Podocarpaceae Endl. is the second largest extant conifer family in terms of number of genera and it exhibits the greatest amount of morphological diversity (Taylor et al., 2009). Leaves of most podocarps are univeined, and it is a major limitation on overall leaf size and shape (Hill, Brodribb, 1999). In such leaves, water conduction occurs through the so-called transfusion tissue (TT). It is common for many species of gymnosperms (Frank, 1864; Esau, 1977; Hu, Yao, 1981). This adaptation in Podocarpaceae, along with shoot flattening, enables them to compete successfully with angiosperms in the tropics and temperate zones (Hill, Brodribb, 1999; Brodribb, 2011). Podocarpaceae of the temperate zone of south-central Chile include five species (Veblen et al., 2005), four of which possess leaves comparable in size with angiosperms. They are of interest for comparative analysis of water-conducting tissue architecture and water relations in leaves. The aim of the study is to identify water use strategies in the leaves of endemic Podocarpaceae of south-central Chilean temperate rainforest. Plant specimens were collected in Puyehue National Park and Nahuelbuta National Park. The methods used in the study are light and transmission electron microscopy.
The water conducting system in the leaves of Podocarpus salignus D. Don. includes the main vein, conspicuous transfusion tissue and accessory transfusion tissue (ATT). TT cells lie on both sides of the midvein and have an irregular isodiametric shape, sometimes elongate. They bear scalariform thickenings or circular bordered pits. Pit apertures are circular or slitlike. The border is pronounced. One may describe the TT tracheids in P. salignus leaves as specialized. They differ from wood tracheids in the disordered pit arrangement that cannot be classified as either alternate or opposite. Primary cell walls of transfusion tracheids are loose. Accessory transfusion tissue is composed of long tracheids, which extend perpendicularly to the main vein and almost reach the edge of leaf lamina. They have conspicuous cavities and slitlike bordered pits. Primary cell walls of ATT tracheids are dense. There are many prismatic or irregular crystals on the cell surface and in the primary cell wall. Parenchyma cells between ATT and mesophyll also bear numerous crystals on the walls contacting with intercellular spaces.
The water conducting system in the leaves of Prumnopitys andina (Poepp. ex Endl.) de Laub. includes the main vein with transfusion tissue located on both sides of it. TT consists of circular-elongated tracheids with reticulated thickenings, some of them with a prominent border. Primary cell walls of transfusion tracheids are loose. In the TT sheath cells there are occasional large prismatic crystals in the middle lamella. Sometimes they can be found on the cell walls contacting with intercellular spaces. Mesophyll is multilayered and isolateral.
The water conducting system in the leaves of Podocarpus nubigenus Lindl. includes the main vein with scarce transfusion tissue located on both sides of it. TT includes isodiametric tracheids of irregular shape bearing scalariform and reticulate thickenings, which are occasionally located in the same cell. Primary cell wall in the TT tracheids is heterogeneous: there are areas of loose and dense structure. A significant part of the mesophyll volume in this species is taken up by water-storage tissue (water-storing parenchyma). Its cells are less specialized and similar in shape and size to spongy mesophyll cells.
The water conducting system in the leaves of Saxegothaea conspicua Lindl. includes the main vein with extremely scarce transfusion tissue located on both sides of it. TT consists of isodiametric tracheids of irregular shape with reticulate cell wall thickening. The border is not prominent. Primary cell walls of transfusion tracheids are heterogeneous. A significant part of the mesophyll volume is taken up by water-storage tissue, which consists of large cells with chloroplasts indistinguishable in the light microscope.
Accessory transfusion tissue, which is characteristic of the P. salignus leaves, was described in broadleaf podocarps (Griffith, 1957; Brodribb et al., 2007; Locosselli, Ceccantini, 2012). ATT is considered to be an adaptation designed to enhance hydraulic conductance (Kleaf) through leaf lamina in the regions remote from the main vein (Brodribb, Holbrook, 2005; Brodribb et al., 2007). ATT cell function is analogous to regular xylem tracheids. The efficiency of this adaptation is quite obvious in that it enables single-vein leaves to achieve leaf widths that are orders of magnitude wider than predicted if they possessed unmodified mesophyll (Brodribb et al., 2007). ATT is an alternative to the dense venation system common in most angiosperms and Gnetaceae. It enables P. salignus to produce leaves with area more than 3 times larger than in podocarps lacking ATT. Pronounced transfusion tissue with loose primary cell walls permeable by water also reflects the specialization of this species in water conduction through leaf. P. salignus has a tendency to occur in gravelly riversides and moist canyons (Debreczy, Rácz, 2012), where it is not necessary to save water. P. andina leaves do not possess accessory transfusion tissue, because of which their size is significantly restricted. However, Brodribb et al. (2014) revealed that P. andina Kleaf is about 4 mmol/(m2sMPa). This value exceeds the P. salignus hydraulic conductance (2.3 mmol/(m2sMPa) measured in the same study. Multilayered isolateral mesophyll in P. andina leaves apparently greatly increases its conductivity to water. We assumed that P. andina leaves have a capacity to conduct water apoplastically through the mesophyll, which is sufficient given the small leaf size. Primary cell walls in transfusion tracheids in P. andina leaves are loose, allowing unimpeded water movement. Crystals in the P. andina and P. salignus leaves are likely to be calcium oxalate and are the result of the elevated water flux through the leaves.
S. conspicua and P. nubigenus possess another strategy, which is based on the accumulation, conservation and economical use of the water. It is realized through water-storage tissue (hydrenchyma) well-developed in both species. Water-storing cells in S. conspicua leaves are larger in size compared with chlorenchyma cells. They contain poorly developed chloroplasts and are simplastically isolated from spongy mesophyll cells. Water-storage tissue takes up the entire central part of the leaf in both species. This tissue in the P. nubigenus leaves is less specialized and contains well-developed chloroplasts. Water-storing cells are similar to spongy mesophyll cells and are connected with them by numerous plasmodesmatal connections. Transfusion tissue is poorly developed in both species. It is represented by single cells. At the same time, primary cell walls in transfusion tracheids contain dense areas, which may potentially impair their water permeability. S. conspicua leaf hydraulic conductance varies from 1.6 mmol/(m2sMPa) (Brodribb et al., 2005) to 2.5 mmol/(m2sMPa) (Brodribb et al., 2014). P. nubigenus leaves have not yet been studied for this parameter, but it is reasonable to assume that it is within a similar range.
We have identified two water relation strategies in the podocarpaceous leaves of the studied community. The first is based on the intensification of water movement through the leaf. It manifests itself in the presence of specialized transfusion tissue tracheids and tracheids of accessory transfusion tissue (P. salignus) or in the tight placement of mesophyll cells, creating conditions for a continuous water path through apoplast (P. andina). The second strategy aims to accumulate water and use it sparingly. It is characteristic of species with water-storage tissue and poorly developed transfusion tissue tracheids (P. nubigenus and S. conspicua).
The authors are grateful to the Core Centre ‘Cell and Molecular Technology in the Plant Science’ at the Komarov Botanical Institute and the Resource Center for Molecular and Cell Technologies and Resource Center “Chromas” of SPBU Research park. This study was supported by the RFBR (grant No. 17-04-01213A to AAP) and government assignment of the Komarov Botanical Institute (АААА-А18-118031690084-9 “Structural-functional basis of higher plants development and adaptation”).