathology and Genetics Research Unit, Davis, CA 95616
Department of Plant Sciences/Pomology, University of California, Davis, CA 95616
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Summary:
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Xylem flow into the fruit declines at the onset of ripening and losses in grape pedicel
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hydraulic conductivity could be attributed to xylem vessel blockages.
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Footnotes:
This work was supported by the U.S. Department of Agriculture/Cooperative State
Research, Education, and Extension Service (grant no. 2006-35100-17440). The Advanced
Light Source is supported by the Director, Office of Science, Office of Basic Energy
Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231.
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Present address: Institute des Sciences de la Vigne et du Vin, Bordeaux, France
*Corresponding authors; e-mail [email protected], [email protected]
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ABSTRACT
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Xylem flow of water into fruits declines during fruit development, and literature indicates a
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corresponding increase in hydraulic resistance in the pedicel. However, it is unknown how
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pedicel hydraulics change developmentally in relation to xylem anatomy and function. In this
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study on grape (Vitis vinifera L.), we determined pedicel hydraulic conductivity (kh) from
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pressure-flow relationships using hydrostatic and osmotic forces, and investigated xylem
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anatomy and function using fluorescent light microscopy and x-ray computed
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microtomography (microCT). Hydrostatic kh (xylem pathway) was consistently four orders of
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magnitude greater than osmotic kh (intracellular pathway), but both declined before veraison
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by ~40 % and substantially over fruit development. Hydrostatic kh declined most gradually for
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low (< 0.08 MPa) pressures, and for water in- and out-flow conditions. Specific hydraulic
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conductivity (kh per xylem area) decreased in a similar fashion to kh despite substantial
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increases in xylem area. MicroCT images provided direct evidence that losses in pedicel kh
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were associated with blockages in vessel elements, whereas embolisms were negligible.
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However, vessel elements were well inter-connected and some remained continuous post-
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veraison suggesting that across the grape pedicel a xylem pathway of reduced kh remains
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functional late into berry ripening.
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Keywords:
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embolism, hydraulic conductivity, hydraulic isolation, ripening, veraison, Vitis, xylem
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occlusion
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INTRODUCTION
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In grapes, fruit growth by water accumulation follows a double sigmoid pattern and is
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influenced by the diurnal and developmental changes in water flows between fruit and parent
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plant (Matthews and Shackel, 2005). Until the onset of fruit ripening (i.e. veraison) water
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enters the fruit predominantly via the xylem and thereafter mainly through the phloem
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(Greenspan et al., 1994, 1996). Choat et al. (2009) showed that the hydraulic conductance
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(i.e. 1/resistance) of the grape berry and pedicel declines substantially at latter ripening
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stages predominantly due to a decline in pedicel conductance. Significant developmental
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changes in pedicel hydraulic properties were also reported for tomato, and were found to be
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associated with xylem anatomical changes (Lee et a., 1988; Van Ieperen et al., 2003; Rancic
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et al., 2008, 2010). Due to its position along the vascular transport pathway between fruit
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and parent plant, the pedicel can play an important role in affecting fruit growth (kiwi,
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Mazzeo et al., 2013). However, for grape it needs to be elucidated how pedicel hydraulic
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properties change developmentally in relation to xylem anatomy and function.
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The location and nature of the loss in hydraulic conductance between the parent plant and
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the fruit is unclear and may differ among fruits. For tomato, Malone and Andrews (2001)
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showed that most loss of hydraulic conductance occurs in the fruit per se, but Van Ieperen et
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al. (2003) reported important and decreasing hydraulic conductance in the pedicel
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abscission zone over fruit development. For Citrus, Garcia-Lius et al. (2002) reported that
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xylem vessels in the pedicel remain largely functional late into fruit ripening. For grape,
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although vessel breakage in the berry was thought to lead to xylem dysfunction (Coombe
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and McCarthy 2000), several studies and methods have shown that xylem vessels in the
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fruit remain functional (Rogiers et al. 2001; Bondada et al., 2005; Chatelet et al. 2008a,b). In
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line with these findings, data by Keller et al. (2006) suggest that the pedicel xylem also
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remains at least partially functional in ripening grape berries and can conduct water to and
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from the parent plant. Nevertheless, a reduction in the ability to transport water during
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ripening has been reported for grape (Tyerman et al. 2004; Choat et al. 2009) and other
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fleshy fruits (e.g. apple, Lang and Ryan, 1994; kiwi, Mazzeo et al. 2013), and it still remains
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unclear what causes this loss in xylem hydraulic conductance. For the grape pedicel, Choat
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et al. (2009) detected higher concentrations of xylem solutes post-veraison, and proposed
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that this is related to the deposition of gels into the xylem vessel lumen. However, direct
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evidence for the presence of xylem blockages and/or embolism formations in the grape
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pedicel is missing.
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The present study of the grape (Vitis vinifera 'Cabernet Sauvignon') pedicel was conducted
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with the goal to obtain a comprehensive understanding of how changes in hydraulic
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properties relate to changes in xylem structure and function over fruit development. Over the
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course of fruit development from 20 to 90 days after anthesis, water transport properties of
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pedicels were investigated under osmotic and hydrostatic driving forces using a modified
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pressure-probe system. This was combined with analyses of spatial and temporal changes
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in pedicel xylem anatomy and function using fluorescent light microscopy and x-ray
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computed microtomography (microCT; Brodersen et al., 2010 2013; Rancic et al., 2010).
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RESULTS
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Fruit and pedicel enlargement
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From 20 to 90 DAA, fruit enlargement was evident from an increase in fruit diameter and
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followed three Stages (I, <40 DAA; II, 40-55 DAA; III, >55 DAA) (Fig.2a). The transition from
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Stage II to III coincided with veraison. Fruit solute potential started to decrease before
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veraison during Stage II from on average -0.82 MPa to -1.54 MPa, and continued to
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decrease in Stage III to on average -3.97 MPa (Fig.2a). Enlargement of the pedicel
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receptacle and stem portion ceased before or around veraison, respectively (Fig. 2b,c).
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Pedicel receptacle diameters increased mainly in Stage I from on average 2.9 mm to 3.4
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mm, but changed little thereafter (Fig. 2b). Pedicel stem diameters increased until the end of
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Stage II from on average 1.3 mm to 1.7 mm (Fig.2c). Pedicel length remained at ~4 mm
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throughout fruit development (Fig.2d).
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Spatial and temporal arrangement of xylem
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Transverse light microscopy images of the pedicel stem and receptacle portion show that the
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vascular system consisted of heavily lignified xylem tissue that was separated by less
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lignified ray cells (Fig.3). The enlarged image of the stem portion highlights that phloem
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tissue was located in close proximity to the outer peripheral xylem tissue and was bordered
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by lignified fiber cap cells in the cortex (Fig.3a). Fiber cap cells were most pronounced in
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pedicel stem portion. Over fruit development, in pedicel stem (Fig.3b, panel 1) and
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receptacle portion (Fig.3b, panels 2 and 3) cross-sectional areas of heavily lignified xylem
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tissue (Ax) increased mainly up to veraison. From 20 to 90 DAA, Ax in the pedicel stem
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portion increased by ~2.7-fold and in the receptacle portion by ~4.0-fold. Comparing both
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pedicel portions, Ax of the stem portion was more than 2-fold smaller as compared to the
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receptacle portion. A unique feature of the receptacle portion in proximity to the fruit was
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radial branches of lignified xylem tissue stretching into the cortex (Fig.3b, panel 3), which
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potentially connect with peripheral vascular bundles in the fruit.
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Hydraulic properties
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For pedicels analyzed between 20 to 90 DAA, kh was derived from pressure-flow
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relationships as measured under hydrostatically and osmotically driven water flows (Fig.4).
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The rate of volume flow (Q) induced hydrostatically ramped up when ΔP exceeded 0.08 MPa
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(Fig.4a), and the pressure-flow relationship was typically composed of two linear parts of ΔP
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= -0.05 to 0.08 MPa and ΔP >0.08 MPa (see also Fig.S3 for pressure-flow characteristics of
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two additional pedicels). In osmotic experiments, Q was much smaller than in hydrostatic
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experiments, and for Δπ of 0.6 to 3.1 MPa the pressure-flow relationship was assumed to be
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linear (Fig.4b). Pedicel kh under osmotically and hydrostatically driven water flows were
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derived from the slopes of the corresponding linear regression lines.
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Pedicel hydrostatic and osmotic kh declined by ~40 % until veraison (~55 DAA) and
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substantially over fruit development (Fig.5). Hydrostatic kh as measured from multiple and
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low ΔPs (≤0.08 MPa) declined gradually and significantly by ~3-fold from ~32 DAA (Stage I)
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to ~62 DAA (early Stage III) (Fig.5a). In comparison, hydrostatic kh as measured from high
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ΔPs (>0.08 MPa) was typically ~2-fold greater, and corresponding losses in pedicel kh were
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less gradual (Fig.5a). Similarly to hydrostatic kh, osmotic kh declined significantly by ~2-fold
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from ~30 DAA to ~76 DAA, but was consistently 3- to 4-orders of magnitude lower than
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hydrostatic kh (Fig.5a). Based on measurements using low ΔPs, pedicel hydrostatic kh was
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also determined exclusively for water inflow (i.e. 0 to 0.08 MPa) and outflow (i.e. -0.05 to 0
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MPa) conditions (Fig.5b). Hydrostatic kh values for both directions of water flow were
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comparable but under water outflow conditions early development changes in kh were most
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pronounced. When hydrostatic kh as measured from low ΔPs was normalized for Ax of
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pedicel stem or receptacle portion (Fig.5c), hydrostatic ks declined significantly from ~32
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DAA (Stage I) to ~62 DAA (early Stage III), for both pedicel portions, which was similar to
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the developmental reduction in kh.
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Xylem structure and function
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Transverse microCT images of representative pedicels show that xylem tissue was water-
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filled both pre- and post-veraison (Fig.6a-c). Air-filled tissue was only detected in the pith of
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pedicel receptacle (Fig.6a,b) and stem (Fig.6c) portion; if any a few xylem vessel elements
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(n < 5) were air-filled close to the pith. For comparison, the microCT image of a dried pedicel
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shows the large number of air-filled elements (n = 1700, see Fig. S2) in the pedicel stem
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xylem portion (Fig.6d).
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The lumen of vessel elements in the pedicel stem portion became interrupted mainly post-
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veraison (Fig.7). Representative 3-D images show volume renderings of the pedicel surface
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(Fig.7a-c), vessel elements extracted from this portion (Fig.7d-f), and longitudinal 2-D
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images of vessel elements embedded in the pedicel tissue (Fig.7g-i). Early in fruit
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development at 20 and 40 DAA, vessel lumen was mainly continuous (see ‘A’ to ‘E’, ‘G’ to
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‘J’, and ‘L’) and rarely constricted (see 'F') or interrupted (see 'K'). After veraison at 80 DAA,
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the lumen of many vessel elements was interrupted at multiple positions along vessel
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elements (see ‘M’ to ‘O’ and ‘R’); interruptions were 2 to 12 μm in length. A quantitative
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analysis predicted that post-veraison on average 46 % of vessel elements in the pedicel
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stem portion were interrupted by blockages, whereas only 6 % of vessel elements were
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interrupted before veraison (p<0.01; Tab.1).
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Higher resolution 3-D images generated from a post-veraison pedicel at 85 DAA (Fig.8)
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revealed that at positions where vessel elements were constricted (see 'A' at position 1 and
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2; 'C' at position 1) or interrupted (see 'B' at position 3; 'D' at position 5) solid material
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emerged into the vessel lumen and ultimately blocked the interior.
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The structure and spatial arrangement of vessel elements in the pedicel stem portion was
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rather complex (Fig.9). Vessel elements of variable length (range from 204 to 530 μm,
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diameter of 5 to 15 μm; in e.g. tomato, length = 60 to 560 μm, diameter = 8-53 μm; Rancic et
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al., 2008; 2010) were packed very closely (Fig.9a), while vessel elements had a tracheid-like
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shape with oblique end-walls and diameters of vessel elements varied along their length
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(Fig.9b). Vessel elements were not aligned vertically, and continuous vessels through the
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pedicel were detected. Using the maceration technique highlighted that vessel elements
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were of the scalariform type having simple perforation plates (Fig.9c); this could not be
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detected from microCT images. Because blockages within the lumen of vessel elements
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were aligned horizontally and were not in proximity to end-walls (Fig.9b), vessel blockages
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detected in microCT images were not associated with the presence of perforation plates
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(Fig.9c).
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Vessel elements in the pedicel stem portion were well interconnected post-veraison (Fig.10,
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same pedicel as shown in Fig.9). Intervessel connections were detected all along vessel
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elements ‘A’ to ‘E’ with n = 15 to 34 connections per vessel element (Fig.10b). In theory,
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intervessel connections facilitate the axial flow of water across the pedicel between vessel
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elements and in the presence of vessel elements with blockages.
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DISCUSSION
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For grape, our data show that pedicel hydraulic conductivity (kh) under both hydrostatically
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and osmotically driven water flow starts to decline before veraison and is reduced
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substantially in early ripening stages (~65 DAA). Gradual changes in pedicel hydrostatic kh
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from 30 to 90 DAA were most pronounced when measured from multiple and low ΔP steps.
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Previous studies only reported significant changes in pedicel hydraulic properties for latter
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stages of ripening (Tyerman et al., 2004; Choat et al. 2009, >80 DAA), when most water
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transport to the berry is already via the phloem (Greenspan et al., 1994).The losses in
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pedicel kh over fruit development, as found here, occurred despite a 3- and 4-fold increase in
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xylem cross sectional area of pedicel stem and receptacle portion, respectively, pointing to
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important changes in xylem function. MicroCT images provided direct evidence that
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decreasing pedicel kh was associated with the formation of blockages in the lumen of water
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conducting vessel elements (i.e. occlusions, as proposed by Choat et al. (2009)), and not air
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embolism formations. A quantitative analysis showed that post-veraison xylem blockages
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were present in ~50% of all vessel elements. However, vessel elements in the pedicel were
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well interconnected post-veraison, suggesting that a functional xylem flow path still exists
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late into berry ripening.
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The diameter of the pedicel receptacle and stem portion increased until 42 and 55 DAA,
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respectively. These increases were accompanied by developmental increases in xylem
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cross-sectional area in both pedicel portions. Due to the fact that pedicel kh was greatest in
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Stage I (<40 DAA) when Ax was the smallest, losses of pedicel kh during development
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cannot be attributed to xylem area related limitations of water transport, as reported for
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tomato (Lee, 1989); this is also evident from the decline in pedicel ks (i.e. kh per Ax) over fruit
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development. The negligible effect of developmental changes in xylem area dimensions on
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pedicel hydraulic properties is also evident when comparing hydraulic characteristics of
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pedicel receptacle vs. stem portion: even though Ax of the receptacle is ~2-fold greater than
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that of the stem portion, the limiting hydraulic conductance along the pedicel is located in the
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receptacle (Choat et al., 2009; see our ks estimates). For apple, developmental increases in
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pedicel xylem area are largely due to proliferation of non-conducting xylem elements (Lang
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and Ryan, 1994; Horbens et al., 2013), and this may also be the case for grape in order to
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support higher fruit weight during ripening.
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The present data revealed that pedicel kh in grape declines before veraison (i.e. by ~40 %)
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and substantially in early stages of berry ripening. This was the case when measured
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osmotically and hydrostatically, under both low (-0.05 to 0.08 MPa) and high (>0.08 MPa)
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ΔP. Due to the fact that pedicel length did not change developmentally, it can be predicted
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that in the intact plant pedicel osmotic and hydrostatic hydraulic conductance (in m3 s-1 MPa-
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to III was most gradual when measured under low ΔP. Because water flow through the
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pedicel ramped up for ΔP >0.08 MPa, our data suggest that under high ΔP water flow is
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induced not exclusively through xylem vessel elements but also through the apoplastic
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space. Similar findings were reported by Van Ieperen et al. (2003) for tomato and Mazzeo et
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al. (2013) for kiwi. This would explain why pedicel hydrostatic kh obtained from high ΔP was
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always greater than the one from low ΔP. Choat et al. (2009) analyzed grape (V. vinifera
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'Chardonnay') pedicel hydraulics from a single and high ΔP of 0.1 MPa without having
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knowledge about the pressure-flow relationship, and found that hydraulic properties only
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change in latter stages of berry ripening. In comparison to our study on 'Cabernet
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Sauvignon', this may indicate that grape pedicels from different V. vinifera varieties differ in
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their developmental changes in hydraulic properties. But on the other hand, pedicel
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hydraulics measured from a single and high ΔP most likely lack the accuracy necessary to
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resolve the early developmental changes in kh . Hence, it is recommended that
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developmental changes in grape pedicel hydraulics should be measured from pressure-flow
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relationships established using multiple ΔPs, and low (<0.08 MPa) ΔP steps should be used
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to minimize apoplastic bypass flows that may potentially mask real developmental changes
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in xylem hydraulic properties.
) changes in a similar fashion. However, the reduction in hydrostatic pedicel kh from Stage I
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Water flow across the pedicel is driven by a gradient in water potential between parent plant
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and berry. Developmental changes in berry skin transpiration and sugar accumulation
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impact the hydrostatic and osmotic pressure component, respectively, of berry water
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potential (Matthews and Shackel, 2005). However, gradients in hydrostatic and osmotic
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pressure can induce water flow along different pathways (Steudle, 2000). In theory,
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hydrostatically-driven water flow follows predominantly an extracellular low-resistance
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pathway whereas osmotically-driven water flow follows mainly a high-resistance pathway
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that involves crossing of osmotic barrier(s) (e.g. cell membranes, apoplastic barriers). In
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order to obtain a complete picture of pedicel hydraulic properties, pedicel kh was determined
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under both driving forces. Based on the composite transport model of water flow, it can be
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concluded that pedicel kh obtained from low ΔP (-0.05 to 0.08 MPa) represent mainly the
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hydraulic property of a hydraulic pathway along xylem vessel elements, whereas high ΔP
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(>0.08) induces an additional apoplastic bypass flow through the cell wall space. A
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predominant water flow driven osmotically requires the presence of osmotic barrier(s) that
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prevent the free diffusion of solutes. In this study, depositions of osmotic barriers in the
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pedicel apoplast, such as casparian band-like structures, were not detected (data not
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shown); the apoplastic space between vessel elements is in biophysical terms an inefficient
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osmotic barrier and therefore osmotically driven flow, if any, is relatively small. Hence,
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pedicel osmotic kh represents mainly the hydraulic property of a high-resistance membrane-
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bound pathway through cortex, pith, and phloem tissue excluding a predominate flow
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through vessel elements. The loss in pedicel osmotic kh over fruit development may be
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associated with a decline in aquaporin activity in the pedicel, as it has been found for some
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aquaporin isoforms in the berry (Choat et al. 2009, Fouquet et al. 2008). Moreover, a
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membrane-bound transport pathway through the pedicel may also be affected
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developmentally by structural changes of cells (Nii and Coombe, 1983). Based on findings
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that cells in 'healthy' berries and rachis remain largely viable post-veraison (Krasnow et al.,
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2008; Hall et al., 2011), it can be speculated that a membrane-bound pathway through the
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pedicel of reduced hydraulic conductivity most likely exists in the pedicel late into fruit
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ripening. However, since pedicel osmotic kh was 3- to 4-orders of magnitude smaller than
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hydrostatic kh from 20 to 90 DAA, membrane-bound water flows through the pedicel may
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only contribute substantially to overall fruit water uptake when water enters the fruit
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predominantly via the phloem or when xylem flows, if present, are primarily in the reverse
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direction (i.e. post-veraison; Greenspan et al., 1994).
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For grape, several physiological parameters of the fruit (e.g. growth, sugar accumulation,
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turgor, firmness) change before veraison (Thomas et al., 2008; Matthews et al., 2009; Wada
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et al., 2008, 2009), which is in line with the early developmental changes in pedicel kh. The
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decrease in berry growth from Stage I to II, that is a normal part of their development, may
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directly be related to the reduction in hydrostatic and osmotic pedicel kh which can reduce
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water transport capacity along the extra- and intracellular pathways, respectively, between
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parent plant and fruit. Developmental changes in fruit water relations, e.g. by a change of
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turgor of berry mesocarp cells and a drop in solute potential from Stage I to III (Wada et al.,
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2008; Matthews et al., 2009), may aid water flow into the fruit when pedicel kh declines.
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Although reductions in water transport capacity of grapevine stems (Brodersen et al., 2010,
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2013) and petioles (Zufferey et al., 2011) are attributable to xylem embolisms, our microCT
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images provide direct evidence that in grape pedicels of well-watered vines water transport
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capacity is not affected by embolism formations pre- and post-veraison. Hydraulic
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measurements are in support of this observation, which show that both kh values obtained
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under low and high ΔP declined in a similar fashion over fruit development. For example, if
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significant amounts of xylem embolisms would have been present in the pedicel then kh as
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derived from high ΔP > 0.08 MPa should have remained constant over fruit development
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because high pressures would flush embolized vessels. But this was not the case.
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It could be argued that during kh measurements ΔP applied initially was already too high,
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and naturally occurring embolisms in the pedicel were already removed at the onset of the
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measurement. If this would be true, the threshold value for a change of the pressure-flow
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relationship should be <<0.08 MPa. We tested this hypothesis by application of an initial ΔP
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of as small as <10 kPa (i.e. including embolisms; Cochard et al., 2013), and subjected the
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pedicel to increasing ΔP steps of > 0.2 MPa (i.e. flushing embolisms), before returning to the
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initial ΔP and applying negative ΔP steps (Fig.S3). We found that in both pedicels harvested
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pre- and post-veraison 1) the pressure threshold remained at 0.08 MPa, 2) the flow rate
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measured initially was similar to the one measured at a comparable ΔP following >0.2 MPa,
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and 3) flow rates under -ΔP were similar to +ΔP of comparable absolute magnitude (i.e.
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negative pressure did not remove potential embolisms either). This proved the validity of the
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pressure-flow relationship, and further supports that effects of xylem embolisms on pedicel
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kh are negligible during the course of fruit development. Moreover, there is a possibility that
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embolized vessels in the pedicel may have refilled naturally during the time period of harvest
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and measurement, as it has been reported for intact grapevines after 10 h (Brodersen et al.,
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2010), but this time period was <1 h when pedicel kh was determined and therefore very
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unlikely. If grape pedicels possess the ability to repair xylem embolisms in a very short time
413
period needs to be investigated in the future.
414
415
Three-dimensional microCT images provided clear evidence that losses in pedicel kh
416
between Stage I and III were associated with the formation of constrictions and ultimately
417
blockages inside the lumen of xylem vessel elements (i.e. occlusions). Grapevine stems
418
produce xylem blockages by tyloses (i.e. spherical shape) and gels in response to wounding
419
(Sun et al., 2008). Even though Kasimatis (1957) thought that tyloses may exist in the
420
pedicel post-veraison, there is no direct evidence for tyloses in the berry (Chatelet et al.,
421
2008a,b) and the grape pedicel (Choat et al., 2009). Based on 3-D microCT images, xylem
422
blockages in the pedicel exhibited irregular shapes, which suggest that they are not tyloses
423
or remaining water menisci after dehydration, and rather composed of material such as
424
polysaccharides (pectins and callose). The deposition of polysaccharide-like material in
425
vessel elements of the pedicel might be associated with the developmental increase in sugar
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426
transport from parent plant into the berry. Xylem blockages can explain the substantial
427
reduction in pedicel kh between Stage I and Stage III, but might not solely explain the early
428
developmental changes in pedicel kh, because at 20 to 45 DAA only ~6 % of vessel
429
elements were occluded (see Tab.1). Those early changes may be related to additional
430
functional and spatial changes of the pedicel xylem network, i.e. the clogging of
431
interconnections between vessel elements (see Fig. 10). It has been reported that
432
intervessel pit resistance accounts for ~80 % of the total hydraulic axial transport resistance
433
in angiosperm trees (Choat et al., 2006), and variability in xylem hydraulic resistance has
434
been associated with changes in vessel-to-vessel resistance (Zwieniecki et al., 2001). Due
435
to the lack of relatively long and continuous vessels, it can be speculated that axial water
436
movement through the pedicel strongly depends on the functional status of intervessel pit
437
connections. Further work on the grape pedicel is needed to investigate (i) the exact timing
438
of changes in pedicel kh and xylem network functionality, (ii) the chemical composition of
439
material that blocks the lumen of vessel elements and potentially clogs intervessel pit
440
connections, and (iii) the mechanism involved in the formation of blockages with respect to
441
activity of living cells adjacent to vessel elements.
442
443
The importance of incorporating pedicel hydraulic properties in fruit growth models has been
444
recognized by Hall et al. (2013), but the xylem tension (i.e. negative pressure) that manifests
445
across the pedicel and in turn ΔΨ (i.e. water potential gradient) is unknown (the tension is
446
related to Ψ of plant and fruit that acts on both sides of the pedicel). For well-watered plants
447
as used here, we did not measure xylem tension directly but we calculated that a ΔΨ (= [ Q
448
x (1 / (kh/length))] ) of only <5 kPa would be required across the pedicel to support xylem
449
flows as measured in vivo between parent plant and fruit by Greenspan et al. (1994) (see
450
Table S3). This is a small fraction of ΔΨ of ~50 to 300 kPa as measured between parent
451
plant and berry cluster (Greenspan et al., 1996). Whether or not a physical hydraulic barrier
452
exists upstream of the pedicel (i.e. toward the rachis) that reduces the propagation of xylem
453
tensions from the parent plant to the fruit remains unknown. In tomato, a fruit abscission
454
zone is located in the ‘knuckle’ midway along the pedicel with a large hydraulic resistance
455
(Lee, 1989; Van Ieperen et al., 2003), and it may be that for grape a structure similar in its
456
hydraulic property is located somewhere in the rachis. Hence, in the context of the intact
457
plant small tensions most likely exist across the pedicel xylem, at least under well-watered
458
conditions, which may explain the negligible amounts of xylem embolisms. However, it
459
needs to be shown if embolism formations in the pedicel remain negligible also under
460
drought conditions, and if hydraulic architecture of the pedicel is an adaption to minimize
461
embolism susceptibility.
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462
463
It has been proposed that maintenance of a partially functional xylem path is important to
464
recycle excess water that enters the fruit via the phloem during ripening (Greenspan et al.
465
1994; Garcia-Lius et al., 2002; Keller et al., 2006). Post-veraison, the amount of daily xylem
466
backflow in grape was estimated to be ~33 % of fruit volume and relatively high (Choat et al.,
467
2009). Our microCT data highlight that xylem vessel elements in the pedicel are well
468
interconnected post-veraison, which most likely secures a xylem flow path even in the
469
presence of xylem blockages. Based on observations of Choat et al. (2009), inter-vessel
470
connection as detected here are most likely bordered pits. Moreover, we can exclude that a
471
rectification of xylem flow resides in the grape pedicel and contributes to the changing water
472
flow dynamics between parent plant and fruit over development, as suggested by Tilbrook
473
and Tyerman (2009), because inflow kh was similar to outflow kh.
474
475
Conclusion
476
Grape pedicel hydraulic conductivity declines before veraison and gradually over fruit
477
development which is associated with the formation of blockages in the lumen of vessel
478
elements. However, due to the fact that post-veraison ~50 % of vessel elements in the
479
pedicel stem portion remained functional and were not occluded and vessel elements
480
remained well interconnected, our data suggest that a functional xylem flow pathway of
481
reduced hydraulic conductivity still exists late into berry ripening (100 DAA). The present
482
findings provide a basis for further anatomical, physiological, and molecular research into the
483
role of the grape pedicel in affecting berry growth and development.
484
485
MATERIAL AND METHODS
486
487
Plant material
488
Own-rooted grapevines (Vitis vinifera 'Cabernet Sauvignon') were grown in a greenhouse
489
(temperature of 20 to 30 oC; relative humidity of 40 to 70 %, and natural light with a daily
490
maximum of 1200 μmol photons m-2 s-1 PAR) at the University of California, Davis. Vines
491
were about 2-years old and maintained in 2 gallon plastic pots (depth ~46 cm, width ~20 cm)
492
filled with a soil mix of one-third peat, one-third sand and one-third redwood compost. In total
493
n = 15 plants were used for experiments. Each plant had two to three shoots that were
494
vertically trained to approximately 1.5 m (n = 3 - 4 berry clusters per shoot, >100 berries per
495
cluster). Pots were drip irrigated three times a day with water supplemented with calcium (90
496
μg mL-1), magnesium (24 μg mL-1), potassium (124 μg mL-1), nitrogen as NH4+ (6 μg mL-1),
497
nitrogen as NO3- (96 μg mL-1), phosphorus (26 μg mL-1), sulfur (16 μg mL-1), iron (1.6 μg mL-
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498
1
), manganese (0.27 μg mL-1), copper (0.16 μg mL-1), zinc (0.12 μg mL-1), boron (0.26 μg mL-
499
1
500
of the cluster was flowering (i.e. day of anthesis).
), and molybdenum (0.016 μg mL-1) at pH 5.5 to 6.0. Berry clusters were tagged when 50 %
501
502
Hydraulic measurements
503
In the greenhouse, n =2 - 3 fruits located midway along the berry cluster were cut off under
504
water with scissors from the rachis, placed in a moist plastic container, and transported
505
within 30 min to the laboratory. In the laboratory, the pedicel end was re-cut under water with
506
a fresh razor blade, and subsequently the pedicel was excised from the fruit at the
507
receptacle end (Fig.1). Pedicel diameter and length, as well as fruit diameter, were
508
measured with an electronic handheld caliper (Y513446, Fisher scientific, USA). Solute
509
potential of berry sap was measured using osmometry (5500 vapor pressure osmometer,
510
Wescor Inc.). Hydraulic properties of excised pedicels were measured using a modified
511
pressure-probe system together with a computer-vision based meniscus tracker (Wong et
512
al., 2009) (Fig.S1). ). Typically a reliable measure of pedicel hydraulic property was obtained
513
for one out of three pedicels from the same berry cluster, and pedicels measured at different
514
days after anthesis were sampled randomly from different vines and berry clusters.
515
516
For hydrostatic experiments, ~2 mm of the pedicel stem portion was inserted into a 10 mm
517
long adapter piece that was attached to a glass microcapillary (i.d. 0.75mm, Stoelting Co.,
518
Wood Dale, IL, USA). The pedicel stem portion was sealed with super-glue (Loctite gel,
519
Henkel, USA). The microcapillary was half-filled with diH20 and backfilled with silicone oil
520
(Dimethyl silicone fluid, Thomas Scientific, NJ, USA), which created an oil-water meniscus.
521
The microcapillary was connected to the probe via a compression seal, and the pedicel
522
receptacle was submerged into a diH2O bathing solution. Hydrostatic water flows across the
523
pedicel were induced by application of multiple step changes in hydrostatic pressure (P, -
524
0.05 to 0.22 MPa) while each P-step was clamped for about 5 to 15s; step changes of
525
increasing and decreasing P applied for the same pedicel showed that water flow rates were
526
reversible. During each P-step, the displacement of the meniscus (d) was followed with a
527
digital camera, recorded every 0.133s, and changes in volume (V) were derived from
528
changes in d (i.e. V = 3.14·x (radius of capillary)2 x d). The volume flow rate (Q) under each
529
applied pressure gradient across the pedicel was determined from the slope of the linear
530
regression line of V versus ΔP. Positive applied pressures induced water flow from pedicel
531
stem to receptacle end ('inflow') and negative applied pressure in the reverse direction
532
('outflow' or ‘backflow’). When water leaked through any connections of pedicel and
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
533
microcapillary, or air bubbles were present in the microcapillary, measurements were
534
discarded.
535
536
For osmotic experiments, the excised pedicel segment was fixed to the glass microcapillary
537
as described for hydrostatic experiments; the microcapillary was half-filled with diH2O and
538
the pedicel receptacle end was bathed in different sucrose concentrations with an osmotic
539
pressure (π) of 0.63, 1.88, and 3.13 MPa. Osmotic pressures of sucrose solutions were in
540
the range of π of extracted fruit sap. Each sucrose solution (i.e. π-step) was applied for 10 to
541
15 min before being exchanged to another one of higher concentration, and finally the
542
sucrose solution of lowest concentration was applied again to check for reversibility in flow.
543
Due to the much longer time required to accurately measure V in osmotic (~15 min) as
544
compared to hydrostatic (<15 s) experiments, only one π-step was applied twice. During
545
each π-step the displacement of the air/H2O meniscus was followed with the digital camera,
546
and Q was determined as described for hydrostatic experiments.
547
548
Hydraulic properties of the pedicel were defined according to the terminology of Tyree and
549
Ewers (1991). From both hydrostatic and osmotic experiments, pedicel hydraulic
550
conductivity (kh) was determined from the slope (m) of the linear part of the pressure-flow
551
relationship and normalized for pedicel length (l) according to kh = [ m x l ]. In hydrostatic
552
experiments the relationship of ΔP vs. Q was typically linear for ΔP ≤ 0.08 and ΔP > 0.08
553
(Fig.4a), and subsequently kh was determined separately for both low and high pressure
554
ranges. When for ΔP > 0.08 MPa a reliable Q was obtained only from a single ΔP, due to
555
problems of water leakage at higher applied pressures, kh was determined by [(Q/ ΔP) l].
556
Pedicel hydrostatic kh under inflow and outflow conditions was determined from m
557
corresponding to ΔP of -0.05 to 0 MPa and ΔP of 0 to 0.0.8 MPa, respectively.
558
559
Pedicel specific hydraulic conductivity (ks) was determined by normalizing hydrostatic kh as
560
measured under low ΔP of -0.05 to 0.08 MPa for cross-sectional area of heavily lignified
561
xylem (Ax) of either pedicel stem or receptacle portion according to ks = [kh / Ax ]. To obtain ks
562
for each pedicel, Ax was derived from diameter measurements of pedicel stem (Ax = 0.159 x
563
(diameter/2)2 x 3.14; R2=0.91, p<0.0001) and receptacle portion (Ax = 0.0866 x (diameter/2)2
564
x 3.14; R2=0.83, p<0.0001) after hydraulic analysis. For both pedicel portions, the
565
relationship of Ax versus diameter was established by measuring Ax from free-hand cross-
566
sections (as described below) and corresponding pedicel diameters (n = 28 pedicels
567
analyzed between 20 to 90 DAA).
568
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569
Light microscopy
570
Free-hand cross-sections were made with a fresh razor blade at three different positions
571
along the pedicel (Fig.1). Unstained sections were mounted on a slide in diH2O and
572
observed under violet fluorescence light (excitation filter 400-410 nm peak emission,
573
dichromatic mirror 455 nm, and barrier filter 455 nm) with an Olympus Vanox-AHBT
574
(Olympus America) compound microscope connected to a Pixera 600ES digital camera
575
(Pixera Corporation). Lignified tissue emitted a bright blue auto-fluorescence signal. Images
576
were analyzed using Fiji image software (www.fiji.sc, ImageJ) and heavily lignified xylem
577
area (Ax) was determined using the ‘Polygon selection’ tool and number and diameter of
578
xylem elements were determined using the ‘Analyze particle’ tool (Fig.S2).
579
580
Maceration was used as described in Chatelet et al. (2008a) to investigate vessel anatomical
581
structure. Briefly, cross-sections ~2 mm thick of a pre-veraison pedicel were immersed in
582
maceration solution (30 % hydrogen peroxide, acetic acid, distilled water), placed in an oven
583
at 57 oC for up to 24 h, subsequently shaken to loosen tissue, stained with Safranin O that
584
strongly binds to lignin, and finally the solution was pipetted onto a slide and viewed with the
585
Olympus Vanox-AHBT compound microscope (see above).
586
587
X-ray computed microtomography (microCT)
588
Pedicels were imaged at the x-ray microtomography facility at the Lawrence Berkeley
589
National Laboratory Advanced Light Source (ALS, Beamline 8.3.2). To determine if
590
embolized xylem conduits existed in the pedicel, entire berry clusters were cut off with
591
scissors under water at the rachis, placed in a moist aluminum covered plastic bag, and
592
transported by car from the University of California to the ALS. Within 20 h after harvest, a
593
pedicel was extracted under water with a fresh razor blade from fruit and rachis, as
594
described for hydraulic measurements, fixed on a solid stage at the receptacle end with
595
adhesive putty, and rotated in the 17- to 19-keV synchrotron x-ray beam in 0.176°
596
increments over 180° yielding 1025 two-dimensional projection images with a 1.78-μm voxel
597
resolution captured on a scientific CMOS camera (PCO edge, 2560 x 2160 pixels, PCO AG,
598
Kehlheim, Germany) (Brodersen et al. 2010, 2011). For a more detailed analysis of vessel
599
structure and spatial arrangement, excised pedicels were dehydrated for 24 h (as shown in
600
Fig.8-10) and 4 days (as shown in Fig.7) at ~30 oC prior to scanning. For imaging, the
601
receptacle end was fixed with epoxy glue (20445 Flow-Mix 5-Minute Epoxy, Devcon,
602
Danvers, MA, USA) to a 2-cm long metal rot (diameter 2 mm) which was inserted into a
603
rotating drill chuck. This minimized vibrations and lateral pedicel movements during
604
scanning. Dehydrated pedicels were imaged in the stem portion at either a 1.78-μm or 0.89-
17
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
605
μm voxel resolution. We focused on the pedicel stem and not the receptacle portion because
606
along the length of the stem portion the arrangement of xylem vessel was most uniform.
607
After a scan was completed, the acquired two-dimensional projection images were
608
reconstructed into a stack of transverse images using a custom software plug-in for Fiji
609
imaging-processing software (www.fiji.sc, ImageJ) that used Octopus 8.3 software (Institute
610
for Nuclear Sciences, Ghent University, Belgium) in the background. After uploading the
611
entire stack of TIF images into AVIZO 6.2 software (VSG Inc., Burlington, MA, USA), 3-D
612
images were generated of the pedicel surface using the ‘Volume rendering’ module and of
613
xylem vessel elements using the ‘Label-field’ segmentation editor and ‘SurfaceGen’ volume
614
rendering module. In order to determine the frequency of vessel elements with blockages
615
(i.e. occlusion) in the pedicel, a transverse images slice within the stack of 2-D images of the
616
pedicel stem portion was selected and n= 40-60 vessel elements intersecting this slice were
617
generated in 3-D (as described above). Generated vessel elements that provided sufficient
618
spatial and visual resolution were included in this analysis, and the lumen of vessel elements
619
was evaluated for the presence of blockages along their entire axial distance.
620
.
621
Data analysis
622
Data were analyzed using SAS (version 9.2; SAS Institute, Cary, NC, USA) and SigmaPlot
623
(version 8.0, Systat Software Inc., San Jose, CA, USA). For linear regression analyses the
624
'PROC REG' procedure was used. For fitting of smoothed trendlines the ‘PROC
625
TRANSREG’ procedure was used. Average values and standard deviations were
626
determined using the 'PROC MEANS' procedure. For statistical analysis, data were
627
subjected to the 'PROC GLM' procedure followed by Tukey-Kramer test using SAS or
628
Student t-test using SigmaPlot.
629
630
Supplemental Data
631
Supplemental Figure S1. Schematic presentation of the modified pressure-probe system
632
used to measure pedicel hydraulic properties.
633
Supplemental Figure S2. Image of the pedicel stem portion showing lignified xylem tissue
634
excluding rays cells and diameter distribution of xylem elements.
635
Supplemental Figure S3. Pedicel hydraulic characteristics in response to a series of applied
636
pressure steps as measured with a bellows-pressure-probe..
637
Supplemental Table S1. Estimation of the water potential gradient across the pedicel.
638
639
Acknowledgements
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
640
We want to thank David Chatelet, Brown University, for providing the image of the
641
macerated xylem vessel. We thank Judy Jernstedt, University of California Davis, for her
642
help with anatomy. The authors kindly thank D. Parkinson and A. MacDowell for their
643
assistance at the Lawrence Berkeley National Laboratory Advanced Light Source beamline
644
8.3.2 microtomography facility.
645
646
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Wada H, Matthews MA, Shackel KA (2009) Seasonal pattern of apoplastic solute
745
accumulation and loss of cell turgor during ripening of Vitis vinifera fruit under field
746
conditions. Journal of Experimental Botany 60: 1773-1781.
21
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
747
Wong ES, Slaughter DC, Wada H, Matthews MA, Shackel KA (2009) Computer vision
748
system for automated cell pressure probe operation. Biosystems Engineering 103:
749
129-136.
750
Zufferey V, Chochard H, Ameglio T, Spring JL, Viret O (2011) Diurnal cycles of embolism
751
formation and repair in petioles of grapevine (Vitis vinifera cv. Chasselas). Journal of
752
Experimental Botany 62: 3885-3894.
753
754
Zwieniecki MA, Melcher PJ, Holbrook NM (2001) Hydrogel control of xylem hydraulic
resistance in plants. Science 291: 1059-1062.
755
756
FIGURES LEGENDS
757
758
Figure 1. Schematic drawing of rachis, pedicel and fruit in grapevine. The vascular system
759
(blue lines) of fruit and rachis via the pedicel is illustrated. The pedicel is composed of a
760
narrower stem portion and wider receptacle portion. In this study, pedicel anatomy and
761
xylem function was analyzed at up to three positions along the pedicel as indicated by
762
numbers. Hydraulic properties were measured on excised pedicels (dashed lines indicate
763
position of cuts) using a modified pressure-probe system.
764
765
Figure 2. Developmental changes in (a) fruit diameter and solute potential, (b) pedicel
766
diameter of receptacle portion, (c) pedicel diameter of stem portion, and (d) pedicel length.
767
The dashed lines indicate the estimated boundaries between Stage I and II and Stage II and
768
III in fruit expansion, and the arrow indicates ‘veraison’ (i.e. 50 % changes in skin color).
769
Data are means ± SE (n= 7 - 31).
770
771
Figure 3. Free-hand cross-sections of pedicels imaged unstained under violet-fluorescence
772
light over fruit development. Lignified tissue appears in bright blue color. (a) Enlarged image
773
of the pedicel stem portion indicating the presence of various tissue types (x, lignified xylem
774
tissue; r, less lignified ray cells; p, phloem; fc, fiber cap cells; c, cortex; pe, periderm, pi, pith).
775
(b) Spatial and temporal changes in pedicel dimensions and xylem cross-sectional area of
776
stem portion (#1, i.e. position 1 in Fig.1) and receptacle portion (#2 and #3, i.e. position 2
777
and 3 in Fig.1, respectively) (V, veraison; DAA, days after anthesis). In the receptacle portion
778
close to the fruit radial branches of xylem tissue (xb) into the cortex are visible (#3), which
779
increase in intensity over fruit development. (c) Developmental changes in heavily lignified
780
xylem cross-sectional area (Ax) in stem portion (#1, left panel) and receptacle portion (#2,
781
right panel). Dashed lines are 75 % smoothed trendlines. The arrow indicates veraison.
782
22
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783
Figure 4. Representative pressure-flow relationships of an excised pedicel as measured
784
with a modified pressure-probe system (see also Figs.S1 and S3). A volume flow of water
785
was induced across the pedicel by application of (a) hydrostatic or (b) osmotic pressure
786
forces. Solid lines are linear regression lines fitted to linear parts of the pressure-flow
787
relationship (i.e. low ΔP = -0.05 to 0.08 MPa (R2 = 0.90), high ΔP > 0.08 MPa (R2 = 0.99),
788
and Δπ = 0.6 to 3.3 MPa (R2 = 0.93)).
789
790
Figure 5. Developmental changes in pedicel hydraulic properties as measured directly on
791
excised pedicels using a modified pressure-probe system. (a) Pedicel hydraulic conductivity
792
(kh) was determined for hydrostatically and osmotically induced water flows (note the
793
difference in Y-scales). Based on the pressure-flow characteristics of the pedicel, hydrostatic
794
kh was derived separately from low ΔP (-0.05 to 0.08 MPa) and high ΔP (>0.08) steps. (b)
795
Pedicel hydrostatic kh as determined under water inflow and outflow conditions using low
796
ΔPs. (c) Specific hydraulic conductivity (ks) as determined by normalizing hydrostatic kh
797
measured under low ΔP for cross-sectional area of heavily lignified xylem tissue (Ax) of
798
either stem or receptacle portion. (a-c) Data are means ± SE (in a, n = 4, 5, 13, 5 (closed
799
circles); n = 4, 4, 8, 5 (gray squares); n = 5, 3, 9 (open diamonds); in b, n = 4, 5, 13, 5 (open
800
squares); n = 3, 5, 6, 4 (closed circles); in c, n = 4, 5, 13, 5) of samples analyzed at <40 DAA
801
(Stage I), 40 to 55 DAA (Stage II), 55 to 75 DAA (Stage III), and 75-90 DAA (Stage III),
802
respectively. When SE is smaller than symbols size, SE is not visible. For each parameter
803
different letters indicate significant differences (p < 0.05) between means (Tukey-Kramer
804
test). The arrow indicates veraison. Dashed lines indicate the estimated boundaries between
805
Stage I and II and Stage II and III in fruit expansion (see Fig.2a).
806
807
Figure 6. Representative transverse microCT images through the pedicel showing water-
808
filled tissue in light gray and air-filled tissue in dark gray. Black arrows indicate some air-filled
809
tissue in the pith. Images show the receptacle portion (i.e. position 2 in Fig.1) of pedicels
810
scanned (a) pre-veraison at 34 DAA and (b) post-veraison at 76 DAA; (c) image of the stem
811
portion scanned at 76 DAA (i.e. position 1 in Fig.1). These images were acquired from
812
pedicels that were scanned immediately after they were cut under water from fruit and
813
rachis. (d) In comparison, an image of the stem portion of a dried pedicel scanned at 85
814
DAA. x, xylem tissue; pi, pith; and c, cortex; r, ray cells.
815
816
Figure 7. Three-dimensional reconstructions of xylem vessel elements in the pedicel stem
817
portion at 20 DAA, 40 DAA, and 80 DAA. Pedicels were scanned after dehydration. (a-c)
818
Volume renderings of the pedicel surface. (d-f) Reconstructed vessel elements (n = 6 each)
23
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
819
within that pedicel portion. Panels to the right show enlarged versions of these reconstructed
820
vessel elements. For better visualization, pairs of vessel elements that were located in close
821
proximity to each other are given the same color (i.e. red, turquoise, green). (g-i) 2-D images
822
of reconstructed vessels as they are embedded in the pedicel apoplast (=light gray; air-filled
823
vessel lumen, dark gray as indicated by white arrows). The position of the longitudinal 2-D
824
image slice is indicated in (a-b) (c, cortex; pi, pith). Asterisks indicate constricted vessel
825
lumen. Yellow arrows indicate positions where the continuity of the vessel lumen is
826
interrupted. Scale bar = 100 μm.
827
828
Figure 8. Three-dimensional reconstructions of xylem vessel elements in the pedicel stem
829
portion at 85 DAA (i.e. post-veraison). Images were obtained from a dehydrated pedicel
830
scanned at high resolution. At positions where vessel elements are constricted ( ‘A’ at
831
position 1 and 2, and ‘C’ at position 4) or interrupted ( ‘B’ at position 3, and ‘D’ at position 5)
832
deposition of some material are visualized inside the vessel lumen. Yellow arrows indicate
833
the position of blockages in the lumen of vessel elements.
834
835
Figure 9. (a) Structure and spatial arrangement of xylem vessel elements in the pedicel
836
stem portion of a post-veraison berry (85 DAA, vessels ‘A’ to ‘D’ are identical to Fig.8).
837
Vessel elements are not aligned vertically. For positions #1 to #5 along the pedicel, images
838
on the right show the location of vessel elements in 2-D (c, cortex; pi, pith). (b) Enlarged
839
image of vessel elements 'C' and 'D' showing their tracheid-like structure with oblique end-
840
walls (ew). The yellow arrow indicates an interruption of vessel lumen. (c) In comparison, an
841
image of a pedicel vessel element extracted with the maceration technique showing oblique
842
end-walls (ew) and perforation plates (pp).
843
844
Figure 10. (a) Examples of inter-vessel connections (white arrows) as detected in a
845
transverse microCT image of the pedicel shown in Fig.9 (c, cortex; r, ray cells). (b) Based on
846
analysis of the entire stack of transverse microCT images (n > 1000), a map of intervessel
847
connectivity was generated. Each circle represents the location of a connection along vessel
848
elements. Solid lines indicate direct connections between vessel elements ‘A’ to ‘F’.
849
Positions of xylem blockages is indicated by black discs within the lumen of vessel elements.
850
The blue line indicates a possible flow path of water through the pedicel even when some
851
vessel elements are blocked locally.
852
853
854
24
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
855
TABLES
856
857
Table 1. Quantification of the frequency of vessel elements with blockages (i.e. occlusions)
858
in the grape pedicel stem portion from microCT images. For each pedicel, vessel elements
859
that intersected the same transverse image slice were generated in 3-D and evaluated along
860
their axial distance for the presences of blockages. Different letters indicate significant
861
differences (p<0.01) between means (Student t-test).
862
Developmental
stage
Sample
1 (20 DAA, Fig.7)
2 (40 DAA, Fig.7)
3 (35-45 DAA)
Total number
of vessel
elements
analyzed
47
46
49
Number of
vessel
elements with
blockages
2
3
4
Pre-veraison
Post-veraison
4 (80 DAA, Fig.7)
5 (85 DAA, Figs. 8-10)
6 (90-100 DAA)
59
49
46
28
22
21
Percentage of
vessel elements
with blockages
4%
7%
8%
a
6.3 ± 2.0 %
(mean ± SD)
47 %
45 %
46 %
b
46.0 ± 1.3 %
(mean ± SD)
863
25
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