Plant, Cell and Environment (2008) 31, 1007–1016
doi: 10.1111/j.1365-3040.2008.01813.x
Photosynthesis in aquatic adventitious roots of the halophytic stem-succulent Tecticornia pergranulata (formerly Halosarcia pergranulata) SARAH M. RICH1, MARTHA LUDWIG2 & TIMOTHY D. COLMER1 1 School of Plant Biology (M084), Faculty of Natural and Agricultural Sciences and 2School of Biomedical, Biomolecular and Chemical Sciences (M310), Faculty of Life and Physical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia
ABSTRACT In flood-tolerant species, a common response to inundation is growth of adventitious roots into the water column. The capacity for these roots to become photosynthetically active has received scant attention. The experiments presented here show the aquatic adventitious roots of the floodtolerant, halophytic stem-succulent, Tecticornia pergranulata (subfamily Salicornioideae, Chenopodiaceae) are photosynthetic and quantify for the first time the photosynthetic capacity of aquatic roots for a terrestrial species. Fluorescence microscopy was used to determine the presence of chloroplasts within cells of aquatic roots. Net O2 production by excised aquatic roots, when underwater, was measured with varying light and CO2 regimes; the apparent maximum capacity (Pmax) for underwater net photosynthesis in aquatic roots was 0.45 mmol O2 m-2 s-1. The photosynthetic potential of these roots was supported by the immunolocalization of PsbA, the major protein of photosystem II, and ribulose-1-5-bisphosphate carboxylase/ oxygenase (Rubisco) in root protein extracts. Chlorophyllous aquatic roots of T. pergranulata are photosynthetically active, and such activity is a previously unrecognized source of O2, and potentially carbohydrates, in flooded and submerged plants. Key-words: adventitious roots; aquatic roots; flooding; halophyte; inland salt marsh; root aeration; root photosynthesis; samphire; underwater photosynthesis; waterlogging. Abbreviations: a, apparent quantum yield of photosynthesis; DIC, dissolved inorganic carbon; G, CO2 compensation point; GI, light compensation point; KS, apparent half saturation constant; Pmax, maximum photosynthetic rate; PN, net photosynthesis.
INTRODUCTION Flooding is an environmental stress faced by many terrestrial plants (Armstrong et al. 1991; Blom & Voesenek 1996; Correspondence: T. D. Colmer. Fax: +61 8 6488 1108; e-mail:
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Voesenek et al. 2006). After inundation, soils quickly become anaerobic, as soil water is depleted of dissolved O2 through the respiration of roots and micro-organisms (Drew & Lynch 1980). Re-oxygenation effectively ceases because of the lower solubility and approximately 10 000fold slower diffusion rate of O2 in water than in air (Grable 1966). Inundation also results in reduced availability of CO2 to submerged photosynthetic organs (Smith & Walker 1980; Sand-Jensen & Frost-Christensen 1998). Soil anoxia and limited access to CO2 for photosynthesis, when shoots are also submerged, usually result in internal O2 deficits, inhibition of respiration and depletion of energy reserves (Armstrong 1979; Voesenek et al. 2006). Plant survival during flooding is dependent on a range of morphological, anatomical and metabolic mechanisms, allowing plants to either avoid internal O2 deficits or to function with anoxic tissues (Armstrong 1979; Crawford & Braendle 1996; Vartapetian & Jackson 1997; Greenway & Gibbs 2003). A common response to flooding is the formation of adventitious roots (Jackson & Drew 1984); these roots arise from a previously lateralized root axis or a site on the plant which is not a root, for example, stems (Barlow 1994). In flooded conditions, many plants produce adventitious roots from the submerged portion of stems (Hook, Brown & Kormanik 1971; Jackson & Drew 1984; Kozlowski 1997). These roots can grow into the soil (sediment roots) or be suspended in the water column (aquatic roots), allowing the plants to exploit resources from both environments (Waisel & Agami 1996). Adventitious roots of wetland species form aerenchyma and may have a barrier to radial O2 loss, features that enhance internal O2 supply, aiding avoidance of tissue anoxia (Armstrong 1979; Colmer 2003). Another feature that has received scant attention is the ability, in some species, for aquatic adventitious roots to develop photosynthetically active chloroplasts that would provide an internal source of O2. Plastid development in soil roots does not usually result in production of chloroplasts. The pathway of chloroplast development is blocked; chlorophyll is not synthesized, and thylakoid extension is limited (Whatley 1983). Chloroplast development within roots can, in many species, be experimentally stimulated through exposure to light (Powell 1925; 1007
1008 S. M. Rich et al. Whatley 1983; Armstrong & Armstrong 1994); however, as roots rarely occur naturally in illuminated environments, it is only in a few species that chloroplast biosynthesis occurs under natural conditions. These include species in which part of the root system occurs above-ground, such as aerial roots of some epiphytic orchids (Benzing & Ott 1981; Hew et al. 1984; Cockburn, Goh & Avadhani 1985; Aschan & Pfanz 2003), mangrove pneumatophores (Dromgoole 1988), and floating aquatic species (Mollenhauer 1967; Wroblewski 1973; Whatley & Gunning 1981; Ishimaru et al. 1996). Root chloroplasts are generally very similar in structure to chloroplasts in leaves, showing well-differentiated thylakoid membranes and starch grains (Whatley 1983; Flores et al. 1993). However, this does not necessarily indicate that these organelles can support light harvesting and/or CO2 assimilation. Björn (1963) showed that pigment synthesis within root chloroplasts can be extremely slow, and it is possible that the photosynthetic components and pathways do not develop completely in all roots that appear green (Fadeel 1962; Whatley 1983). Few investigations have examined the photosynthetic capacity of ‘green’ roots, and none of these has evaluated aquatic roots formed by a terrestrial species. Rates of CO2 uptake have been measured in laboratory-induced green roots of both wheat (Triticum sp.) and flax (Linum usitatissimum) (Fadeel 1963), in pneumatophores of Avicennia marina (Dromgoole 1988) and in the aerial roots of several orchids (Dycus & Knudson 1957; Benzing et al. 1983; Hew et al. 1984; Cockburn et al. 1985). Rates of O2 evolution were assessed for the filamentous roots of the aquatic Trapa bispinosa (Ishimaru et al. 1996). The experiments presented here tested the hypothesis that aquatic adventitious roots of the halophyte Tecticornia pergranulata (J. M. Black) K. A. Sheph. & Paul G. Wilson ssp. pergranulata (syn. Halosarcia pergranulata ssp. pergranulata) (Shepherd & Wilson 2007) contain photosynthetically active chloroplasts. Firstly, chloroplast presence and transverse distribution in adventitious roots were evaluated using fluorescence microscopy. Secondly, chloroplast-containing roots were characterized by measuring chlorophyll concentrations, examining root protein extracts for the presence of PsbA and ribulose-1-5bisphosphate carboxylase/oxygenase (Rubisco) and quantifying net underwater photosynthesis by measuring O2 evolution by roots under varying light and CO2 regimes. Photosynthetic O2 evolution within roots of flooded plants would provide a source of O2 in addition to that available from the flood water or internally via the aerenchyma. The ability of these organs to fix CO2 would also be beneficial to the carbon balance of the plants.
MATERIALS AND METHODS Study site and plant material Tecticornia pergranulata ssp. pergranulata (hereafter referred to as T. pergranulata) is a succulent C3 plant that
grows on the margins of salt lakes and brackish swamps across southern Australia and is well adapted to high salinity levels and seasonal inundation (Short & Colmer 1999; Pedersen, Vos & Colmer 2006). Tecticornia pergranulata grows as an erect subshrub, rarely higher than 0.5 m, with articulated, succulent stems (Wilson 1980). When flooded, larger individuals of T. pergranulata grow an extensive system of aquatic adventitious roots from the woody basal stem regions (Fig. 1). Smaller plants, such as those used by Pedersen et al. (2006), do not form aquatic roots, but do form adventitious roots in the sediments. Aquatic adventitious roots formed by T. pergranulata exhibit two distinctive growth forms; the most abundant roots are exclusively aquatic, floating in the water column (hereafter referred to as aquatic roots, Fig. 1). These roots are usually superficially pink, but also can be brownish/ green, especially in the basal regions, and typically have diameters 5 kPa) in waterlogged-flooded soils and possible effects on root growth and metabolism. Annals of Botany 98, 9–32 Grossman A.R., Bhaya D., Apt K.E. & Kehoe D.M. (1995) Lightharvesting complexes in oxygenic photosynthesis: diversity, control, and evolution. Annual Review of Genetics 29, 231– 288. Hew C.S., Ng Y.W., Wong S.C., Yeoh H.H. & Ho K.K. (1984) Carbon dioxide fixation in orchid aerial roots. Physiologia Plantarum 60, 154–158. Hook D.D., Brown C.L. & Kormanik P.P. (1971) Inductive flood tolerance in swamp tupelo (Nyssa sylvatica var. biflora (Walt.) Sarg.). Journal of Experimental Botany 22, 78–89. Ishimaru K., Kubota F., Saitou K. & Nakayama M. (1996) Photosynthetic response and carboxylation activity of enzymes in leaves and roots of water chestnut Trapa bispinosa Roxb. Journal of the Faculty of Agriculture, Kyushu University 41, 57–65. Jackson M.B. & Drew M.C. (1984) Effects of flooding on growth and metabolism of herbaceous plants. In Flooding and Plant Growth (ed. T.T. Kozlowski), pp. 47–111. Academic Press Inc., London, UK. Jassby A.D. & Platt T. (1976) Mathematical formulation of the relationship between photosynthesis and light for phytoplankton. Limnology and Oceanography 21, 540–547. Kettunen R., Tyystjarvi E. & Aro E.-M. (1996) Degradation pattern of photosystem II reaction center protein D1 in intact leaves. Plant Physiology 111, 1183–1190. Kozlowski T.T. (1997) Responses of woody plants to flooding and salinity. Tree Physiology Monograph 1, 1–29. Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. Lerman A. (1978) Chemical exchange across sediment–water interface. Annual Review of Earth and Planetary Sciences 6, 281– 303. Marder J.B., Mattoo A.K. & Edelman M. (1986) Identification and characterization of the psbA gene product: the 32-kDa chloroplast membrane protein. Methods in Enzymology 118, 384– 396. Mollenhauer H.H. (1967) A comparison of root cap cells of epiphytic, terrestrial and aquatic plants. American Journal of Botany 54, 1249–1259. Mommer L. & Visser E.J.W. (2005) Underwater photosynthesis in flooded terrestrial plants: a matter of leaf plasticity. Annals of Botany 96, 581–589. Pedersen O., Vos H. & Colmer T.D. (2006) Oxygen dynamics during submergence in the halophytic stem succulent Halosarcia pergranulata. Plant, Cell & Environment 29, 1388–1399. Porra R.J., Thompson W.A. & Kriedemann P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic adsorption spectroscopy. Biochimica et Biophysica Acta 975, 384–394. Powell J. (1925) The development and distribution of chlorophyll in roots of flowering plants grown in the light. Annals of Botany 39, 503–513.
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