Exit control: the role of Arabidopsis hydathodes in auxin storage and nutrient recovery

Abstract

Hydathodes are organs on the leaves of all vascular plants. They regulate the secretion of fluids derived from the xylem sap (Bellenot et al., 2022; Cerutti et al., 2019). When stomata are closed at night and the humidity level levels are too high, the xylem delivers excess water from the roots, which is secreted at the hydathodes in a process called guttation (Figure 1a) (Singh, 2020). Hydathodes are composed of an epidermal surface layer with water pores, and an inner parenchyma, called the epithem, which is highly vascularized and constitutes a direct connection between leaf surface and xylem vessels (Figure 1b) (Bellenot et al., 2022). Hydathodes were first described by the German botanist Anton de Bary in 1877, and named by the Austrian botanist Gottlieb Haberlandt in 1897, from the Greek ‘hyda’ (water) and ‘hodos’ (way) (Bellenot et al., 2022). When Jean-Marc Routaboul, the corresponding author of the highlighted publication, joined Laurent Noël's team at INRAE, France, in 2018, he was surprised to find that hydathodes and the process of guttation were not well understood at the molecular level. Therefore, Routaboul and his colleagues set out to test two long-standing hypotheses about hydathodes: that hydathodes are sites of auxin accumulation, and that they facilitate the withholding of nutrients from guttation fluids (Routaboul et al., 2024).

These hypotheses are based on genes expressed in hydathodes, including those for auxin biosynthesis, transport, and signalling. Moreover, the presence of auxin in hydathodes was detected by antibodies and by using the auxin signalling reporter DR5 (Aloni et al., 2003). Other hydathode-specific genes encode membrane transporters for amino acids, sugar or ions (Nagai et al., 2013), potentially preventing nutrient loss through guttation. For their study, Routaboul et al. combined RNAseq of hydathode-enriched tissue by deep sequencing with a detailed metabolomic analysis of guttation fluids.

First, the authors compared the transcriptome of macro-dissected leaf margins containing hydathodes with the transcriptome of leaf blade tissue of mature Arabidopsis leaves. They found higher expression of genes related to auxin metabolism, stress, DNA, plant cell wall, transport, RNA and lipids in the hydathode-enriched tissue. Genes related to glucosinolate synthesis and transport, the sulfation pathway, metal handling or photosynthesis were more highly expressed in the leaf blade. Because many genes related to auxin biosynthesis were expressed in hydathodes, the authors measured the accumulation of free auxin in hydathode-enriched tissue and leaf blades with liquid chromatography/mass spectrometry (LC/MS) and found nearly 40% more free auxin in hydathode-enriched tissue than in leaf blades. Reporter gene expression confirmed that genes encoding the key auxin biosynthetic enzymes Tryptophan Aminotransferase of Arabidopsis 1 (TAA1), YUCCA2, YUCCA5, YUCCA8 and YUCCA9 were specifically expressed in hydathodes. This suggests that the auxin concentration in hydathodes was high because of localised auxin biosynthesis. Two GH3 IAA-amido synthetases that contribute to maintaining auxin homeostasis by conjugating excess IAA to amino acid conjugates were also more highly expressed in the hydathode-enriched fraction. Therefore, the authors measured oxindole-3-AIA, a downstream product of that pathway. Higher oxindole-3-AIA in hydathodes suggested that hydathodes have a high auxin storage capability. Auxin signalling and response genes like Auxin Response Factors (ARFs) and AUXIN/INDOLE ACETIC ACIDs (Aux/IAAs) were also more highly expressed in hydathodes, altogether suggesting that hydathodes are active sites of auxin synthesis and signalling.

As shown before (Krouk et al., 2010; Misson et al., 2004), the authors found high expression of many genes encoding transporters of water, ions (nitrate, phosphate, sulphate, calcium, zinc, iron, copper, chloride and boron arsenate), hormone transporters (ABA, GA, auxin and cytokinins) and transporters of sugars, peptides, waxes and other organic compounds in hydathodes, suggesting that hydathodes could be sites that actively modify guttation fluid. Therefore, the authors sampled xylem fluid exuding from the petiole and compared its composition to that of pre-hydathode fluid sampled from leaves with excised hydathodes and to guttation fluid collected from hydathodes, using gas chromatography/MS. Leaf tissues captured 91% of the metabolites before they reached the hydathode. From the pre-hydathode fluid, 78% of the remaining metabolites were captured before the fluid got released by the hydathodes. The concentration of 23 metabolites (including amino acids, organic acids, sugars and myo-inositol) was lower in the guttation fluid than in the pre-hydathode fluid. Colorimetric assays showed that inorganic phosphate (Pi) and nitrate were taken up by the hydathode. Additionally, the authors used inductively coupled plasma-optical emission spectrometry (ICP-OES) and found lower concentrations of phosphorus, calcium and magnesium in the guttation fluid than in the pre-hydathode fluid. This suggests that hydathodes capture specific organic and mineral compounds before guttation.

The authors focused on nitrate transporter NRT2.1 and Pi transporter PHT1;4, which are highly expressed in hydathodes and well-studied in roots (Little et al., 2005; Shin et al., 2004). Higher nitrate and Pi levels in the guttation fluid of nrt2.1 and pht1;4 mutants suggested that the transporters contribute to the uptake of Pi and nitrate from the guttation fluid. PHT1;4 expression matched Pi accumulation sites in leaves, with more Pi in leaf margins than blades (Figure 1c). The margins of pht1;4 mutants contained less Pi than WT at the margins, suggesting that Pi is actively captured by PHT1;4 from the guttation fluid. Metabolites and minerals taken up from the guttation fluid can be recycled back into the leaves: an autoradiogram of a barley leaf fed with ³²Pi at hydathodes showed an ingress of Pi from the hydathode after 2 h followed by diffusion to the entire leaf tip after 1 day (Nagai et al., 2013). In conclusion, Routaboul et al. speculate that hydathodes act analogously to nephrons in kidneys, filtering the guttation fluid and retrieving valuable nutrients.

Hydathodes are essential for maintaining plant water status and to get rid of excess water that could lead to mesophyll flooding, lower photosynthesis/respiration and leaf necrosis. Beside their role in metabolite scavenging, hydathodes can also mediate excretion of undesired or toxic compounds such as boron (Sutton et al., 2007) or nanoparticles (Zhang et al., 2011). They are also the main entry for several plant pathogens (Cerutti et al., 2017). Understanding hydathode physiology could thus have implications for adapting plant performance in stressful conditions such as flooding, drought, immunity or phytoremediation.