Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.Numerous studies have illustrated the relevant role of dissolved organic matter (DOM) present in soil solution and aquatic reservoirs (lakes, rivers, etc.) in the biological and chemical evolution of both natural and anthropogenic ecosystems (Stevenson, 1994; Tipping, 2002; Chen et al., 2004; Trevisan et al., 2011; Berbara and García, 2014; Canellas and Olivares, 2014; Mora et al., 2014a, 2014b). In many studies, DOM fractionation is made by using the methodology proposed by the International Humic Substances Society. Fractions obtained are operationally named humic acid (HA), fulvic acid, humin, and nonhumic fraction, which includes more hydrophilic compounds (polycarboxylic acids, aminoacids, sugars, etc.; Swift, 1996). Many studies have reported that HAs obtained from either organic materials (soils, soil sediments, composted wastes, etc.) or water reservoirs (rivers, lakes, etc.), extracted with alkaline water solutions, or isolated by resin fixation, reverse osmosis, or ultrafiltration (Alberts and Takács, 2004) affected the development of diverse plant species (for instance, cucumber Cucumis sativus], tomato Solanum lycopersicum], maize Zea mays], wheat Triticum aestivum], Arabidopsis Arabidopsis thaliana], and rapeseed Brassica Napus]) through common signaling pathways, which involved key phytoregulators, such as indole acetic acid (IAA)-nitric oxide (NO; Zandonadi et al., 2010; Canellas et al., 2011; Trevisan et al., 2011; Mora et al., 2012, 2014a), ethylene, and abscisic acid (ABA) in roots (Mora et al., 2012, 2014a) as well as cytokinins in shoots (Mora et al., 2010, 2014b). Recently, Mora et al., 2014a showed that the HA ability to enhance both shoot growth and ABA root concentration in cucumber was regulated by IAA and NO root signaling pathways. However, despite all of this information, the nature of a possible primary, common action on plant roots of HAs with diverse origin and structure remains elusive.Recently, Asli and Neumann (2010) described a new mechanism by which high concentrations of HAs extracted from diverse organic sources decreased shoot plant growth. This mechanism involved the reduction of root hydraulic conductivity (Lp_r) resulting from the fouling of root cell wall pores because of the accumulation and aggregation of HA molecules at root surface. Although the concentration of HAs used by Asli and Neumann (2010) (1 g L⁻¹) is much higher than that related to HA plant growth promotion ability (50–250 mg L⁻¹; Rose et al., 2014), the results do raise the hypothesis that the primary, still unknown event emerging from the interaction of humic substances with root surface cells might involve an unspecific, physical action on root permeability and water uptake. This event might trigger a chain of secondary events in the root that, in turn, would affect specific hormone signaling pathways, which may regulate shoot and root growth. This HA action on plant development would be positive (increasing) or negative (decreasing) depending on HAs concentration in the rhizosphere.To explore the suitability of this hypothesis, we have tested the potential role of Lp_r in the main mechanism by which HAs promote shoot growth in cucumber. To this end, we used a well-characterized and modeled sedimentary humic acid (SHA) at a concentration (100 mg of SHA organic carbon C] L⁻¹) that was associated with plant shoot growth promotion in previous studies (Mora, 2009; Mora et al., 2014a, 2014b). We also investigated the functional relationships between these effects of SHA on Lp_r and shoot growth as well as in some shoot water-related parameters (leaf stomatal conductance G_s] and ABA) and those caused by SHA on IAA-NO and ABA root signaling pathways. Finally, taking into account that root plasma membrane aquaporins (plasma membrane intrinsic proteins PIPs]) are involved in the ABA regulation of Lp_r in other plant systems, we also studied the role of PIPs in SHA effects on plant shoot growth.The results obtained here show that SHA enhances shoot growth in cucumber through ABA-dependent increases in both Lp_r and root PIPs (CsPIPs) gene up-regulation.