Anatomy of epithemal hydathodes in four monocotyledon plants of economic and academic relevance

Hydathode is a plant organ responsible for guttation in vascular plants, i.e. the release of droplets at leaf margin or surface. Because this organ connects the plant vasculature to the external environment, it is also a known entry site for some vascular pathogens. In this study, we present a detailed microscopic examination of monocot hydathodes for three crops (maize, rice and sugarcane) and the model plant Brachypodium distachyon. Our study highlights both similarities and specificities of those epithemal hydathodes. These observations will serve as a foundation for future studies on the physiology and the immunity of hydathodes in monocots.


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Hydathodes are organs found on leaves, sepals and petals of all vascular plants and are responsible 36 for guttation. This phenomenon is the release of fluids usually observed in conditions where stomata 37 are closed and humidity high. Guttation is supposed to play an important role in plant physiology to 38 promote water movement in planta in specific conditions [1,2], to detoxify plant tissues by exporting 39 excessive salts or molecules [3,4] and to specifically capture some solutes from xylem sap before   dieffenbachiae [10,11], in bacterial canker of tomato caused by Clavibacter michiganensis subsp. 56 michiganensis [12] and in bacterial leaf blight of rice caused by X. oryzae pv. oryzae (Xoo) [13][14][15][16].

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Certain pathogens are thus adapted to colonize the hydathode niche and access plant vasculature. Though hydathodes were first described over a century ago, their anatomy is still poorly described.

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Most published studies use single microscopic techniques and provide descriptions of either surface 61 or inner organizations so that a global overview of the organ is difficult to capture. Because most of 62 the anatomic studies were performed before the 80s, literature search engines such as Pubmed will 63 not lead you to such publications. Anatomy of arabidopsis hydathodes has only been recently reported 64 [9]. Only scarce descriptions are available for monocot hydathodes, and none in the model plant 65 Brachypodium distachyon. In rice (Oryza sativa) hydathodes, the large vessel elements are not 66 surrounded by a bundle sheath but included in a lacunar mesophyll facing water pores [17,18]. In 67 barley (Hordeum vulgare), a single hydathode is also found at the leaf tip and water pores are reported 68 to be very close to vascular elements [19]. In wheat (Triticum aestivum), an ultrastructural study 69 showed that intercellular space directly connects vessel elements with water pores [17]. Determining 70 or refining the anatomy of hydathodes in those or other monocots is a thus a prerequisite to study the 71 physiology and immunity of those organs.

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In this study, we report on anatomy of hydathodes in four species of monocots, such as rice, 74 sugarcane, maize and the model plant Brachypodium distachyon using a combination of optical and 75 electron microscopy on fresh or fixed tissues. Our study highlights both similarities and specificities 76 of those epithemal hydathodes and provides a comprehensive overview of their anatomy.   164 Some levels of variation in this organization pattern can be observed (See schematic drawings in Fig.   165 1I, 2H, 3J, 4J). In maize and rice, the epithem occupies with the vasculature the whole inner space of 166 the hydathodes. In rice, the epithem is sometimes so reduced (Fig. 2F-H) so that the connection 167 between the water pores and the vascular elements is sometimes direct (Fig. 2G). In sugarcane and

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In order to better observe the cell wall of xylem vessels within hydathodes, we used transmission 177 electron microscopy (TEM) coupled to PATAg labelling of cell wall polysaccharides (S2 Fig.).  In monocots, guttation is always observed at leaf tips and sometimes at leaf margins such as in maize 190 and sugarcane (Fig. 4)

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Several markers for stomatal identity or differentiation cannot be used to differentiate water pores for 205 stomata either [9], thus suggesting a common origin of both cell types. Yet, several morphological 206 differences can distinguish water pores from stomata. Water pores are often inserted deeper in the 207 epidermis. Also, the subsidiary cells known to be important for stomatal movement [25] could not be 208 observed around water pores. Yet, the transition from water pores to stomata is not as dramatic in 209 monocots as in dicots [9,26] and it is thus sometimes difficult to locate the hydathode boundaries in 210 monocots. We could observe a developmental gradient between water pores and stomata along the 211 leaf longitudinal axis with morphological traits of water pores being more pronounced at leaf apex.

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If the chemical nature of such gradient is unknown, auxin maxima and expression of auxin 213 biosynthetic genes is observed in rice hydathodes [27] similar to dicots [28,29]. Because auxin was 214 recently described as a negative regulator of stomatal differentiation [30], it remains to be tested 215 whether auxin accumulating at hydathodes could impact water pore differentiation and be responsible 216 for the observed developmental gradient.