Water Uptake along the Length of Grapevine Fine Roots: Developmental Anatomy,Tissue-Specific Aquaporin Expression,and Pathways of Water Transport

To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lp_r) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lp_r and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lp_r, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.In woody perennial root systems, the majority of water uptake is often attributed to unsuberized fine roots (Kramer and Boyer, 1995), even though woody portions can constitute the vast majority of root surface area for these plants at maturity (Nightingale, 1934; Kramer and Bullock, 1966). This assumption has likely been reinforced by the fact that most studies investigating root water uptake have been done with herbaceous species, whose roots function more like the tips of woody perennials. Although unsuberized fine roots typically have a greater ability to absorb water (i.e. they are more conductive per unit of surface area), it has been shown that older suberized portions of woody taproots can still contribute significantly to root system water uptake (Kramer and Bullock, 1966; Queen, 1967; Chung and Kramer, 1975; MacFall et al., 1990, 1991). Despite this knowledge and the fact that unsuberized roots of many woody perennials are scarce or absent during periods of the growing season when peak transpiration requires much water (MacFall et al., 1991), we still know little about how suberized portions of perennial rooting systems contribute to radial water absorption across species.The composite transport model (Steudle, 2001) is a conceptual framework describing water transport into plant roots. This model posits that water is able to flow into the root via multiple parallel pathways, traveling either in the cell walls (apoplastic) and/or from cell to cell (symplastic and/or transcellular). Transport across the cell-to-cell pathway can involve water crossing plasma membranes; thus, the rate of water uptake can be influenced by the abundance and activity of aquaporins (i.e. water channels). The contribution of aquaporins to root water uptake has been the focus of numerous studies, and the absolute magnitude of this contribution appears to be highly variable, ranging from 20% to 90% across species (for review, see Javot and Maurel, 2002). Steudle (2000) suggested that radial water flow would be dominated by aquaporin regulation in heavily suberized roots, as flow through the apoplast would be minimized. The localization of aquaporins should play a critical role in defining their impact on radial water uptake across suberized and unsuberized roots. For herbaceous species, peak aquaporin mRNA and/or protein levels have been found in root tips and the endodermis, pericycle, phloem, and xylem tissues (Schäffner, 1998; Otto and Kaldenhoff, 2000; Suga et al., 2003; Fraysse et al., 2005; Knipfer et al., 2011). Few aquaporin localization studies have been conducted in woody perennials (Vandeleur et al., 2009). Recent work from our laboratory revealed a precipitous drop in aquaporin expression between the grapevine (Vitis spp. rootstocks) root tips and older root portions (Gambetta et al., 2012). These observations led to this study, where we explore patterns of aquaporin localization in Vitis species fine roots and how they intersect with the structural anatomy and patterns of suberization to affect water uptake along the root length.Hydraulic conductivity (Lp_r) of the apoplastic pathway can be altered through changes in cell wall chemistry, especially through the deposition of suberin. Suberized apoplastic barriers in plant roots include the Casparian band of the endodermis and the suberin lamella of the endodermis, exodermis, and periderm in woody species (Esau, 1977). Casparian bands and suberin lamella are solute impermeable (for review, see Peterson and Enstone, 1996), but across studies, the extent to which they impede the flow of water is highly variable (Peterson et al., 1993; Steudle et al., 1993; Peterson and Enstone, 1996; Schreiber et al., 2005). Regardless, studies support the idea that in roots there is always some flow across the cell-to-cell pathway due to apoplastic barriers and/or an osmotic component to the driving gradient (Steudle et al., 1993; Miyamoto et al., 2001; Knipfer and Fricke, 2011). In the cell-to-cell pathway, Lp_r can be altered by intrinsic plasma membrane properties, plasmodesmata (Oparka and Prior, 1992; Roberts and Oparka, 2003), and/or the abundance and activity of aquaporins. Changes in aquaporin gene expression and protein activity remain potentially dynamic and can occur within hours, while alterations of suberized apoplastic barriers are permanent and would manifest over longer developmental time frames.The total water potential gradient across a fine root can be composed of both osmotic (ΔΨOs) and hydrostatic (ΔΨHy) pressure gradients. A purely ΔΨOs requires that some portion of the pathway be cell to cell. A purely ΔΨHy should drive flow through both pathways, and the proportion of flow through the two pathways will be determined by their Lp_r. Experimentally, Lp_r generated under ΔΨHy is typically greater than Lp_r generated under ΔΨOs, typically ranging from 2-fold to more than 100-fold greater (Steudle et al., 1987; Hallgren et al., 1994; Miyamoto et al., 2001; Knipfer and Fricke, 2011). In some cases, Lp_r is nearly equal under both types of gradients (Miyamoto et al., 2001; Knipfer and Fricke, 2011). These results suggest that if Lp_r through the apoplast were to be reduced by the presence of an apoplastic barrier, this would force flow across a cell-to-cell pathway regardless of the driving gradient (Steudle, 2000).In this study, we sought to provide a more detailed understanding of the localization of aquaporin expression and its contribution to radial water uptake in different zones of grapevine fine roots, from the unsuberized actively growing root tip to portions of the fine root undergoing secondary growth and containing a developed periderm. We characterized the developmental anatomy along the length of the fine root, including the localization of suberized structures, and quantified tissue-specific mRNA levels of plasma membrane aquaporin isogenes via a combination of laser-capture microdissection (LCM) and quantitative PCR. Finally, we determined the Lp_r of root tips and secondary growth root zones under both ΔΨOs and ΔΨHy while investigating the contribution of aquaporin activity to Lp_r via chemical inhibition.